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Latest Articles of Bio-Synthesis Inc.

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    Stable isotopes can be used to study metabolic pathways and turnover rates of biological molecules such as proteins, peptides, RNA and others.

    Stable isotopes were used to define metabolic pathways and turnover of biological molecules in the body shortly after the discovery of deuterium in 1931, the heavy isotope of hydrogen, by Harold Urey. Urey received a Nobel Prize in 1934 for this discovery. Already in 1935 Rudolf Schoenheimer used isotopic tracer techniques in metabolic research. His work, for the first time, provided clear experimental evidence of the dynamics of the body’s metabolism. Schoenheimer’s research group defined synthesis and degradation pathways of many compounds in the following years. The availability of enriched 15N, the heavy isotope of nitrogen, enabled demonstrating that protein had a dynamic life cycle in that they were both continually being synthesized and degraded. However, all of this early work was performed using crude methods that involved the preparation of labeled compounds, followed by tedious measurements using isotope ratio mass spectrometry (IRMS). In addition, for the measurements of the isotopic enrichment, all compounds investigated needed to be reduced to the simple gases carbon dioxide (CO2), hydrogen (H2), and nitrogen (N2). After World War II, the radioisotopes tritium (3H) and carbon 14 (14C) were mostly used for many years to come. Improvements made in instrument development, specifically for gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) instruments, allowed the use of stable isotopes for metabolic and proteomic studies with more ease in recent decades. The increasing trend for the use of isotopes, in particular the use of stable isotopes for metabolic or proteomic studies, is reflected in the increased publication volume of papers covering these applications which can be seen in figure 1. More than 18,500 publications for isotope labeling have been published since 1953 and can be found in PubMed. 



    Figure 1: Timeline of papers in PubMed describing various isotope labeling methods and applications. As can been seen the use of isotopes, in particular stable isotopes, has increased in recent years.


    Figure 2: The helium, as an example, has 2 protons, 2 neutrons, and 2 electrons. Helium has two isotopes but it consists almost entirely of He-4 with natural He only containing little more than 0.0001% of He-3.

     

    Let us briefly review the basic chemistry and physical properties of isotopes. Elements contain nuclei each with their own unique physicochemical characteristics. According to the “atomic theory”, each atomic nucleus can be defined by its mass, its number of constituent elementary particles, its spin rate, its magnetic strength, its electric charge, and its multitude of existing forms. The uniqueness of elements is reflected in their characteristic ratio of isotopes. This phenomenon has allowed geological scientists to study the geological record of the earth’s crust revealing its history. Carbon dioxide (CO2) and methane (CH4) in the atmosphere record its history and the impact of human beings on the Earth’s atmosphere which can be studied from ice cores, for example taken from glaciers or eternal ice fields, in the Alps, in Greenland or in the Antarctic. Atoms are the smallest units of elements that exist in nature. Every atom consists of protons, neutrons, and electrons. Nuclides are a specific type of atoms or nuclei. Every element, E, or AZ, can be defined by its atomic number, Z. This so-called “atomic number”, Z, is the number of protons in the atomic nucleus. In electrically neutral atoms it is also the number of electrons. However, the atomic mass or atomic weight is the sum of the masses of protons, neutrons, and electrons. The atomic mass or the mass number of heavy particles made up of neutrons also called nucleons, can be defined as A = Z + N + ne, where A is the “mass number” , Z is the number of protons, N is the number of neutrons, and ne is the number of electrons. Since electrons are ~1,836 times lighter than the hydrogen atom A is usually expressed as the sum of Z and N. 

    A few definitions as they relate to isotopes follow.


    Mass number:            A: A = Z + N


    Protein:           The proton is a nuclear particle with the charge number +1, a spin quantum number ½, and a rest mass of 1.007276470(12) u.

     

    Neutron:    The neutron is a nuclear particle of zero charge, a spin quantum number ½, and a mass of 1.008664905(14) u.

     

    Electron:       The electron is an elementary particle that is not affected by the strong force. The electron has a spin quantum number ½, a negative charge and a rest mass of 0.00054879903(13) u.

     

    Atomic mass constant:         

     

    The atomic mass constant, u or mu, is defined as one 12 (1/12) of a carbon 12 atom in its nuclear and electronic ground state. u = mu = 1.6605402(10) x 10-27 kg = amu, atomic mass constant. Therefore the mass of a proton equals 1.007276 amu, the mass of a neutron equals 1.008665 amu, and the mass of an electron equals 0.00054858 amu.

     
    Atomic mass or weight:      

     

    Usually the atomic mass or weight is the average mass of all atoms in an element calculated from the relative abundance of the naturally occurring isotopes of the element.

     

    Atomic mass unit:     

     

    The atomic mass unit (symbolized AMU or amu) is defined as precisely 1/12 the mass of an atom of carbon-12. The carbon-12 (C-12) atom has six protons and six neutrons in its nucleus.


    In imprecise terms, one AMU is the average of the proton rest mass and the neutron rest mass. This is approximately 1.67377 x 10 -27 kilogram (kg), or 1.67377 x 10 -24 gram (g). The mass of an atom in AMU is roughly equal to the sum of the number of protons and neutrons in the nucleus. For example, in the case of oxygen, each isotope of this element has the same number of protons, Z = 8. Oxygen has three stable isotopes, A = 16, A = 17, and A = 18. From the sum of nucleons it is evident that these contain N = 8, N = 9, and N = 10 neutrons, respectively, within their nuclei, so that they produce the three mass numbers for oxygen. The notations of these are written with the mass number as a preceding superscript: 16O, 17O and 18O.

    Table : Isotopes and their natural abundance commonly used as tracers in biological experiments

    Z

    Name

    Symbol

    Mass of Atom (u)

    Abundance in %

    1

    Hydrogen

    Deuterium

    Tritium

    1H

    2H

    3H

      1.007825

      2.014102

      3.016049

    99.9885

    0.0115

    *

    6

    Carbon

    12C

    13C

    14C

    12.000000

    13.003355

    14.003242

    98.93

    1.07

    *

    7

    Nitrogen

    14N

    15N

    14.003074

    15.000109

    99.632

    0.368

    8

    Oxygen

    16O

    17O

    18O

    15.994915

    16.999132

    17.999160

    99.757

    0.038

    0.205

    15

    Phosphorus

    31P

    30.973762

    100

    16

    Sulphur

    32S

    33S

    34S

    36S

    31.972071

    32.971458

    33.967867

    35.967081

    94.93

    0.78

    4.29

    0.02


    The heavy isotopes most often used for metabolic studies are deuterium, 2H, heavy carbon, 13C and heavy nitrogen, 15N. Labeled amino acids and peptides plus tagging molecules are now commercially available. Virtually almost any labeled peptide can be synthesized using Fmoc-chemistry based automated synthesis.


    IRMS

     

    Isotope ratio mass spectrometry (IRMS) is a technique that has found an increasingly widespread use in archaeology, medicine, geology, biology, food authenticity, and forensic science. IRMS instruments have the ability to accurately and precisely measure variations in the abundance of isotopic ratios of light elements such as 13C/12C, 18O/16O, D/1H, 15N/14N, and 34S/32S. The ratios of these isotopes always need to be measured relative to an isotopic standard in order to eliminate any bias or systematic error that can occur during the measurements. IRMS provides information about the geographic, chemical, and biological origins of substances. The relative isotopic abundances of elements in the studied material allow determination of the source of an organic substance. Isotope ratios of elements, such as carbon, hydrogen, oxygen, sulfur, and nitrogen, can become locally enriched or depleted through a variety of kinetic and thermodynamic factors. The isotope ratios allow differentiation of samples which otherwise share identical chemical compositions.

     

    Labeling of cell compartments with stable isotopes avoids the use of radioactive tracer or labeling compounds. However, similar to radioactive isotopes, stable isotopes can be incorporated into bio-molecules without any changes in their chemical structure. Stable isotopes commonly used include deuterium, 2H, heavy nitrogen, 15N, heavy carbon, 13C and heavy oxygen, 18O. After incorporation into metabolic molecules, for example by metabolic labeling of cells, compounds containing these stable isotopes are detected in a mass spectrometer (MS) due to their increased mass when compared to natural light isotopes.

    During the last decades classical protein analysis has evolved into proteomics resulting in a flood of new improved technologies and approaches for the study of cells and their compartments. Even though genes encode and regulate proteins, it is the proteins that are responsible for most life functions and usually make up the majority of structures in cells or tissue. These large complex molecules are synthesized from smaller subunits called amino acids. The chemical nature of the 20 natural amino acids found in proteins determine their specific three-dimensional (3D) structures and define the particular function of a protein in the cell. The proteome refers to the entire set or complement of proteins that is or can be expressed by a cell, tissue, an organism or an organelle. Unlike the genome the proteome is more dynamic and changes from minute to minute in response to external and internal chemical cues. The chemistry of a protein is determined by its sequence as well as by the number and nature of other proteins or molecules it interacts with in the cell. Due to its complexity, proteomics, the study to explore protein structures and activities in an organism, will be the focus of research for many years to come. The Human Genome Project generated a flood of genomic and ultimately proteomic data which analyses will continue for many years.

    The Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of Energy (DOE) and the National Institutes of Health that was completed in 2003. The goal of this project was to identify all the approximately 20,500 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project. [Source:http://web.ornl.gov/sci/techresources/Human_Genome/publicat/jmmbbag.pdf;http://genomics.energy.gov/http://www.proteinatlas.org/.]

    Presently, there is no clear consensus among scientists which isotope labeling strategy is the best one to use or is considered to be the “best labeling practice”. Ultimately, the selection of the isotope labeling technique will dependent upon experimental design, the scope of a particular analysis and the sample or system being analyzed. However, quantitative analysis of proteins and peptides is a very important issue in mass spectrometry based proteomics. During the last decade several isotope labeling techniques have been developed and introduced. These labeling techniques allow studying protein structures with the help of mass spectrometry based approaches or nuclear magnetic resonance (NMR) based experiments or techniques. Improvements made in both technologies have greatly expanded the range of biological applications that can now be studied.

    Types of isotope labeling methods that were developed since the 1960s are:

    • Uniform labeling with the stable isotopes 13C, 15N, and 2H.
    • Differential labeling of proteins and peptides.
    • Amino acid type selective labeling.
    • Site-specific labeling.
    • Random or fractional labeling.
    • Specific protonated amino acids against a deuterated background.
    • Selective incorporation of 15N-labeld amino acids against an unlabled (12C/14N) background.
    • Methyl specific protonation.
    • Segmental labeling.
    • Stereo-arrayed labeling.

     

    Recombinant proteins have now become an important part of medicine. These therapeutic proteins are used for the treatment of many different diseases. Unfortunately, proteins can have posttranslational modifications (PTMs) that may occur during manufacturing and storage. The result is product-related impurities. With improvements made in mass spectrometry instrumentation mass spectrometry is now being widely used for the characterization of recombinant proteins and their PTMs. The used of liquid chromatography coupled with mass spectrometry (LC-MS) and search algorithms makes it possible to identify hundreds of PTMs in a single LC-MS analysis. However, the quantification of PTMs using mass spectrometry is challenging. To address this problem many approaches using stable isotope-labeled internal standards have been developed. The goal is to provide accurate and precise quantification of proteins. In addition, many different approaches can be used to introduce stable isotopes into the sample.   

    Reference

    Atreya, Hanudatta S. (Ed.); Isotope labeling in Biomolecular NMR. Series: Advances in Experimental Medicine and Biology, Vol. 992, 2012, VIII, 219 p. 75 illus., 45 illus. in color.

    Cambridge Isotope Laboratories, Inc.: http://www.isotope.com/

    Donald Clayton; Handbook of Isotopes in the Cosmos. Hydrogen to Gallium. August 2007. Cambridge University Press. www.cambridge.org.  isbn: 9780521530835

    Xinzhao Grace Jiang, Izydor Apostol, Quanzhou Luo, Jeffrey Lewis, Ronald Keener III, Shun Luo,

    Matthew Jerums, Xin Zhang, Jette Wypych, Gang Huang; Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. I: Principles and applications. Analytical Biochemistry 421 (2012) 506–516.


    Link to the Pandey Lab: http://www.silac.org/


    Zeland Muccio and Glen P. Jackson; Isotope ratio mass spectrometry. First published as an Advance Article on the web 14th November 2008. DOI: 10.1039/b808232d.


    Ray H. Liu, Dennis V. Canfield, Sheng-Meng Wang;  Quantitation and Mass Spectrometric Data of Drugs and Isotopically Labeled Analogs. Published: August 5, 2009 by CRC Press.


    Karen A. Sap and Jeroen A. A. Demmers (2012); Labeling Methods in Mass Spectrometry Based Quantitative

    Proteomics, Integrative Proteomics, Dr. Hon-Chiu Le ung (Ed.), ISBN: 978-953-51-0070-6, InTech, Available

    from: http://www.intechopen.com/books/integrative-proteomics/labeling-methods-in-mass-spectrometry-bas

    ed-quantitative-proteomics

    Warscheid, Bettina (Ed.); Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods and Protocols. Methods in Molecular Biology, Vol. 1188, 2014, XIV, 372 p. 65 illus., 27 illus. in color. Humana Press.

    D. Rodriguez, G. Audi, J. Aystö, D. Beck, K. Blaum, et al.. Accurate mass measure-

    ments on neutron-defcient krypton isotopes. Nuclear Physics A, Elsevier, 2006, 769, pp.1-15.10.1016/j.nuclphysa.2006.02.001.in2p3-00025070


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  • 12/12/14--00:00: Dual labeled probes
  •  Dual labeled probes

     

    Methods that use fluorescence are widely used in biological sciences including biochemistry, biophotonics, biophysics, cell biology, clinical chemistry, histochemistry, and molecular medicine. Molecules in electronically excited states emit light and fluoresce. Many biological molecules display intrinsic fluorescence, or can be labeled with molecules that exhibit fluorescence. Fluorescent molecules used as labels are also called fluorophores. Fluorescently labeled molecules are also called molecular probes. Typically, fluorescent molecules are aromatic compounds that display light absorption in the ultraviolet to visible regions of the electromagnetic spectrum. UV: 250 to 400 nm; Visible: 400 to 700 nm.


    Fluorescently labeled probes are useful for many biochemical assays for monitoring of specific molecular events such as binding, cleavage or conformational changes of oligonucleotides, proteins, and peptides. Dual labeled probes containing a fluorophore and a quencher molecule have many applications in genetic analysis.
    Dual labeled probes are often used for quantitative polymer chain reaction (qPCR). Dual labeled probes are single-stranded oligonuclotides labeled with two different dyes, a fluorophore and a quencher molecule. Dual labeled probes are hybridization probes that can be synthesized using standard automated oligonucleotide chemistry and are also referred to as hydrolysis probes. Dual labeled probes function by reporting the presence of specific nucleic acids in homogenous samples in solution. Usually, a reporter dye is located at the 5’-end and a quencher molecule located at the 3’-end. The quencher molecule inhibits or decreases the fluorescence intensity of a sample or fluorophore via fluorescence resonance energy transfer (FRET). When the primer is elongated by the polymerase during PCR the dual labeled probe can bind to the amplified specific DNA template. Hydrolysis releases the reporter molecule from the probe or target hybrid causing an increase in fluorescence and the measured fluorescence signal is directly proportional to the amount of target DNA.

    What is FRET?

    Resonance energy transfer or RET occurs when an energy quantum is transmitted from its site of absorption to the site of its utilization in a molecule, or system of molecules. This phenomenon occurs between chromophores over distances greater than interatomic, without conversion to thermal energy, and without kinetic collision of the donor and acceptor.

     

    The donor is the dye that initially absorbs the energy.

    The acceptor is the chromophore or dye to which the energy is transferred.

     

    Fluorescence resonance energy transfer or FRET is now widely used in many fluorescence based applications. In the past FRET has been widely used to measure dimensions and distances within or between molecules over distances of 10 to 100 Å. This distance range is well suited to probe structures of oligonucleotides, proteins and peptides. The range of applications includes medical diagnostics, DNA analysis as well as optical imaging and many others. Favorable distances for energy transfer are typically in the size of proteins, DNA or RNA or the thickness of a membrane. Furthermore, the extent of FRET is predictable from spectral properties of the selected fluorophors. These properties allow for the design of experiments based on the known sizes and structural features of the studied sample.

     

    FRET is an electromagnetic phenomenon that can be explained using the laws of classical physics. FRET occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state. Typically, the donor molecule emits at shorter wavelengths that overlap with the absorption spectrum of the acceptor molecule. The energy transfer occurs without the appearance of a photon as a result of long-range dipole-dipole interactions between donor and acceptor. The extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and the acceptor molecule determine the rate of energy transfer.  

     
    Figure 1: The classical Jablonski diagram, shown her in the left panel, illustrates electronic states of a molecule as well as photo induced processes related to absorption and emission of energy. A simplified energy-level diagram illustrating resonance energy transfer is shown on the right panel. An asterisks denotes an excited state.  

    What is fluorescence quenching?

    Fluorescence quenching is any process that decreases the fluorescence intensity of a sample. Many molecular interactions can result in quenching including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching. The efficiency of fluorescence quenching is distance dependent. If the reporter fluorophore and quencher are far apart, fluorescence occurs. However, if the reporter and quencher are close together in space fluorescence is suppressed and does not occur. In oligonucleotide probes, the reporter and quencher are typically placed such that a change in distance will produce a maximal change in fluorescence. The observed fluorescent signal monitors the event, for example, a hybridization or nuclease activity. In this case the oligonucleotide sequence acts as a flexible tether or link between the fluorescent reporter and quencher. Since, many dyes are known to aggregate, self-associate, form dimers, trimers, or polymers, the tendency for dyes to aggregate is the basis of the static quenching mechanism.

    In static quenching or contact quenching a reporter such as FAM and a quencher such as BHQ-1 label can bind together to form a new, nonfluorescent intramolecular dimer. Furthermore, the efficiency of static quenching is dependent on the affinity of the reporter and quencher for each other. Often the reporter and quencher are planar, hydrophobic molecules that stack together to avoid contact with water.

     

    Reference


    dos Remedios CG, Moens PD.; Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor.
     J Struct Biol. 1995 Sep-Oct;115(2):175-85. 

    Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers. Authors: Johansson, M.K. Book: Methods in Molecular Biology, v. 335; V.V. Didenko, Ed; Humana Press: Totowa, NJ,
    2006; pp 17-29. 

    Intramolecular Dimers: A New Design Strategy for Fluorescence-Quenched Probes.
      Authors: Johansson, M.K.; Cook, R.M. Journal: Chem.-Eur. J. 2003, 9, 3466. 

    Intramolecular Dimers: A
    New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes. Authors: Mary Katherine Johansson, Henk Fidder, Daren Dick and Ronald M. Cook. Journal: J. Am. Chem. Soc. 2002, 124, 6950.

    Joseph R. Lakowicz; Principles of Fluorescence Spectroscopy. Editors: ISBN: 978-0-387-31278-1 (Print) 978-0-387-46312-4 (Online)

    Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Authors: Salvatore A. E. Marras, Fred Russell Kramer and Sanjay Tyagi. Journal: Nucleic Acids Research, 2002, 30, e122.

    Peng L, Minbo H, Fang C, Xi L, Chaocan Z.;The interaction between cholesterol and human serum albumin. Protein Pept Lett. 2008;15(4):360-4.


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    Can cancer cells or their microenvironment be targeted selectively to treat tumors?

    Yes, is appears that this is possible.


    A number of peptides have been reported to specifically target tumor and tumor associated microenvironments, such as the tumor vasculature, after their systematic delivery. These peptides are known as “tumor-specific internalizing peptides” (TSIPs) or “tumor homing peptides” (THPs).

    Similar to anti-cancer peptides tumor-specific internalizing peptides are usually short peptides in sequence lengths of 3 to 15 amino acids that specifically recognize and bind to tumor cells or tumor vasculature. Since 1998 a number of these peptides have been identified using in vitro and in vivo phage display technology. Phage display is a molecular biology technology in which proteins or peptides are displayed on the surface of a phage as a fusion with one of the phage coated proteins. Phage display has been used intensively for the screening for protein-protein interactions. This screening method allowed for the identification of tumor-specific or tumor homing peptides that target specific tumor cells or tumor vasculature.

    According to the International Agency for Research on Cancer, an agency of the World Health Organization, cancer is now the world’s biggest killer. The “World Cancer Report” showed that there were 8.2 million deaths from cancer in 2012 and predicts that cancer cases worldwide will rise by 75 % over the next two decades. By then it is estimated that up to 25 million people may be suffering from cancer worldwide. Unfortunately, despite progress made in our understanding of the molecular basis of cancer and improvements made in treatment options, mortality rate is still high. This suggests that the availability of new types, more selective drugs that fight cancer would be of great benefit to humans.

    Tumor-specific internalizing peptides or tumor homing peptides have common sequence motifs like RGD, or NGR, which specifically bind to a surface molecule on tumor cells or tumor vasculature. The best known examples are the short peptides RGD and NGR. The RGD (Arg-Gly-Asp) peptide is known to bind α integrins and NGR (Asn-Gly-Arg) is known to bind to a receptor aminopeptidase N present on the surface of tumor endothelial cells, also called tumor angiogenic markers. It is no wonder that tumor-specific internalizing peptides are being used in cancer diagnosis and treatment. So far, many anti-cancer and imaging agents have been targeted to tumor sites in mice models by conjugation them to tumor-specific peptides. A database called “TumorHoPe” provides comprehensive information about experimentally validated tumor homing peptides and their target cells (http://crdd.osdd.net/raghava/tumorhope/). This is a manually curated database containing 744 entries of experimentally characterized tumor homing peptides that recognize tumor tissues and tumor associated micro environment, including tumor metastasis.


    A list of some tumor homing peptide motifs

     

    Motif

    Action

    NGR (Asn-Gly-Arg)

    Binds aminopeptidase N

    GSL (Gly-Ser-Leu)

    Inhibition of tumor homing

    RGD (Arg-Gly-Asp)

    Binds selectively to integrins which are overexpressed on endothelial cell surface in the cancer and facilitate cancer cell migration

    TSPLNIHGQKL

    Hn-1 appears to be HNSCC specific. Targeted drug delivery into solid tumors.

     

    Models of tumor homing peptides




    The specific internalization of peptides that target tumor cells has been evaluated for targeted siRNA delivery into human cancer cells. Un et al. in 2012 investigated the internalization of the HN-1TYR-anti-hRRM2 siRNAR peptide conjugate in human head and neck or breast cancer cells to establish its utility for targeted siRNA delivery into human cancer cells. The researchers used a FITC-HN-1TYR-anti-hRRM2 siRNAR construct to image its successful internalization into a human cancer cell line. For the synthesis of the fluorescent siRNA delivery vehicle, FITC-HN-1TYR-anti-hRRM2 siRNAR, a tyrosine and a FITC was added to the N-terminal end. Next, a synthetic anti-hRRM2 siRNA was synthesized with fluorine, incorporated at its 2’-OH position, to avoid degradation by RNases in vivo, and conjugated to the 5’-end of the antisense strand using a hexynyl phophoramidite linker. The selected HN1 peptide, a 12mer peptide that was isolated by peptide display library screening using a M13 phage library, contains the sequence TSPLNIHNGQKL. It has the ability to translocate drugs across the cell membrane into the cytosol, its uptake occurs in a tumor-specific manner, and it is capable of penetrating solid tumors. Ribonucleotide Reductase (RR), composed of the subunits hRRM1 and hRRM2, catalyses the conversion of ribonucleotides to their corresponding deoxy forms need for DNA replication. The researchers choose an anti-hRRM2 siRNA to allow for the degradation of hRRM2’s mRNA to suppress tumorgenesis.

    To conclude, tumor-specific internalizing peptides or tumor homing peptides appear to be future drug candidates for targeted siRNA delivery into human cancer cells that may enable a more selective treatment of tumors with less site effects.

    References

    Kapoor P, Singh H, Gautam A, Chaudhary K, Kumar R, et al. (2012); TumorHoPe: A Database of Tumor Homing Peptides. PLoS ONE 7(4): e35187. doi:10.1371/journal.pone.0035187.

    FRANK UN, BINGSEN ZHOU and YUN YEN; The Utility of Tumor-specifically Internalizing Peptides for Targeted siRNA Delivery into Human Solid Tumors. ANTICANCER RESEARCH 32: 4685-4690 (2012).

     


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    Control templates for molecular DNA/RNA diagnostics

    As the number and scope of disease-producing pathogens and their genetic variants that cause human disease have continued to increase, there has been a commensurate and rapid increase in the use of nucleic acid based tests for routine clinical diagnosis. Due to the complex nature of nucleic acids, these molecular tests must be fully controlled to accurately ascertain their specificity and sensitivity. However, the success of molecular diagnostics is often impeded by the availability of DNA- or RNA-based positive controls with the same or similar number of mutations as the organism being screened, for example, in the case of a pandemic or newly emerging disease, such as Ebola, where it can be difficult to acquire necessary positive controls.

    DNA or RNA standards allow a researcher to determine if an assay accurately represents the composition or quantities of known input as well as to derive standard calibration curves. This allows to relate read-out counts of analyte concentrations in the studied samples to accurate amounts or quantities. Furthermore, the use of control standards allows for direct measurement of error rates, coverage biases, and other veriables that can affect downstream analysis, such as the analysis of various isoforms.   

     
     

    As Good Laboratory Practices, government agencies, and organizations that establish standards and control require diagnostic laboratories to use stringent quality controls (QCs) guidelines that include calibrating equipment against control samples and performing tests of patient samples in tandem with consistent references, it is critical that reference samples be used in a manner that provides comprehensive evaluation of every component in these highly complex procedures and reagent mixtures. The need for these controls and/or standards became particularly acute with the widespread use of high complexity and high volume DNA- or RNA-based real time testing platforms.

    Bio-Synthesis provides molecular assay services, focused on the design and development of nucleic acid-based, positive control templates (PCT) to monitor the molecular diagnostic testing process, including the extraction, amplification, and detection components of test systems used to measure disease producing organisms. We provide thousands of PCTs to genotype high value polymorphisms for various drug metabolism and transporter genes. These PCTs can be manufactured in our cutting edge molecular diagnostic facilities and significantly shorten your path from RESEARCH to RESULT by providing you with the full development process for control templates that may be used as standard references in the simultaneous detection of mutations in any genome. These laboratory-safe, synthetic or semi-synthetic DNA/RNA Positive Controls can be a relatively cost effective, simple and efficient alternative to difficult-to-acquire controls from infectious samples.


    Our contract services are confidential, fast, efficient and well-documented, with objective to support the improvement of analysis and control of human infectious diseases by providing high quality evaluation materials to aid in the advancement of nucleic acid technologies.

    Advantages


    Select Platform

     DNA/RNA control templates, length >1000 bp

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     Suitable for Real-Time PCR, qPCR, microarray...

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    Optimized preparation for specific applicationsSelect Platform: DNA/RNA control templates, length >1000 bp

     






    BSI's On-demand HPV and HLA controls can be used as positive controls in nucleic acid amplification reactions.

    These quantitative controls can also be used to generate standard curves for qPCR assays.

     


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    Ebola Peptides for Diagnostics and Vaccines


    Peptides derived from Ebola virus proteins can be used to study antigenicity and immunogenicity of Ebola proteins. In addition, these peptide epitopes can be used further to develop sensitive and accurate diagnostic tests using polyclonal or monoclonal antibodies. Another potential use for this type of peptides is for the development of unique peptide-based vaccines. In particular, succesful and potent vaccines could be developed using antigenic peptides derived from proteins of the Ebola virus or other Ebola virus strains. 
     

    Figure 1: Ultra structures and models of the Ebola virus and its genome (Source: Ellis et al. 1978; CDC).  Ellis et al. in 1978 showed that electron microscopy can be used to detect and observe the ultrastructure of the Eboli virus in infected human tissue. The Ebola virus was detected in tissue samples from human liver, kidney, spleen and lung.   

    Infection of a cell by a virus requires the fusion between viral and host membranes. Infection of a cell by the Ebola virus (EboV) begins with the uptake of viral particles into cellular endosomes. Experimental data suggests that the viral envelope glycoprotein (GP) catalyzes the fusion between the viral and host cell membranes. The fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike ultimately resulting in the formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Membrane fusion is mediated by fusion proteins that extrude from the viral membrane. Key components that are in contact with the host cell membrane are fusion peptides, parts of the fusion proteins. The Ebola glycoprotein (GP) is responsible for both receptor binding and membrane fusion. The GP is composed of two sub-domains, GP1 and GP2. The two domains are connected via a disulfide bond. The Ebola fusion peptide (EFP) (G524AAIGLAWIPYFGPAA539) is thought to be in direct contact with the host cell membrane. This peptide is conserved within the virus family. EFP is an internal fusion peptide located 22 residues from the N-terminus of GP2. Experimental data suggests that the EFP peptide in the presence of the membrane has a tendency to form helical structures.

    Figure 2: Model of the Ebola fusion protein in its fusiogenic state as suggested by Jaskierny et al. in 2011. The globular protein GP1 is thought to initiate the binding to the host cell receptor. The GP2 domain contains a helical bundle with the fusion peptide near the N-terminus. Jaskierney et al. studied the monomeric form of the internal fusion peptide from Ebola virus in membrane bilayer and water environments using computer simulations. The wild type Ebola fusion peptide, the W8A mutant form, and an extended construct with flanking residues were examined. The researchers found that the monomeric form of wild type Ebola fusion peptide adopts a coil-helix-coil structure with a short helix from residue 8 to 11 orientate parallel to the membrane surface.

     

    Using circular dichroism (CD) together with infrared (IR) spectroscopy the researchers showed that the EFP peptide has three states:

    A random coil in solution and either an α–helix or a β–sheet when bound to the membrane. Furthermore, the secondary structure of the membrane-bound peptide depends on the presence of Ca2+ and in the presence of Ca2+ a β-sheet structure is preferred while in the absence of Ca2+ helical structures are dominant. A nuclear magnetic resonance (NMR) study of EFP showed that the peptide adopts a random coil structure in aqueous buffers and a more defined structure in the presence of sodium dodecyl sulfate (SDS) micelles. Tryptophan fluorescent emission data suggests that W8 enters the hydrophobic core of SDS micelles. Nuclear Overhauser effect (NOE) measurements obtained from 1H NMR suggested the presence of a short 310 helix form I9 to F12 in the middle of the peptide while the N- and C-termini appear to be less structured.


    Miller et al. in 2011 performed a study using synthetic peptides of the CHR sequence region (C-peptides) to test if these peptides can inhibit the entry of the virus particles. The researchers prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, known to accumulate in endosomes, and found that this peptide specifically inhibits viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. The researchers determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Miller et al. argue that targeting C-peptides to endosomal compartments can serve as an approach to localize inhibitors to sites of membrane fusion.


    To diagnose and control the endemic outbreaks of haemorrhagic fever in humans caused by filioviruses, such as the Ebola and the Marburg virus, rapid, highly sensitive, reliable, and specific assays are required. The identification and characterization of antigenic sites in viral proteins is important for the development of viral antigen detection assays.

    Changula et al. in 2013 generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of the Zaire Ebola virus. The researchers divided the mABs into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. The use of synthetic peptides corresponding to the Ebola virus nucleoprotein (NP) sequence allowed to map mAb binding sites to seven antigenic regions in the C-terminal half of the NP. The mapped antigenic sites included two highly conserved regions present among all five Ebola virus species currently known. In addition, the scientists were successfully in producing species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. These results provide useful information for the development of Ebola virus antigen detection assays and potentially new vaccines for Ebola virus strains.


    Table 1: Ebola virus peptides

    Peptide

    Sequence

    Notes

     

    Fusion Peptide

    Jaskierny et al., 2011.

    EFP

    G524AAIGLAWIPYFGPAA539

    Chain A fusion peptide in SDS micelles at pH 7

     

     

     

     

    C-Peptide Study

    Miller et al. 2011

    Tat-Ebo

    YGRKKRRQRRR-GSG-IEPHDWTKNITDKIDQIIHDFVDK

    Ebola virus chain A fusion peptide

    Lys-Ebo

           KKKK-GSG-IEPHDWTKNITDKIDQIIHDFVDK

    Ebola virus chain A fusion peptide

    Tat-only

    YGRKKRRQRRR

     

    Tat-Scram

    YGRKKRRQRRR-GSG-HTEHINFQDDTIKIWPDVIKIKDD

     

    Tat-ASLV

    YGRKKRRQRRR-GSG-FNLSDHSESIQKKFQLMKEHVNKIG

     

     

     

     

     

    Peptide epitopes of mABs against EBOV NP

    Changula et al. 2013

    ZNP31-1-8

    ZNP41-2-4

    YDDDDDIPFP, aa 421–430

    NP protein

    ZNP74-7

    YDDDDDIPFPGPINDDDNPG, aa 421–440

    NP protein

    ZNP24-4-2

    QTQFRPIQNVPGPHRTIHHA, aa 521–540

    TPTVAPPAPVYRDHSEKKEL, aa 601–620

    NP protein

    ZNP106-9

    DTTIPDVVVD, aa 451–460a

    NP protein

    ZNP98-7

    MLTPINEEADPLDDADDETS, aa 561–580

    NP protein

    ZNP35-16-3-5

    DDEDTKPVPNRSTKGGQQKN, aa 491–510

    NP protein

    ZNP62-7

    YRDHSEKKELPQDEQQDQDH, aa 611–630

    NP protein

     

    Ebola virus NucleoProtein (NP) sequence

    >gi|158341892|gb|ABW34756.1| nucleoprotein, partial [Zaire ebolavirus]

    RQIQVHAEQGLIQYPTAWQSVGHMMVIFRMMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSG

    LLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLF

    PQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKK

    NEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQDTT

    IPDVVVDPDDGSYGEYQSYSENGMNAPDDLVLFDLDEDDEDTKPVPNRLTKGGQQKNSQKGHHTEGRQTQ

    SRPTQNVPGPRRTIHHASAPLTDNDRGNEPSGSTSPRMLTPINEEADPLDDADDETSSLPPLESDDEEQD

    RDETSNRTPTVAPPAPVYRDHSEKKELPQDEQQDQDHTQEARNQDSDNTQPEHSFEEMYRHIL


    The location of the Zaire envelope protein (ZNP) peptides are highlighted in red and magenta within the amino acid sequence of Ebola virus nucleoprotein.

    Table 2: Observed mutations for the QTQFRPIQNVPGPHRTIHHA, aa 521–540, peptide.


    Models of Ebola virus peptides and proteins

    Figure 3: NMR structure of the Ebola virus chain A fusion peptide, GAAIGLAWIPYFGPAA.


    Figure 4: Crystal structure models of the Ebola virus membrane fusion subunit, GP2 envelope glycoprotein ectodomain.

    Table 3: Peptides used for the production of rabbit antisera by Changula et al. 2013.


    Virus Protein

    Peptide

    Amino Acids

    EBOV NP

    QDHTQEARNQD

    628-638

    SUDV NP

    QGSESEALPINSKK

    631-644

    TAFV NP

    NQVSGSENTDNKPH

    630-643

    BDBV NP

    QSNQTNNEDNVRNN

    628-641

    RESTV NP

    TSQLNEDPDIGQSK

    630-643

    MARV NP

    RVVTKKGRTFLYPNDLLQ

    635-652

     

    Legend: BDBV = Bundibugyo virus; EBOV = Ebola virus; MATV = Marburg virus; RESTV = Reston virus; SUDV = Sudan virus; TAFV = Tai Forest Ebola virus.


    The membrane proximal external region (MPER) peptide


    Regula et al. in 2013 investigated the role of the membrane proximal external region (MPER) that precedes the transmembrane domain of glycoprotein 2 (GP2) of Ebola virus strains. Earlier research indicated that an infection by a filovirus requires membrane fusion between the host and the virus. The fusion process is facilitated by the two subunits of the envelope glycoprotein, the surface subunit GP1and the transmembrane subunit GP2. A sequence region called the membrane proximal external region (MPER) is a tryptophan (Trp, W) rich peptide segment located immediately in front of the transmembrane domain of GP2. In the human immunodeficiency virus 1 (HIV-1) glycoprotein gp41, the MPER is known to be critical for membrane fusion. In addition, this amino acid sequence was also identified as a target for several neutralizing antibodies. Regula et al. characterized the properties of GP MPER segment peptides of Ebola virus and Sudan virus. The study used  micelle-forming surfactants and lipids, at pH 7 and pH 4.6. The researchers employed circular dichroism (CD) spectroscopy and tryptophan fluorescence to determine if GP2 MPER peptides bind to micelles of sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC). Nuclear magnetic resonance (NMR) spectroscopy was used to reveal that residues 644 to 651 of the Sudan virus MPER peptide interacted directly with DPC. This interaction enhanced the helical conformation of the peptide. The scientists found that the Sudan virus MPER peptide moderately inhibited cell entry by a GP-pseudotyped vesicular stomatitis virus. However, it did not induce leakage of a fluorescent molecule from large unilamellar vesicle comprised of 1-palmitoyl-2-oleoylphostatidyl choline (POPC) or cause hemolysis. The analysis performed by this research group suggested that the filovirus GP MPER binds and inserts shallowly into lipid membranes.


    GP2 MPER Peptides


    Table 4: Alignment of GP2 MPER peptides from different viruses.

    Virus Strain

    GP2 MPER Peptide

    Amino Acids

    EBOV

        DKTLPDQGDNDNWWTGWRQW

    632 to 651

    BDBV

        DKPLPDQTDNDNWWTGWRQW

    632 to 651

    SUDV

        DNPLPNQDNDDNWWTGWRQW

    632 to 651

    TAFV

        DNNLPNQNDGSNWWTGWKQW

    632 to 651

    RESTV

        DNPLPDHGDDLNNWTGWRQW

    633 to 652

    FIV

        LQKWEDWVGWIGNIPQYLKG

    767 to 786

    HIV-1

    LLELDKWASLWNWFDITNWLWYIK

    660 to 683

     

    Table 4 shows the amino acid alignment of GP2 MPER regions from different members of the five Ebola virus species. Many residues that are identical in at least four of the viruses. For comparison, the MPER segments of FIV and HIV-1 gp41 are included.

     

    Legend: BDBV Bundbuyo virus, EBOV Ebola virus, FIV filio virus, HIV-1 human immunodeficiency virus 1, RESTV Reston virus, SUDV Sudan virus, TAFV Thai Forest virus.


    Alignments of GP2 MPER peptides from various virus strains.


    Location of the GP2 MPER peptides within the GP2 protein of the Ebola virus

    Figure 5: The location of the MPER peptides is highlighted in yellow in the crystal structure of the Ebola virus membrane fusion subunit, GP2 envelope glycoprotein ectodomain. The amino acid of the peptide shown in gray where not observed in the crystal indicating that this part of the peptide may take up a random coil structure in the crystal.


    Regula et al. used EBOV and SUDV MPER peptides for their study because both viruses are the most prevalent and pathogenic among the ebolaviruses. Synthetic peptides corresponding to the MPER region for EBOV and SUDV were used. The N-termini were blocked with an acetyl group and the C-termini contained an amide group.

     

    The study revealed three characteristics of the GP2 MPER peptides:



    • As a peptide, the GP2 MPER binds to micelle-forming surfactants in a pH-independent manner with higher affinity for zwitterionic micelles; 
    • A large conformational change to a more predominantly helical state occurs for the tryptophan-rich region of this peptide upon micelle-binding;
    • These peptides have modest viral entry inhibitory activity but do not induce leakage from LUVs.

     

    The study observed inhibitory activity for the S-MPER peptide which suggests that addition of this peptide may interfer with the viral entry process. For the FIV MPER peptide it was observed that a WX2WX2W motif is required for the membrane interaction responsible for its inhibitory activity.

    This peptide motif, WTGWRQW, is strictly conserved among all species.

    Results of the study indicated that the MPER peptide segments of EBOV and SUDV bind membrane surfaces which induces a conformational change in the Trp-rich peptide segment. This behavior suggests a role for the EBOV and SUDV MPER in membrane fusion.

     

    Reference



    http://www.cdc.gov/vhf/ebola/

    D. S. ELLIS, D. I. H. SIMPSON, D. P. FRANCIS, J. KNOBLOCH, E. T. W. BOWEN, PACIFICO LOLIK, AND ISAIAH MAYOM DENG; Ultrastructure of Ebola virus particles in human Liver. Journal of Clinical Pathology, 1978, 31, 201-208.

    Katendi Changula
    , Reiko Yoshidac, Osamu Noyoric, Andrea Marzid, Hiroko Miyamotoc, Mari Ishijimac, Ayaka Yokoyamac, Masahiro Kajiharac,Heinz Feldmannd, Aaron S. Mweenea, Ayato Takadaa; Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein.  Virus Research 176  (2013) 83– 90.

    Thomas Hoenen, Allison Groseth, and Heinz Feldmann; Current Ebola vaccines. Expert Opin Biol Ther. 2012 July; 12(7): 859–872.  oi:10.1517/14712598.2012.685152.

    Adam J. Jaskierny
    , Afra Panahi, and Michael Feig; Effect of flanking residues on the conformational sampling of the internal fusion peptide from Ebola virus. Proteins. 2011 April ; 79(4): 1109–1117. doi:10.1002/prot.22947.

    Emily Happy Miller, Joseph S. Harrison, Sheli R. Radoshitzky, Chelsea D. Higgins, Xiaoli Chi, Lian Dong, Jens H. Kuhn, Sina Bavari, Jonathan R. Lai, and Kartik Chandran; Inhibition of Ebola Virus Entry by a C-peptide Targeted to Endosome J Biol Chem. May 6, 2011; 286(18): 15854–15861. Published online Mar 16, 2011. doi:  10.1074/jbc.M110.207084. PMCID: PMC3091195.

    Lauren K. Regula, Richard Harris, Fang Wang, Chelsea D. Higgins, Jayne F. Koellhoffer, Yue Zhao, Kartik Chandran, Jianmin Gao, Mark E. Girvin, and Jonathan R. Lai; Conformational Properties of Peptides Corresponding to the Ebolavirus GP2 Membrane-Proximal External Region in the Presence of Micelle-Forming Surfactants and Lipids. Biochemistry. 2013 May 21; 52(20): . doi:10.1021/bi400040v.




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    MicroRNAs or miRNAs are a class of endogenous small RNAs approximately 22 nucleotides in size found in plants and animals including humans. More and more research data indicate that in humans microRNAs or miRNAs are useful indicator molecules for different cancer types which may be useful as cancer biomarkers.


    Increasingly, it has become apparent that microRNAs take part in the development of cancer. This observation has made miRNAs potential biomarkers for cancer diagnosis and prognosis. Therefore it can be reasoned that microRNAs or miRNAs are good candidates as cancer biomarker. Because many studies now suggest that the pattern of microRNA expression in tissues reflects the disease status in this tissue, miRNA expression levels may serve as potential biomarkers with multiple applications in clinical diagnostics. miRNAs can be successfully isolated from biological fluids allowing for the development of biofluid biopsies or diagnostics. This type of biomarker diagnostics promises to allow for the development of minimal invasive assays, to save cost and simplify complex invasive procedures.

    miRNA's processing occurs from approximately 70 nucleotides in size hairpin precursor RNAs by the protein Dicer. miRNA have been shown to regulate their target messengerRNA (mRNA) by destabilizing mRNA molecules and translational repression.

    Figure 1: Pre-miRNA nuclear export machinery.


    Okada et al in 2009. solved the structure of the "pre-miRNA nuclear export machinery" formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP) at 2.9 angstrom. The data showed that RNA recognition by Exp-5:RanGTP does not depend on RNA sequence. This implys that Exp-5:RanGTP can recognize a variety of pre-miRNAs.

    Figure 2: The molecules present in the structure and their interactions are shown here.


    [Source: 
    http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=78532]


    During a study of the nematode Caenorhabditis elegans (C. elegans) development involving the gene lin-14 Victor Ambros, Rosalind Lee and Rhonda Feinbaum first discovered miRNAs in 1993. However, at the time the researcher speculated that these molecules could be a nematode idiosyncrasy. In 2000, it was shown that let-7 represses lin-41, lin-14, lin28, lin42 and daf12 mRNA during transition in developmental stages in C. elegans. At this time miRNAs were recognized as small regulatory RNAs. Furthermore, it became clear that miRNAs are conserved in many species.  In addition, it was noted that short non-coding RNAs, first identified in 1993, were part of a wider phenomenon. For example, Lagos-Quintana et al. in 2001 referred to 22- and 21-nucleotide (nt) RNAs as small temporal RNAs (stRNAs). These RNAs functioned as key regulators in developmental timing. The Tuschl lab in 2001 showed that many 21- and 22-nt expressed RNAs exist in invertebrates and vertebrates. Furthermore, some of these RNAs, similar to let-7 stRNA, are highly conserved. This discovery led to the conclusion that sequence-specific, posttranscriptional regulatory mechanisms as mediated by small RNAs are more general than was previously appreciated. Over 4000 miRNAs have been found so far in all studied eukaryotes. More than 700 miRNAs have already been identified in humans. In addition, more than and over 800 are predicted to exist. V. Ambros in 2001 reported that these microRNAs are diverse in sequence and expression patterns. The observation that these molecules are evolutionarily widespread suggests that they may participate in a wide range of genetic regulatory pathways. Figure 3 shows the dramatic increase in publications involving miRNAs and miRNA research. 

    Figure 3: Increase in miRNA publications in Pubmed.


    Animal miRNAs derived from longer primary transcripts carry hairpin structures. The processing of these precursor hairpin RNA structures proceeds in a stepwise fashion catalyzed by the RNase III enzymes Drosha and Dicer. Drosha cleaves these RNA molecules near the hairpin base to release the pre-miRNA hairpin. This reaction occurs in the nucleus. Next, the pre-miRNA hairpin is exported into the cytoplasm and Dicer cleaves on the loop side of the hairpin.  The result is a miRNA:miRNA* duplex. In the next step, one strand of this complex is preferentially incorporated into a silencing complex.

    Recently an alternative nuclear pathway for miRNA biogenesis was identified in invertebrates. Researchers found that short introns with hairpin potential, termed mirtrons, can be spliced and debranched into pre-miRNA hairpin mimics that appear to bypass Drosha cleavage. Debranched mirtrons access the canonical miRNA pathway during nuclear export and are then cleaved by Dicer and incorporated into silencing complexes. As pointed out by Brezikow et al. in 2007, mirtrons are alternative precursor molecules for microRNA biogenesis present in invertebrates. Splicing allows these short hairpin introns to bypass Drosha cleavage. Drosha cleavage is essential for the generation of canonical animal microRNAs. With the help of computational and experimental strategies Brezikow et al. establish that mammals have mirtrons as well. Therefore, mirtrons are miRNAs located in the introns of mRNA encoding genes. Brezikow et al. identified three (3) well conserved mirtrons expressed in diverse mammals. In addition, 16 primate-specific mirtrons, and 46  mirtron candidates, as supported by limited cloning, are suspected to be present in primates as well.

     


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    Disease dependent differentially expressed miRNAs

     

    MicroRNAs (miRNAs) are known to play important roles in diseases pathology such as infections and cancer. The recent development of high-throughput technologies for the global measurement of miRNAs these molecules have now emerged as a new class of cancer biomarkers. Already many studies have explored associations between miRNAs and different cancer features. Often real-time polymerase chain (rt-PCR) reaction is used to measure the expression of miRNAs in various tissues.Analyzing global miRNA gene expression using complementary DNA microarrays allows for the examination of differentially expressed miRNAs. This type of assays allows finding disease specific miRNA associations which hopefully will reveal how miRNAs regulate their target genes. For example, miRNA profiling of different cancer tissue has the potential to allow determination of lineage and differentiation state of tumors. The following table contains a list of differentially regulated miRNAs in various diseases.


    miRNAs

    Disease Types

    Up/Down-regulated

    Reference

    let-7a, let-7b, let-7c, let-7d, let-7g, miR-16, miR-23a, miR-23b, miR-26a, miR-92, miR-99a, miR-103, miR-125a, miR-125b, miR-143, miR-145, miR-195, miR-199a, miR-199a, miR-221, miR-222, miR-497

    prostate cancer

    down-regulated

    Porkka KP, Pfeiffer MJ, Waltering KK, Vessella RL, Tammela TL, Visakorpi T: MicroRNA expression profiling in prostate cancer. Cancer Res 2007, Jul 1; 67(13):6130-5.

    http://cancerres.aacrjournals.org/content/67/13/6130.abstract

     

    miR-202, miR-210, miR-296, miR-320, miR-370, miR-373, miR-498, miR-503

    prostate cancer

    up-regulated

    Porkka KP, Pfeiffer MJ, Waltering KK, Vessella RL, Tammela TL, Visakorpi T: MicroRNA expression profiling in prostate cancer. Cancer Res 2007, Jul 1; 67(13):6130-5.       

    miR-16, miR-92a, miR-103, miR-107, miR-197, miR-34b, miR-328, miR-485-3p, miR-486-5p, miR-92b, miR-574-3p, miR-636, miR-640, miR-766, miR-885-5p

    prostate cancer

    up-regulated

    Lodes MJ, Caraballo M, Suciu D, Munro S, Kumar A, Anderson B: Detection of cancer with serum miRNAs on an oligonucleotide microarray. PLoS One 2009, Jul 14; 4 (7):e6229.Published: July 14, 2009.

     

    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006229

     

    miR-21, miR-155, miR-221

    Pancreatic cancer

    up-regulated

    Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, Liu CG, Bhatt D, Taccioli C, Croce CM: MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis JAMA 2007, 297(17):1901-1908. (doi:10.1001/jama.297.17.1901). http://www.ncbi.nlm.nih.gov/pubmed/17473300

     

    miR-155, miR-21

    colon, lung, breast,stomach, prostate

    up-regulated

    Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri A, Musiani P, Volinia S, Nenci I, Calin GA, Querzzoli P: MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005, 65:7065–7070. http://www.ncbi.nlm.nih.gov/pubmed/16103053

     

    Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 2006, Feb 14; 103(7):2257-2261. Epub 2006 Feb 3.

    http://www.ncbi.nlm.nih.gov/pubmed/16461460

     

    miR-142-5p, miR-369-3p, miR-215

    lung cancer

    up-regulated

    Baffa R, Fassan M, Volinia S, O'Hara B, Liu CG, Palazzo JP, Gardiman M, Rugge M, Gomella LG, Croce CM, Rosenberg A: MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets. J Pathol 2009, Jun 1.

    http://www.ncbi.nlm.nih.gov/pubmed/19593777.

     

    miR-373

    lung cancer

    down- regulated

    Baffa et al. 2009

    miR-30d, miR-125b, miR-26a, miR-30a-5p

     

    thyroid anaplastic carcinomas

    down- regulated

    Visone R, Pallante P, Vecchione A, Cirombella R, Ferracin M, Ferraro A, Volinia S, Coluzzi S, Leone V, Borbone E, Liu CG, Petrocca F, Troncone G, Calin GA, Scarpa A, Colato C, Tallini G, Santoro M, Croce CM, Fusco A: Specific microRNAs are down regulated in human thyroid anaplastic carcinomas. Oncogene 2007, Nov 29; 26(54):7590-7595. Epub 2007 Jun 11.

    http://www.ncbi.nlm.nih.gov/pubmed/17563749.

     

    miR-10b, miR-125b, miR-145

    breast cancer

    down- regulated

    Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri A, Musiani P, Volinia S, Nenci I, Calin GA, Querzzoli P: MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005, 65:7065–7070.

    http://www.ncbi.nlm.nih.gov/pubmed/16103053.

     

    miR-27a, miR-96, miR-182

     

    breast cancer

    up-regulated

    Guttilla IK, White BA: Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells. J Biol Chem. 2009 Aug 28;284(35):23204-16.

    miR-21, miR-155

    breast cancer

    up-regulated

    Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri A, Musiani P, Volinia S, Nenci I, Calin GA, Querzzoli P: MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005, 65:7065–7070.

    http://www.ncbi.nlm.nih.gov/pubmed/16103053

     

    miR-21

    breast cancer

    up-regulated

    Huang GL, Zhang XH, Guo GL, Huang KT, Yang KY, Shen X, You J, Hu XQ: Clinical significance of miR-21 expression in breast cancer: SYBR-Green I-based real-time RT-PCR study of invasive ductal carcinoma. Oncol Rep. 2009, Mar 21; (3):673-9.

    http://www.ncbi.nlm.nih.gov/pubmed/19212625       

     

    miR-30b, miR148a

    breast cancer

    up-regulated

    Baffa R, Fassan M, Volinia S, O'Hara B, Liu CG, Palazzo JP, Gardiman M, Rugge M, Gomella LG, Croce CM, Rosenberg A: MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets. J Pathol. 2009, 219(2):214-21.

     

    http://www.ncbi.nlm.nih.gov/pubmed/19593777

     

    miR-205

    breast cancer

    down-regulated

    Baffa et al. 2009.

    miR-142-5p, miR-29b, miR-30b

    bladder cancer

    up-regulated

    Baffa et al. 2009.

    miR-145, miR-143, miR-320

    bladder cancer

    down- regulated

    Baffa et al. 2009.

    miR-138, miR-125b

    colon cancer

    up-regulated

    Baffa et al. 2009.

    miR-17, miR-106a

    colon cancer

    down- regulated

    Baffa et al. 2009.

    hsa-miR-205

    head and neck cancer

    up-regulated

    Tran N, McLean T, Zhang X, Zhao CJ, Thomson JM, O’Brien C, Rose B: MicroRNA expression profiles in head and neck cancer cell lines. Biochem Biophys Res Commun 2007, 358:12–17.

    http://www.ncbi.nlm.nih.gov/pubmed/17475218.

     

    miR-21, miR-221

    brain cancer

    up-regulated

    Ciafre, S.A., Galardi, S., Mangiola, A., Ferracin, M., Liu, C.G., Sabatino, G., Negrini, M., Maira, G., Croce, C.M., Farace, and M.G: Extensive modulation of a set of microRNAs in primary glioblastomas. Biochem Biophys Res Commun 2005, 334:1351–1358.

    http://www.sciencedirect.com/science/article/pii/S0006291X0501481

     

    miR-9-2, miR-10b, miR-21, miR-25, miR-123, miR-125b-1, miR-125b-2,  miR-130a, miR-221

    glioblastomas

    up-regulated

    Ciafre et al., 2005

    miR-10b, miR-21, miR-26a, miR-383, miR-451, miR-486, miR-516-3p, miR-519d

    glioblastomas

    up-regulated

    Godlewski J, Nowicki MO, Bronisz A, Williams S, Otsuki A, Nuovo G, Raychaudhury A, Newton HB, Chiocca EA, Lawler S: Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res 2008, 68:9125–9130. doi:10.1158/0008-5472.CAN-08-2629.

    http://www.ncbi.nlm.nih.gov/pubmed/19010882

     

    miR-7, miR-29b, miR-31, miR-101, miR-107, miR-124, miR-124-2, miR-128-1, miR-129, miR-132, miR-133a , miR-133b, miR-137

    glioblastomas

    down- regulated

    Silber J, Lim DA, Petritsch C, Persson AI, Maunakea AK, Yu M, Vandenberg SR, Ginzinger DG, James CD, Costello JF, Bergers G, Weiss WA, Alvarez-Buylla A, Hodgson JG: miR-124 and miR-137 inhibit proliferation of glioblastoma multiform cells and induce differentiation of brain tumor stem cells. BMC Med 2008, 6:14. doi:10.1186/1741-7015-6-14.

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2443372/

     

    miR-138, miR-139, miR-149, miR-153, miR-154, miR-185, miR-187, miR-203, miR-218, miR-323, miR-328, miR-330

    glioblastomas

    down- regulated

    Gal H, Pandi G, Kanner AA, Ram Z, Lithwick-Yanai G, Amariglio N, Rechavi G, Givol D: MIR-451 and Imatinib mesylate inhibit tumor growth of glioblastoma stem cells. Biochem Biophys Res Commun 2008, 376:86–90. doi:10.1016/j.bbrc. 2008.08.107.

     

    http://www.ncbi.nlm.nih.gov/pubmed/18765229

     

    miR-15, miR-16

    chronic lymphocytic leukemia (CLL)

    down- regulated

    Calin GA: Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002, 99: 15524–15529.

    http://www.ncbi.nlm.nih.gov/pubmed/12434020       

     

    miR-150, miR-155           

    CLL

    up-regulated

    Bartels CL, Tsongalis GJ: MicroRNAs: novel biomarkers for human cancer. Clin Chem 2009, Apr; 55(4):623-31. Epub 2009 Feb 26. http://www.ncbi.nlm.nih.gov/pubmed/19246618

     

    miR-143, miR-145

    colorectal neoplasia

    down- regulated

    Michael MZ, O´Connor SM, Van Holst Pellekaan NG, Young GP, James RJ: Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res 2003, 1:882-891.

    http://www.ncbi.nlm.nih.gov/pubmed/14573789

     

    miR-18, miR-224

    hepato. Carcinoma

    up- regulated

    Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K: Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-timorous tissues. Oncogene 2006, 25:2537–2545.

    http://www.ncbi.nlm.nih.gov/pubmed/16331254

     

    miR-199, miR-195, miR-200, miR-125

    hepato. Carcinoma

    down- regulated

    Murakami et al., 2006

    miR-221, miR-222, miR-146, miR-181

    papillary thyroid carcinoma

    up-regulated

    He H, Jazdzewski K, Li W, Liyanarachchi S, Nagi R, Volinia S, Calin GA: The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci USA 2005b, 102:19075-19080.

    http://www.ncbi.nlm.nih.gov/pubmed/16365291

    Pallante P, Visone R, Ferracin M, Ferraro A, Berlingieri MT, Troncone G, Chiappetta G, Liu CG, Santoro M, Negrini M: Deregulation in human thyroid papillary carcinomas. Endocr Relat Cancer 2006, 13:497–508.

    http://www.ncbi.nlm.nih.gov/pubmed/16728577

     

    miR-372, miR-373

    testicular germ cell tumors

    up-regulated

    Voorhoeve PM, le Sage C, Schrier M: A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 2006, 124:1169-1181.

    http://www.ncbi.nlm.nih.gov/pubmed/16564011

     

    miR-31, miR-96, miR-135b, miR-183

    colorectal cancer

    up-regulated

    Bartels and Tsongalis 2009.

    miR-48, miR-135b, miR-133b

    colorectal cancer

    down- regulated

    Bartels and Tsongalis 2009.

    let-7b, let-7 g, miR-9, miR-21, miR-26a, miR-30a-3p, miR-30a-5p, miR-31, miR-96, miR-124b, miR-132, miR-135a, miR-135b, miR-141, miR-142-3p, miR-142-5p, miR-181a, miR-181b, miR-182, miR-183, miR-194, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-215, miR-219, miR-320, miR-338, miR-372

    colorectal cancer

    up-regulated

    Yang L, Belaguli N, Berger DH: MicroRNA and Colorectal Cancer. World J Surg 2009, 33:638–646.

     

    let-7a, miR-10a, miR-15b, miR-23a, miR-25, miR-27a, miR-27b, miR-30c, miR-107, miR-124a, miR0125a, miR-125b, miR-127, miR-130a, miR-133a, miR-133b, miR-134, miR-137, miR-143, miR-145, miR-147, miR-154, miR-191, miR-199a, miR-199b, miR-214, miR-296, miR-299, miR-337, miR-339, miR-342, miR-368, miR-370, miR-582

    colorectal cancer

    down- regulated

    Yang L, Belaguli N, Berger DH: MicroRNA and Colorectal Cancer. World J Surg 2009, 33:638–646.

    miR-224, miR-18 and pre-miR-P18, miR-221

    Hepatocellular cancer

    up-regulated

    Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K: Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-timorous tissues. Oncogene 2006, 25:2537–2545.

    http://www.ncbi.nlm.nih.gov/pubmed/16331254

    Fornari F, Gramantieri L, Ferracin M, Veronese A, Sabbioni S, Calin GA, Grazi GL, Giovannini C, Croce CM, Bolondi L, Negrini M: MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma.  Oncogene 2008, Sep 25; 27(43):5651-61 Epub 2008 Jun 2.

    http://www.ncbi.nlm.nih.gov/pubmed/18521080

     

    miR-199a, miR-199a*, miR-200a, miR-125a, miR-195, miR-125b

    Hepatocellular cancer

    down- regulated

    Murakami et al., 2006.

    Li W, Xie L, He X, Li J, Tu K, Wei L, Wu J, Guo Y, Ma X, Zhang P, Pan Z, Hu X, Zhao Y, Xie H, Jiang G, Chen T, Wang J, Zheng S, Cheng J, Wan D, Yang S, Li Y, Gu J: Diagnostic and prognostic implications of microRNAs in human hepatocellular carcinoma. Int J Cancer 2008, Oct 1; 123(7):1616-22.

    http://www.ncbi.nlm.nih.gov/pubmed/18649363

     


    Additional reference

    Stefanie S Jeffrey; Cancer biomarker profiling with microRNAs. Nature Biotechnology26, 400 - 401 (2008)
    doi:10.1038/nbt0408-400. http://www.nature.com/nbt/journal/v26/n4/full/nbt0408-400.html


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    Stable isotope labeling can be used to study metabolic pathways and turnover rates of biological molecules such as proteins, peptides, RNA and others.

    Early on stable isotope labeling was used to define metabolic pathways and turnover rates of biological molecules such as amino acids and peptides in the body. Stable isotope labeling was used shortly after the discovery of deuterium in 1931, the heavy isotope of hydrogen, by Harold Urey. Urey received a Nobel Prize in 1934 for this discovery. Already in 1935 Rudolf Schoenheimer used isotopic tracer techniques in metabolic research for stable isotope labeling experiments. His work, for the first time, provided clear experimental evidence of the dynamics of the body’s metabolism. Schoenheimer’s research group defined synthesis and degradation pathways of many compounds in the following years. The availability of enriched 15N, the heavy isotope of nitrogen, enabled demonstrating that protein had a dynamic life cycle in that they were both continually being synthesized and degraded. However, all of this early work was performed using crude methods that involved the preparation of labeled compounds, followed by tedious measurements using isotope ratio mass spectrometry (IRMS). In addition, for the measurements of the isotopic enrichment, all compounds investigated needed to be reduced to the simple gases carbon dioxide (CO2), hydrogen (H2), and nitrogen (N2). After World War II, the radioisotopes tritium (3H) and carbon 14 (14C) were mostly used for many years to come. Improvements made in instrument development, specifically for gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) instruments, allowed the use of stable isotopes for metabolic and proteomic studies with more ease in recent decades. The increasing trend for the use of isotopes, in particular the use of stable isotopes for metabolic or proteomic studies, is reflected in the increased publication volume of papers covering these applications which can be seen in figure 1. More than 18,500 publications for isotope labeling have been published since 1953 and can be found in PubMed. 



    Figure 1: Timeline of papers in PubMed describing various isotope labeling methods and applications. As can been seen the use of isotopes, in particular stable isotopes, has increased in recent years.


    Figure 2: The helium, as an example, has 2 protons, 2 neutrons, and 2 electrons. Helium has two isotopes but it consists almost entirely of He-4 with natural He only containing little more than 0.0001% of He-3.

     

    Let us briefly review the basic chemistry and physical properties of isotopes. Elements contain nuclei each with their own unique physicochemical characteristics. According to the “atomic theory”, each atomic nucleus can be defined by its mass, its number of constituent elementary particles, its spin rate, its magnetic strength, its electric charge, and its multitude of existing forms. The uniqueness of elements is reflected in their characteristic ratio of isotopes. This phenomenon has allowed geological scientists to study the geological record of the earth’s crust revealing its history. Carbon dioxide (CO2) and methane (CH4) in the atmosphere record its history and the impact of human beings on the Earth’s atmosphere which can be studied from ice cores, for example taken from glaciers or eternal ice fields, in the Alps, in Greenland or in the Antarctic. Atoms are the smallest units of elements that exist in nature. Every atom consists of protons, neutrons, and electrons. Nuclides are a specific type of atoms or nuclei. Every element, E, or AZE, can be defined by its atomic number, Z. This so-called “atomic number”, Z, is the number of protons in the atomic nucleus. In electrically neutral atoms it is also the number of electrons. However, the atomic mass or atomic weight is the sum of the masses of protons, neutrons, and electrons. The atomic mass or the mass number of heavy particles made up of neutrons also called nucleons, can be defined as A = Z + N + ne, where A is the “mass number” , Z is the number of protons, N is the number of neutrons, and ne is the number of electrons. Since electrons are ~1,836 times lighter than the hydrogen atom A is usually expressed as the sum of Z and N. An interactive chart of the nuclides can be reviewed here or here or here.

    A few definitions as they relate to isotopes follow.


    Mass number:            A: A = Z + N


    Protein:           The proton is a nuclear particle with the charge number +1, a spin quantum number ½, and a rest mass of 1.007276470(12) u.

     

    Neutron:    The neutron is a nuclear particle of zero charge, a spin quantum number ½, and a mass of 1.008664905(14) u.

     

    Electron:       The electron is an elementary particle that is not affected by the strong force. The electron has a spin quantum number ½, a negative charge and a rest mass of 0.00054879903(13) u.

     

    Atomic mass constant:         

     

    The atomic mass constant, u or mu, is defined as one 12 (1/12) of a carbon 12 atom in its nuclear and electronic ground state. u = mu = 1.6605402(10) x 10-27 kg = amu, atomic mass constant. Therefore the mass of a proton equals 1.007276 amu, the mass of a neutron equals 1.008665 amu, and the mass of an electron equals 0.00054858 amu.

     
    Atomic mass or weight:      

     

    Usually the atomic mass or weight is the average mass of all atoms in an element calculated from the relative abundance of the naturally occurring isotopes of the element.

     

    Atomic mass unit:     

     

    The atomic mass unit (symbolized AMU or amu) is defined as precisely 1/12 the mass of an atom of carbon-12. The carbon-12 (C-12) atom has six protons and six neutrons in its nucleus.


    In imprecise terms, one AMU is the average of the proton rest mass and the neutron rest mass. This is approximately 1.67377 x 10 -27 kilogram (kg), or 1.67377 x 10 -24 gram (g). The mass of an atom in AMU is roughly equal to the sum of the number of protons and neutrons in the nucleus. For example, in the case of oxygen, each isotope of this element has the same number of protons, Z = 8. Oxygen has three stable isotopes, A = 16, A = 17, and A = 18. From the sum of nucleons it is evident that these contain N = 8, N = 9, and N = 10 neutrons, respectively, within their nuclei, so that they produce the three mass numbers for oxygen. The notations of these are written with the mass number as a preceding superscript: 16O, 17O and 18O.

    Table : Isotopes and their natural abundance commonly used as tracers in biological experiments

    Z

    Name

    Symbol

    Mass of Atom (u)

    Abundance in %

    1

    Hydrogen

    Deuterium

    Tritium

    1H

    2H

    3H

      1.007825

      2.014102

      3.016049

    99.9885

    0.0115

    *

    6

    Carbon

    12C

    13C

    14C

    12.000000

    13.003355

    14.003242

    98.93

    1.07

    *

    7

    Nitrogen

    14N

    15N

    14.003074

    15.000109

    99.632

    0.368

    8

    Oxygen

    16O

    17O

    18O

    15.994915

    16.999132

    17.999160

    99.757

    0.038

    0.205

    15

    Phosphorus

    31P

    30.973762

    100

    16

    Sulphur

    32S

    33S

    34S

    36S

    31.972071

    32.971458

    33.967867

    35.967081

    94.93

    0.78

    4.29

    0.02


    The heavy isotopes most often used for metabolic studies are deuterium, 2H, heavy carbon, 13C and heavy nitrogen, 15N. Labeled amino acids and peptides plus tagging molecules are now commercially available. Virtually almost any labeled peptide can be synthesized using Fmoc-chemistry based automated synthesis.


    IRMS

     

    Isotope ratio mass spectrometry (IRMS) is a technique that has found an increasingly widespread use in archaeology, medicine, geology, biology, food authenticity, and forensic science. IRMS instruments have the ability to accurately and precisely measure variations in the abundance of isotopic ratios of light elements such as 13C/12C, 18O/16O, D/1H, 15N/14N, and 34S/32S. The ratios of these isotopes always need to be measured relative to an isotopic standard in order to eliminate any bias or systematic error that can occur during the measurements. IRMS provides information about the geographic, chemical, and biological origins of substances. The relative isotopic abundances of elements in the studied material allow determination of the source of an organic substance. Isotope ratios of elements, such as carbon, hydrogen, oxygen, sulfur, and nitrogen, can become locally enriched or depleted through a variety of kinetic and thermodynamic factors. The isotope ratios allow differentiation of samples which otherwise share identical chemical compositions.

     

    Labeling of cell compartments with stable isotopes avoids the use of radioactive tracer or labeling compounds. However, similar to radioactive isotopes, stable isotopes can be incorporated into bio-molecules without any changes in their chemical structure. Stable isotopes commonly used include deuterium, 2H, heavy nitrogen, 15N, heavy carbon, 13C and heavy oxygen, 18O. After incorporation into metabolic molecules, for example by metabolic labeling of cells, compounds containing these stable isotopes are detected in a mass spectrometer (MS) due to their increased mass when compared to natural light isotopes.

    During the last decades classical protein analysis has evolved into proteomics resulting in a flood of new improved technologies and approaches for the study of cells and their compartments. Even though genes encode and regulate proteins, it is the proteins that are responsible for most life functions and usually make up the majority of structures in cells or tissue. These large complex molecules are synthesized from smaller subunits called amino acids. The chemical nature of the 20 natural amino acids found in proteins determine their specific three-dimensional (3D) structures and define the particular function of a protein in the cell. The proteome refers to the entire set or complement of proteins that is or can be expressed by a cell, tissue, an organism or an organelle. Unlike the genome the proteome is more dynamic and changes from minute to minute in response to external and internal chemical cues. The chemistry of a protein is determined by its sequence as well as by the number and nature of other proteins or molecules it interacts with in the cell. Due to its complexity, proteomics, the study to explore protein structures and activities in an organism, will be the focus of research for many years to come. The Human Genome Project generated a flood of genomic and ultimately proteomic data which analyses will continue for many years.

    The Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of Energy (DOE) and the National Institutes of Health that was completed in 2003. The goal of this project was to identify all the approximately 20,500 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project. 
    [Source:http://web.ornl.gov/sci/techresources/HumanGenome/publicat/jmmbbag.pdf;http://genomics.energy.gov/http://www.proteinatlas.org/.]

    Presently, there is no clear consensus among scientists which isotope labeling strategy is the best one to use or is considered to be the “best labeling practice”. Ultimately, the selection of the isotope labeling technique will dependent upon experimental design, the scope of a particular analysis and the sample or system being analyzed. However, quantitative analysis of proteins and peptides is a very important issue in mass spectrometry based proteomics. During the last decade several isotope labeling techniques have been developed and introduced. These labeling techniques allow studying protein structures with the help of mass spectrometry based approaches or nuclear magnetic resonance (NMR) based experiments or techniques. Improvements made in both technologies have greatly expanded the range of biological applications that can now be studied.

    Types of isotope labeling methods that were developed since the 1960s are:

    • Uniform labeling with the stable isotopes 13C, 15N, and 2H.
    • Differential labeling of proteins and peptides.
    • Amino acid type selective labeling.
    • Site-specific labeling.
    • Random or fractional labeling.
    • Specific protonated amino acids against a deuterated background.
    • Selective incorporation of 15N-labeld amino acids against an unlabled (12C/14N) background.
    • Methyl specific protonation.
    • Segmental labeling.
    • Stereo-arrayed labeling.

     

    Recombinant proteins have now become an important part of medicine. These therapeutic proteins are used for the treatment of many different diseases. Unfortunately, proteins can have posttranslational modifications (PTMs) that may occur during manufacturing and storage. The result is product-related impurities. With improvements made in mass spectrometry instrumentation mass spectrometry is now being widely used for the characterization of recombinant proteins and their PTMs. The used of liquid chromatography coupled with mass spectrometry (LC-MS) and search algorithms makes it possible to identify hundreds of PTMs in a single LC-MS analysis. However, the quantification of PTMs using mass spectrometry is challenging. To address this problem many approaches using stable isotope-labeled internal standards have been developed. The goal is to provide accurate and precise quantification of proteins. In addition, many different approaches can be used to introduce stable isotopes into the sample.   

    Reference

    Atreya, Hanudatta S. (Ed.); Isotope labeling in Biomolecular NMR. Series: Advances in Experimental Medicine and Biology, Vol. 992, 2012, VIII, 219 p. 75 illus., 45 illus. in color. Cambridge Isotope Laboratories, Inc.: http://www.isotope.com/


    Donald Clayton
    ; Handbook of Isotopes in the Cosmos. Hydrogen to Gallium. August 2007. Cambridge University Press.
    www.cambridge.org.  isbn: 9780521530835


    Xinzhao Grace Jiang, Izydor Apostol, Quanzhou Luo, Jeffrey Lewis, Ronald Keener III, Shun Luo,


    Matthew Jerums, Xin Zhang, Jette Wypych, Gang Huang; Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. I: Principles and applications. Analytical Biochemistry 421 (2012) 506–516.



    Link to the Pandey Lab:
    http://www.silac.org/



    Zeland Muccio and Glen P. Jackson; Isotope ratio mass spectrometry. First published as an Advance Article on the web 14th November 2008. DOI: 10.1039/b808232d.



    Ray H. Liu, Dennis V. Canfield, Sheng-Meng Wang;  Quantitation and Mass Spectrometric Data of Drugs and Isotopically Labeled Analogs. Published: August 5, 2009 by CRC Press.



    Karen A. Sap and Jeroen A. A. Demmers (2012); Labeling Methods in Mass Spectrometry Based Quantitative

    Proteomics, Integrative Proteomics, Dr. Hon-Chiu Le ung (Ed.), ISBN: 978-953-51-0070-6, InTech, Available

    from:http://www.intechopen.com/books/integrative-proteomics/labeling-methods-in-mass-spectrometry-bas

    ed-quantitative-proteomics


    Warscheid, Bettina (Ed.); Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods and Protocols.
    Methods in Molecular Biology, Vol. 1188, 2014, XIV, 372 p. 65 illus., 27 illus. in color. Humana Press.


    D. Rodriguez, G. Audi, J. Aystö, D. Beck, K. Blaum, et al.. Accurate mass measure
    ments on neutron-defcient krypton isotopes. Nuclear Physics A, Elsevier, 2006, 769, pp.1-15.10.1016/j.nuclphysa.2006.02.001.in2p3-00025070


    0 0
  • 12/12/14--00:00: Dual labeled probes
  •  Dual labeled probes

     

    Methods that use fluorescence are widely used in biological sciences including biochemistry, biophotonics, biophysics, cell biology, clinical chemistry, histochemistry, and molecular medicine. Molecules in electronically excited states emit light and fluoresce. Many biological molecules display intrinsic fluorescence, or can be labeled with molecules that exhibit fluorescence. Fluorescent molecules used as labels are also called fluorophores. Fluorescently labeled molecules are also called molecular probes. Typically, fluorescent molecules are aromatic compounds that display light absorption in the ultraviolet to visible regions of the electromagnetic spectrum. UV: 250 to 400 nm; Visible: 400 to 700 nm.


    Fluorescently labeled probes are useful for many biochemical assays for monitoring of specific molecular events such as binding, cleavage or conformational changes of oligonucleotides, proteins, and peptides. Dual labeled probes containing a fluorophore and a quencher molecule have many applications in genetic analysis.
    Dual labeled probes are often used for quantitative polymer chain reaction (qPCR). Dual labeled probes are single-stranded oligonuclotides labeled with two different dyes, a fluorophore and a quencher molecule. Dual labeled probes are hybridization probes that can be synthesized using standard automated oligonucleotide chemistry and are also referred to as hydrolysis probes. Dual labeled probes function by reporting the presence of specific nucleic acids in homogenous samples in solution. Usually, a reporter dye is located at the 5’-end and a quencher molecule located at the 3’-end. The quencher molecule inhibits or decreases the fluorescence intensity of a sample or fluorophore via fluorescence resonance energy transfer (FRET). When the primer is elongated by the polymerase during PCR the dual labeled probe can bind to the amplified specific DNA template. Hydrolysis releases the reporter molecule from the probe or target hybrid causing an increase in fluorescence and the measured fluorescence signal is directly proportional to the amount of target DNA.

    What is FRET?

    Resonance energy transfer or RET occurs when an energy quantum is transmitted from its site of absorption to the site of its utilization in a molecule, or system of molecules. This phenomenon occurs between chromophores over distances greater than interatomic, without conversion to thermal energy, and without kinetic collision of the donor and acceptor.

     

    The donor is the dye that initially absorbs the energy.

    The acceptor is the chromophore or dye to which the energy is transferred.

     

    Fluorescence resonance energy transfer or FRET is now widely used in many fluorescence based applications. In the past FRET has been widely used to measure dimensions and distances within or between molecules over distances of 10 to 100 Å. This distance range is well suited to probe structures of oligonucleotides, proteins and peptides. The range of applications includes medical diagnostics, DNA analysis as well as optical imaging and many others. Favorable distances for energy transfer are typically in the size of proteins, DNA or RNA or the thickness of a membrane. Furthermore, the extent of FRET is predictable from spectral properties of the selected fluorophors. These properties allow for the design of experiments based on the known sizes and structural features of the studied sample.

     

    FRET is an electromagnetic phenomenon that can be explained using the laws of classical physics. FRET occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state. Typically, the donor molecule emits at shorter wavelengths that overlap with the absorption spectrum of the acceptor molecule. The energy transfer occurs without the appearance of a photon as a result of long-range dipole-dipole interactions between donor and acceptor. The extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and the acceptor molecule determine the rate of energy transfer.  

     
    Figure 1: The classical Jablonski diagram, shown her in the left panel, illustrates electronic states of a molecule as well as photo induced processes related to absorption and emission of energy. A simplified energy-level diagram illustrating resonance energy transfer is shown on the right panel. An asterisks denotes an excited state.  

    What is fluorescence quenching?

    Fluorescence quenching is any process that decreases the fluorescence intensity of a sample. Many molecular interactions can result in quenching including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching. The efficiency of fluorescence quenching is distance dependent. If the reporter fluorophore and quencher are far apart, fluorescence occurs. However, if the reporter and quencher are close together in space fluorescence is suppressed and does not occur. In oligonucleotide probes, the reporter and quencher are typically placed such that a change in distance will produce a maximal change in fluorescence. The observed fluorescent signal monitors the event, for example, a hybridization or nuclease activity. In this case the oligonucleotide sequence acts as a flexible tether or link between the fluorescent reporter and quencher. Since, many dyes are known to aggregate, self-associate, form dimers, trimers, or polymers, the tendency for dyes to aggregate is the basis of the static quenching mechanism.

    In static quenching or contact quenching a reporter such as FAM and a quencher such as BHQ-1 label can bind together to form a new, nonfluorescent intramolecular dimer. Furthermore, the efficiency of static quenching is dependent on the affinity of the reporter and quencher for each other. Often the reporter and quencher are planar, hydrophobic molecules that stack together to avoid contact with water.

     

    Reference


    dos Remedios CG, Moens PD.; Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor.
     J Struct Biol. 1995 Sep-Oct;115(2):175-85. 

    Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers. Authors: Johansson, M.K. Book: Methods in Molecular Biology, v. 335; V.V. Didenko, Ed; Humana Press: Totowa, NJ,
    2006; pp 17-29. 

    Intramolecular Dimers: A New Design Strategy for Fluorescence-Quenched Probes.
      Authors: Johansson, M.K.; Cook, R.M. Journal: Chem.-Eur. J. 2003, 9, 3466. 

    Intramolecular Dimers: A
    New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes. Authors: Mary Katherine Johansson, Henk Fidder, Daren Dick and Ronald M. Cook. Journal: J. Am. Chem. Soc. 2002, 124, 6950.

    Joseph R. Lakowicz; Principles of Fluorescence Spectroscopy. Editors: ISBN: 978-0-387-31278-1 (Print) 978-0-387-46312-4 (Online)

    Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Authors: Salvatore A. E. Marras, Fred Russell Kramer and Sanjay Tyagi. Journal: Nucleic Acids Research, 2002, 30, e122.

    Peng L, Minbo H, Fang C, Xi L, Chaocan Z.;The interaction between cholesterol and human serum albumin. Protein Pept Lett. 2008;15(4):360-4.


    0 0

    Ebola Peptides for Diagnostics and Vaccines


    Peptides derived from Ebola virus proteins can be used to study antigenicity and immunogenicity of Ebola proteins. In addition, these peptide epitopes can be used further to develop sensitive and accurate diagnostic tests using polyclonal or monoclonal antibodies. Another potential use for this type of peptides is for the development of unique peptide-based vaccines. In particular, succesful and potent vaccines could be developed using antigenic peptides derived from proteins of the Ebola virus or other Ebola virus strains. 
     

    Figure 1: Ultra structures and models of the Ebola virus and its genome (Source: Ellis et al. 1978; CDC).  Ellis et al. in 1978 showed that electron microscopy can be used to detect and observe the ultrastructure of the Eboli virus in infected human tissue. The Ebola virus was detected in tissue samples from human liver, kidney, spleen and lung.   

    Infection of a cell by a virus requires the fusion between viral and host membranes. Infection of a cell by the Ebola virus (EboV) begins with the uptake of viral particles into cellular endosomes. Experimental data suggests that the viral envelope glycoprotein (GP) catalyzes the fusion between the viral and host cell membranes. The fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike ultimately resulting in the formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Membrane fusion is mediated by fusion proteins that extrude from the viral membrane. Key components that are in contact with the host cell membrane are fusion peptides, parts of the fusion proteins. The Ebola glycoprotein (GP) is responsible for both receptor binding and membrane fusion. The GP is composed of two sub-domains, GP1 and GP2. The two domains are connected via a disulfide bond. The Ebola fusion peptide (EFP) (G524AAIGLAWIPYFGPAA539) is thought to be in direct contact with the host cell membrane. This peptide is conserved within the virus family. EFP is an internal fusion peptide located 22 residues from the N-terminus of GP2. Experimental data suggests that the EFP peptide in the presence of the membrane has a tendency to form helical structures.

    Figure 2: Model of the Ebola fusion protein in its fusiogenic state as suggested by Jaskierny et al. in 2011. The globular protein GP1 is thought to initiate the binding to the host cell receptor. The GP2 domain contains a helical bundle with the fusion peptide near the N-terminus. Jaskierney et al. studied the monomeric form of the internal fusion peptide from Ebola virus in membrane bilayer and water environments using computer simulations. The wild type Ebola fusion peptide, the W8A mutant form, and an extended construct with flanking residues were examined. The researchers found that the monomeric form of wild type Ebola fusion peptide adopts a coil-helix-coil structure with a short helix from residue 8 to 11 orientate parallel to the membrane surface.

     

    Using circular dichroism (CD) together with infrared (IR) spectroscopy the researchers showed that the EFP peptide has three states:

    A random coil in solution and either an α–helix or a β–sheet when bound to the membrane. Furthermore, the secondary structure of the membrane-bound peptide depends on the presence of Ca2+ and in the presence of Ca2+ a β-sheet structure is preferred while in the absence of Ca2+ helical structures are dominant. A nuclear magnetic resonance (NMR) study of EFP showed that the peptide adopts a random coil structure in aqueous buffers and a more defined structure in the presence of sodium dodecyl sulfate (SDS) micelles. Tryptophan fluorescent emission data suggests that W8 enters the hydrophobic core of SDS micelles. Nuclear Overhauser effect (NOE) measurements obtained from 1H NMR suggested the presence of a short 310 helix form I9 to F12 in the middle of the peptide while the N- and C-termini appear to be less structured.


    Miller et al. in 2011 performed a study using synthetic peptides of the CHR sequence region (C-peptides) to test if these peptides can inhibit the entry of the virus particles. The researchers prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, known to accumulate in endosomes, and found that this peptide specifically inhibits viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. The researchers determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Miller et al. argue that targeting C-peptides to endosomal compartments can serve as an approach to localize inhibitors to sites of membrane fusion.


    To diagnose and control the endemic outbreaks of haemorrhagic fever in humans caused by filioviruses, such as the Ebola and the Marburg virus, rapid, highly sensitive, reliable, and specific assays are required. The identification and characterization of antigenic sites in viral proteins is important for the development of viral antigen detection assays.

    Changula et al. in 2013 generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of the Zaire Ebola virus. The researchers divided the mABs into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. The use of synthetic peptides corresponding to the Ebola virus nucleoprotein (NP) sequence allowed to map mAb binding sites to seven antigenic regions in the C-terminal half of the NP. The mapped antigenic sites included two highly conserved regions present among all five Ebola virus species currently known. In addition, the scientists were successfully in producing species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. These results provide useful information for the development of Ebola virus antigen detection assays and potentially new vaccines for Ebola virus strains.


    Table 1: Ebola virus peptides

    Peptide

    Sequence

    Notes

     

    Fusion Peptide

    Jaskierny et al., 2011.

    EFP

    G524AAIGLAWIPYFGPAA539

    Chain A fusion peptide in SDS micelles at pH 7

     

     

     

     

    C-Peptide Study

    Miller et al. 2011

    Tat-Ebo

    YGRKKRRQRRR-GSG-IEPHDWTKNITDKIDQIIHDFVDK

    Ebola virus chain A fusion peptide

    Lys-Ebo

           KKKK-GSG-IEPHDWTKNITDKIDQIIHDFVDK

    Ebola virus chain A fusion peptide

    Tat-only

    YGRKKRRQRRR

     

    Tat-Scram

    YGRKKRRQRRR-GSG-HTEHINFQDDTIKIWPDVIKIKDD

     

    Tat-ASLV

    YGRKKRRQRRR-GSG-FNLSDHSESIQKKFQLMKEHVNKIG

     

     

     

     

     

    Peptide epitopes of mABs against EBOV NP

    Changula et al. 2013

    ZNP31-1-8

    ZNP41-2-4

    YDDDDDIPFP, aa 421–430

    NP protein

    ZNP74-7

    YDDDDDIPFPGPINDDDNPG, aa 421–440

    NP protein

    ZNP24-4-2

    QTQFRPIQNVPGPHRTIHHA, aa 521–540

    TPTVAPPAPVYRDHSEKKEL, aa 601–620

    NP protein

    ZNP106-9

    DTTIPDVVVD, aa 451–460a

    NP protein

    ZNP98-7

    MLTPINEEADPLDDADDETS, aa 561–580

    NP protein

    ZNP35-16-3-5

    DDEDTKPVPNRSTKGGQQKN, aa 491–510

    NP protein

    ZNP62-7

    YRDHSEKKELPQDEQQDQDH, aa 611–630

    NP protein

     

    Ebola virus NucleoProtein (NP) sequence

    >gi|158341892|gb|ABW34756.1| nucleoprotein, partial [Zaire ebolavirus]

    RQIQVHAEQGLIQYPTAWQSVGHMMVIFRMMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSG

    LLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLF

    PQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKK

    NEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQDTT

    IPDVVVDPDDGSYGEYQSYSENGMNAPDDLVLFDLDEDDEDTKPVPNRLTKGGQQKNSQKGHHTEGRQTQ

    SRPTQNVPGPRRTIHHASAPLTDNDRGNEPSGSTSPRMLTPINEEADPLDDADDETSSLPPLESDDEEQD

    RDETSNRTPTVAPPAPVYRDHSEKKELPQDEQQDQDHTQEARNQDSDNTQPEHSFEEMYRHIL


    The location of the Zaire envelope protein (ZNP) peptides are highlighted in red and magenta within the amino acid sequence of Ebola virus nucleoprotein.

    Table 2: Observed mutations for the QTQFRPIQNVPGPHRTIHHA, aa 521–540, peptide.


    Models of Ebola virus peptides and proteins

    Figure 3: NMR structure of the Ebola virus chain A fusion peptide, GAAIGLAWIPYFGPAA.


    Figure 4: Crystal structure models of the Ebola virus membrane fusion subunit, GP2 envelope glycoprotein ectodomain.

    Table 3: Peptides used for the production of rabbit antisera by Changula et al. 2013.


    Virus Protein

    Peptide

    Amino Acids

    EBOV NP

    QDHTQEARNQD

    628-638

    SUDV NP

    QGSESEALPINSKK

    631-644

    TAFV NP

    NQVSGSENTDNKPH

    630-643

    BDBV NP

    QSNQTNNEDNVRNN

    628-641

    RESTV NP

    TSQLNEDPDIGQSK

    630-643

    MARV NP

    RVVTKKGRTFLYPNDLLQ

    635-652

     

    Legend: BDBV = Bundibugyo virus; EBOV = Ebola virus; MATV = Marburg virus; RESTV = Reston virus; SUDV = Sudan virus; TAFV = Tai Forest Ebola virus.


    The membrane proximal external region (MPER) peptide


    Regula et al. in 2013 investigated the role of the membrane proximal external region (MPER) that precedes the transmembrane domain of glycoprotein 2 (GP2) of Ebola virus strains. Earlier research indicated that an infection by a filovirus requires membrane fusion between the host and the virus. The fusion process is facilitated by the two subunits of the envelope glycoprotein, the surface subunit GP1and the transmembrane subunit GP2. A sequence region called the membrane proximal external region (MPER) is a tryptophan (Trp, W) rich peptide segment located immediately in front of the transmembrane domain of GP2. In the human immunodeficiency virus 1 (HIV-1) glycoprotein gp41, the MPER is known to be critical for membrane fusion. In addition, this amino acid sequence was also identified as a target for several neutralizing antibodies. Regula et al. characterized the properties of GP MPER segment peptides of Ebola virus and Sudan virus. The study used  micelle-forming surfactants and lipids, at pH 7 and pH 4.6. The researchers employed circular dichroism (CD) spectroscopy and tryptophan fluorescence to determine if GP2 MPER peptides bind to micelles of sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC). Nuclear magnetic resonance (NMR) spectroscopy was used to reveal that residues 644 to 651 of the Sudan virus MPER peptide interacted directly with DPC. This interaction enhanced the helical conformation of the peptide. The scientists found that the Sudan virus MPER peptide moderately inhibited cell entry by a GP-pseudotyped vesicular stomatitis virus. However, it did not induce leakage of a fluorescent molecule from large unilamellar vesicle comprised of 1-palmitoyl-2-oleoylphostatidyl choline (POPC) or cause hemolysis. The analysis performed by this research group suggested that the filovirus GP MPER binds and inserts shallowly into lipid membranes.


    GP2 MPER Peptides


    Table 4: Alignment of GP2 MPER peptides from different viruses.

    Virus Strain

    GP2 MPER Peptide

    Amino Acids

    EBOV

        DKTLPDQGDNDNWWTGWRQW

    632 to 651

    BDBV

        DKPLPDQTDNDNWWTGWRQW

    632 to 651

    SUDV

        DNPLPNQDNDDNWWTGWRQW

    632 to 651

    TAFV

        DNNLPNQNDGSNWWTGWKQW

    632 to 651

    RESTV

        DNPLPDHGDDLNNWTGWRQW

    633 to 652

    FIV

        LQKWEDWVGWIGNIPQYLKG

    767 to 786

    HIV-1

    LLELDKWASLWNWFDITNWLWYIK

    660 to 683

     

    Table 4 shows the amino acid alignment of GP2 MPER regions from different members of the five Ebola virus species. Many residues that are identical in at least four of the viruses. For comparison, the MPER segments of FIV and HIV-1 gp41 are included.

     

    Legend: BDBV Bundbuyo virus, EBOV Ebola virus, FIV filio virus, HIV-1 human immunodeficiency virus 1, RESTV Reston virus, SUDV Sudan virus, TAFV Thai Forest virus.


    Alignments of GP2 MPER peptides from various virus strains.


    Location of the GP2 MPER peptides within the GP2 protein of the Ebola virus

    Figure 5: The location of the MPER peptides is highlighted in yellow in the crystal structure of the Ebola virus membrane fusion subunit, GP2 envelope glycoprotein ectodomain. The amino acid of the peptide shown in gray where not observed in the crystal indicating that this part of the peptide may take up a random coil structure in the crystal.


    Regula et al. used EBOV and SUDV MPER peptides for their study because both viruses are the most prevalent and pathogenic among the ebolaviruses. Synthetic peptides corresponding to the MPER region for EBOV and SUDV were used. The N-termini were blocked with an acetyl group and the C-termini contained an amide group.

     

    The study revealed three characteristics of the GP2 MPER peptides:



    • As a peptide, the GP2 MPER binds to micelle-forming surfactants in a pH-independent manner with higher affinity for zwitterionic micelles; 
    • A large conformational change to a more predominantly helical state occurs for the tryptophan-rich region of this peptide upon micelle-binding;
    • These peptides have modest viral entry inhibitory activity but do not induce leakage from LUVs.

     

    The study observed inhibitory activity for the S-MPER peptide which suggests that addition of this peptide may interfer with the viral entry process. For the FIV MPER peptide it was observed that a WX2WX2W motif is required for the membrane interaction responsible for its inhibitory activity.

    This peptide motif, WTGWRQW, is strictly conserved among all species.

    Results of the study indicated that the MPER peptide segments of EBOV and SUDV bind membrane surfaces which induces a conformational change in the Trp-rich peptide segment. This behavior suggests a role for the EBOV and SUDV MPER in membrane fusion.

     

    Reference



    http://www.cdc.gov/vhf/ebola/

    D. S. ELLIS, D. I. H. SIMPSON, D. P. FRANCIS, J. KNOBLOCH, E. T. W. BOWEN, PACIFICO LOLIK, AND ISAIAH MAYOM DENG; Ultrastructure of Ebola virus particles in human Liver. Journal of Clinical Pathology, 1978, 31, 201-208.

    Katendi Changula
    , Reiko Yoshidac, Osamu Noyoric, Andrea Marzid, Hiroko Miyamotoc, Mari Ishijimac, Ayaka Yokoyamac, Masahiro Kajiharac,Heinz Feldmannd, Aaron S. Mweenea, Ayato Takadaa; Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein.  Virus Research 176  (2013) 83– 90.

    Thomas Hoenen, Allison Groseth, and Heinz Feldmann; Current Ebola vaccines. Expert Opin Biol Ther. 2012 July; 12(7): 859–872.  oi:10.1517/14712598.2012.685152.

    Adam J. Jaskierny
    , Afra Panahi, and Michael Feig; Effect of flanking residues on the conformational sampling of the internal fusion peptide from Ebola virus. Proteins. 2011 April ; 79(4): 1109–1117. doi:10.1002/prot.22947.

    Emily Happy Miller, Joseph S. Harrison, Sheli R. Radoshitzky, Chelsea D. Higgins, Xiaoli Chi, Lian Dong, Jens H. Kuhn, Sina Bavari, Jonathan R. Lai, and Kartik Chandran; Inhibition of Ebola Virus Entry by a C-peptide Targeted to Endosome J Biol Chem. May 6, 2011; 286(18): 15854–15861. Published online Mar 16, 2011. doi:  10.1074/jbc.M110.207084. PMCID: PMC3091195.

    Lauren K. Regula, Richard Harris, Fang Wang, Chelsea D. Higgins, Jayne F. Koellhoffer, Yue Zhao, Kartik Chandran, Jianmin Gao, Mark E. Girvin, and Jonathan R. Lai; Conformational Properties of Peptides Corresponding to the Ebolavirus GP2 Membrane-Proximal External Region in the Presence of Micelle-Forming Surfactants and Lipids. Biochemistry. 2013 May 21; 52(20): . doi:10.1021/bi400040v.



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    MicroRNAs or miRNAs are a class of endogenous small RNAs approximately 22 nucleotides in size found in plants and animals including humans. More and more research data indicate that in humans microRNAs or miRNAs are useful indicator molecules for different cancer types which may be useful as cancer biomarkers.


    Increasingly, it has become apparent that microRNAs take part in the development of cancer. This observation has made miRNAs potential biomarkers for cancer diagnosis and prognosis. Therefore it can be reasoned that microRNAs or miRNAs are good candidates as cancer biomarker. Because many studies now suggest that the pattern of microRNA expression in tissues reflects the disease status in this tissue, miRNA expression levels may serve as potential biomarkers with multiple applications in clinical diagnostics. miRNAs can be successfully isolated from biological fluids allowing for the development of biofluid biopsies or diagnostics. This type of biomarker diagnostics promises to allow for the development of minimal invasive assays, to save cost and simplify complex invasive procedures.

    miRNA's processing occurs from approximately 70 nucleotides in size hairpin precursor RNAs by the protein Dicer. miRNA have been shown to regulate their target messengerRNA (mRNA) by destabilizing mRNA molecules and translational repression.

    Biosynthesis Inc.

    Figure 1: Pre-miRNA nuclear export machinery.


    Okada et al in 2009. solved the structure of the "pre-miRNA nuclear export machinery" formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP) at 2.9 angstrom. The data showed that RNA recognition by Exp-5:RanGTP does not depend on RNA sequence. This implys that Exp-5:RanGTP can recognize a variety of pre-miRNAs.

    Biosynthesis Inc.

    Figure 2: The molecules present in the structure and their interactions are shown here.


    [Source: 
    http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=78532]


    During a study of the nematode Caenorhabditis elegans (C. elegans) development involving the gene lin-14 Victor Ambros, Rosalind Lee and Rhonda Feinbaum first discovered miRNAs in 1993. However, at the time the researcher speculated that these molecules could be a nematode idiosyncrasy. In 2000, it was shown that let-7 represses lin-41, lin-14, lin28, lin42 and daf12 mRNA during transition in developmental stages in C. elegans. At this time miRNAs were recognized as small regulatory RNAs. Furthermore, it became clear that miRNAs are conserved in many species.  In addition, it was noted that short non-coding RNAs, first identified in 1993, were part of a wider phenomenon. For example, Lagos-Quintana et al. in 2001 referred to 22- and 21-nucleotide (nt) RNAs as small temporal RNAs (stRNAs). These RNAs functioned as key regulators in developmental timing. The Tuschl lab in 2001 showed that many 21- and 22-nt expressed RNAs exist in invertebrates and vertebrates. Furthermore, some of these RNAs, similar to let-7 stRNA, are highly conserved. This discovery led to the conclusion that sequence-specific, posttranscriptional regulatory mechanisms as mediated by small RNAs are more general than was previously appreciated. Over 4000 miRNAs have been found so far in all studied eukaryotes. More than 700 miRNAs have already been identified in humans. In addition, more than and over 800 are predicted to exist. V. Ambros in 2001 reported that these microRNAs are diverse in sequence and expression patterns. The observation that these molecules are evolutionarily widespread suggests that they may participate in a wide range of genetic regulatory pathways. Figure 3 shows the dramatic increase in publications involving miRNAs and miRNA research. 

    Biosynthesis Inc.

    Figure 3: Increase in miRNA publications in Pubmed.


    Animal miRNAs derived from longer primary transcripts carry hairpin structures. The processing of these precursor hairpin RNA structures proceeds in a stepwise fashion catalyzed by the RNase III enzymes Drosha and Dicer. Drosha cleaves these RNA molecules near the hairpin base to release the pre-miRNA hairpin. This reaction occurs in the nucleus. Next, the pre-miRNA hairpin is exported into the cytoplasm and Dicer cleaves on the loop side of the hairpin.  The result is a miRNA:miRNA* duplex. In the next step, one strand of this complex is preferentially incorporated into a silencing complex.

    Recently an alternative nuclear pathway for miRNA biogenesis was identified in invertebrates. Researchers found that short introns with hairpin potential, termed mirtrons, can be spliced and debranched into pre-miRNA hairpin mimics that appear to bypass Drosha cleavage. Debranched mirtrons access the canonical miRNA pathway during nuclear export and are then cleaved by Dicer and incorporated into silencing complexes. As pointed out by Brezikow et al. in 2007, mirtrons are alternative precursor molecules for microRNA biogenesis present in invertebrates. Splicing allows these short hairpin introns to bypass Drosha cleavage. Drosha cleavage is essential for the generation of canonical animal microRNAs. With the help of computational and experimental strategies Brezikow et al. establish that mammals have mirtrons as well. Therefore, mirtrons are miRNAs located in the introns of mRNA encoding genes. Brezikow et al. identified three (3) well conserved mirtrons expressed in diverse mammals. In addition, 16 primate-specific mirtrons, and 46  mirtron candidates, as supported by limited cloning, are suspected to be present in primates as well.

     


    0 0

    Stable isotope labeling can be used to study metabolic pathways and turnover rates of biological molecules such as proteins, peptides, RNA and others.

    Early on stable isotope labeling was used to define metabolic pathways and turnover rates of biological molecules such as amino acids and peptides in the body. Stable isotope labeling was used shortly after the discovery of deuterium in 1931, the heavy isotope of hydrogen, by Harold Urey. Urey received a Nobel Prize in 1934 for this discovery. Already in 1935 Rudolf Schoenheimer used isotopic tracer techniques in metabolic research for stable isotope labeling experiments. His work, for the first time, provided clear experimental evidence of the dynamics of the body’s metabolism. Schoenheimer’s research group defined synthesis and degradation pathways of many compounds in the following years. The availability of enriched 15N, the heavy isotope of nitrogen, enabled demonstrating that protein had a dynamic life cycle in that they were both continually being synthesized and degraded. However, all of this early work was performed using crude methods that involved the preparation of labeled compounds, followed by tedious measurements using isotope ratio mass spectrometry (IRMS). In addition, for the measurements of the isotopic enrichment, all compounds investigated needed to be reduced to the simple gases carbon dioxide (CO2), hydrogen (H2), and nitrogen (N2). After World War II, the radioisotopes tritium (3H) and carbon 14 (14C) were mostly used for many years to come. Improvements made in instrument development, specifically for gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) instruments, allowed the use of stable isotopes for metabolic and proteomic studies with more ease in recent decades. The increasing trend for the use of isotopes, in particular the use of stable isotopes for metabolic or proteomic studies, is reflected in the increased publication volume of papers covering these applications which can be seen in figure 1. More than 18,500 publications for isotope labeling have been published since 1953 and can be found in PubMed. 



    Figure 1: Timeline of papers in PubMed describing various isotope labeling methods and applications. As can been seen the use of isotopes, in particular stable isotopes, has increased in recent years.


    Figure 2: The helium, as an example, has 2 protons, 2 neutrons, and 2 electrons. Helium has two isotopes but it consists almost entirely of He-4 with natural He only containing little more than 0.0001% of He-3.

     

    Let us briefly review the basic chemistry and physical properties of isotopes. Elements contain nuclei each with their own unique physicochemical characteristics. According to the “atomic theory”, each atomic nucleus can be defined by its mass, its number of constituent elementary particles, its spin rate, its magnetic strength, its electric charge, and its multitude of existing forms. The uniqueness of elements is reflected in their characteristic ratio of isotopes. This phenomenon has allowed geological scientists to study the geological record of the earth’s crust revealing its history. Carbon dioxide (CO2) and methane (CH4) in the atmosphere record its history and the impact of human beings on the Earth’s atmosphere which can be studied from ice cores, for example taken from glaciers or eternal ice fields, in the Alps, in Greenland or in the Antarctic. Atoms are the smallest units of elements that exist in nature. Every atom consists of protons, neutrons, and electrons. Nuclides are a specific type of atoms or nuclei. Every element, E, or AZE, can be defined by its atomic number, Z. This so-called “atomic number”, Z, is the number of protons in the atomic nucleus. In electrically neutral atoms it is also the number of electrons. However, the atomic mass or atomic weight is the sum of the masses of protons, neutrons, and electrons. The atomic mass or the mass number of heavy particles made up of neutrons also called nucleons, can be defined as A = Z + N + ne, where A is the “mass number” , Z is the number of protons, N is the number of neutrons, and ne is the number of electrons. Since electrons are ~1,836 times lighter than the hydrogen atom A is usually expressed as the sum of Z and N. An interactive chart of the nuclides can be reviewed here or here or here.

    A few definitions as they relate to isotopes follow.


    Mass number:            A: A = Z + N

    Protein:           The proton is a nuclear particle with the charge number +1, a spin quantum number ½, and a rest mass of 1.007276470(12) u.

     

    Neutron:    The neutron is a nuclear particle of zero charge, a spin quantum number ½, and a mass of 1.008664905(14) u.

     

    Electron:       The electron is an elementary particle that is not affected by the strong force. The electron has a spin quantum number ½, a negative charge and a rest mass of 0.00054879903(13) u.

     

    Atomic mass constant:         

     

    The atomic mass constant, u or mu, is defined as one 12 (1/12) of a carbon 12 atom in its nuclear and electronic ground state. u = mu = 1.6605402(10) x 10-27 kg = amu, atomic mass constant. Therefore the mass of a proton equals 1.007276 amu, the mass of a neutron equals 1.008665 amu, and the mass of an electron equals 0.00054858 amu.

     
    Atomic mass or weight:      

     

    Usually the atomic mass or weight is the average mass of all atoms in an element calculated from the relative abundance of the naturally occurring isotopes of the element.

     

    Atomic mass unit:     

     

    The atomic mass unit (symbolized AMU or amu) is defined as precisely 1/12 the mass of an atom of carbon-12. The carbon-12 (C-12) atom has six protons and six neutrons in its nucleus.


    In imprecise terms, one AMU is the average of the proton rest mass and the neutron rest mass. This is approximately 1.67377 x 10 -27 kilogram (kg), or 1.67377 x 10 -24 gram (g). The mass of an atom in AMU is roughly equal to the sum of the number of protons and neutrons in the nucleus. For example, in the case of oxygen, each isotope of this element has the same number of protons, Z = 8. Oxygen has three stable isotopes, A = 16, A = 17, and A = 18. From the sum of nucleons it is evident that these contain N = 8, N = 9, and N = 10 neutrons, respectively, within their nuclei, so that they produce the three mass numbers for oxygen. The notations of these are written with the mass number as a preceding superscript: 16O, 17O and 18O.

    Table : Isotopes and their natural abundance commonly used as tracers in biological experiments

    Z

    Name

    Symbol

    Mass of Atom (u)

    Abundance in %

    1

    Hydrogen

    Deuterium

    Tritium

    1H

    2H

    3H

      1.007825

      2.014102

      3.016049

    99.9885

    0.0115

    *

    6

    Carbon

    12C

    13C

    14C

    12.000000

    13.003355

    14.003242

    98.93

    1.07

    *

    7

    Nitrogen

    14N

    15N

    14.003074

    15.000109

    99.632

    0.368

    8

    Oxygen

    16O

    17O

    18O

    15.994915

    16.999132

    17.999160

    99.757

    0.038

    0.205

    15

    Phosphorus

    31P

    30.973762

    100

    16

    Sulphur

    32S

    33S

    34S

    36S

    31.972071

    32.971458

    33.967867

    35.967081

    94.93

    0.78

    4.29

    0.02


    The heavy isotopes most often used for metabolic studies are deuterium, 2H, heavy carbon, 13C and heavy nitrogen, 15N. Labeled amino acids and peptides plus tagging molecules are now commercially available. Virtually almost any labeled peptide can be synthesized using Fmoc-chemistry based automated synthesis.


    IRMS

     

    Isotope ratio mass spectrometry (IRMS) is a technique that has found an increasingly widespread use in archaeology, medicine, geology, biology, food authenticity, and forensic science. IRMS instruments have the ability to accurately and precisely measure variations in the abundance of isotopic ratios of light elements such as 13C/12C, 18O/16O, D/1H, 15N/14N, and 34S/32S. The ratios of these isotopes always need to be measured relative to an isotopic standard in order to eliminate any bias or systematic error that can occur during the measurements. IRMS provides information about the geographic, chemical, and biological origins of substances. The relative isotopic abundances of elements in the studied material allow determination of the source of an organic substance. Isotope ratios of elements, such as carbon, hydrogen, oxygen, sulfur, and nitrogen, can become locally enriched or depleted through a variety of kinetic and thermodynamic factors. The isotope ratios allow differentiation of samples which otherwise share identical chemical compositions.

     

    Labeling of cell compartments with stable isotopes avoids the use of radioactive tracer or labeling compounds. However, similar to radioactive isotopes, stable isotopes can be incorporated into bio-molecules without any changes in their chemical structure. Stable isotopes commonly used include deuterium, 2H, heavy nitrogen, 15N, heavy carbon, 13C and heavy oxygen, 18O. After incorporation into metabolic molecules, for example by metabolic labeling of cells, compounds containing these stable isotopes are detected in a mass spectrometer (MS) due to their increased mass when compared to natural light isotopes.

    During the last decades classical protein analysis has evolved into proteomics resulting in a flood of new improved technologies and approaches for the study of cells and their compartments. Even though genes encode and regulate proteins, it is the proteins that are responsible for most life functions and usually make up the majority of structures in cells or tissue. These large complex molecules are synthesized from smaller subunits called amino acids. The chemical nature of the 20 natural amino acids found in proteins determine their specific three-dimensional (3D) structures and define the particular function of a protein in the cell. The proteome refers to the entire set or complement of proteins that is or can be expressed by a cell, tissue, an organism or an organelle. Unlike the genome the proteome is more dynamic and changes from minute to minute in response to external and internal chemical cues. The chemistry of a protein is determined by its sequence as well as by the number and nature of other proteins or molecules it interacts with in the cell. Due to its complexity, proteomics, the study to explore protein structures and activities in an organism, will be the focus of research for many years to come. The Human Genome Project generated a flood of genomic and ultimately proteomic data which analyses will continue for many years.

    The Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of Energy (DOE) and the National Institutes of Health that was completed in 2003. The goal of this project was to identify all the approximately 20,500 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project. 
    [Source:http://web.ornl.gov/sci/techresources/HumanGenome/publicat/jmmbbag.pdf;http://genomics.energy.gov/http://www.proteinatlas.org/.]

    Presently, there is no clear consensus among scientists which isotope labeling strategy is the best one to use or is considered to be the “best labeling practice”. Ultimately, the selection of the isotope labeling technique will dependent upon experimental design, the scope of a particular analysis and the sample or system being analyzed. However, quantitative analysis of proteins and peptides is a very important issue in mass spectrometry based proteomics. During the last decade several isotope labeling techniques have been developed and introduced. These labeling techniques allow studying protein structures with the help of mass spectrometry based approaches or nuclear magnetic resonance (NMR) based experiments or techniques. Improvements made in both technologies have greatly expanded the range of biological applications that can now be studied.

    Types of isotope labeling methods that were developed since the 1960s are:

    • Uniform labeling with the stable isotopes 13C, 15N, and 2H.
    • Differential labeling of proteins and peptides.
    • Amino acid type selective labeling.
    • Site-specific labeling.
    • Random or fractional labeling.
    • Specific protonated amino acids against a deuterated background.
    • Selective incorporation of 15N-labeld amino acids against an unlabled (12C/14N) background.
    • Methyl specific protonation.
    • Segmental labeling.
    • Stereo-arrayed labeling.

     

    Recombinant proteins have now become an important part of medicine. These therapeutic proteins are used for the treatment of many different diseases. Unfortunately, proteins can have posttranslational modifications (PTMs) that may occur during manufacturing and storage. The result is product-related impurities. With improvements made in mass spectrometry instrumentation mass spectrometry is now being widely used for the characterization of recombinant proteins and their PTMs. The used of liquid chromatography coupled with mass spectrometry (LC-MS) and search algorithms makes it possible to identify hundreds of PTMs in a single LC-MS analysis. However, the quantification of PTMs using mass spectrometry is challenging. To address this problem many approaches using stable isotope-labeled internal standards have been developed. The goal is to provide accurate and precise quantification of proteins. In addition, many different approaches can be used to introduce stable isotopes into the sample.   

    Reference

    Atreya, Hanudatta S. (Ed.); Isotope labeling in Biomolecular NMR. Series: Advances in Experimental Medicine and Biology, Vol. 992, 2012, VIII, 219 p. 75 illus., 45 illus. in color. Cambridge Isotope Laboratories, Inc.: http://www.isotope.com/


    Donald Clayton
    ; Handbook of Isotopes in the Cosmos. Hydrogen to Gallium. August 2007. Cambridge University Press.
    www.cambridge.org.  isbn: 9780521530835


    Xinzhao Grace Jiang, Izydor Apostol, Quanzhou Luo, Jeffrey Lewis, Ronald Keener III, Shun Luo,


    Matthew Jerums, Xin Zhang, Jette Wypych, Gang Huang; Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. I: Principles and applications. Analytical Biochemistry 421 (2012) 506–516.



    Link to the Pandey Lab:
    http://www.silac.org/



    Zeland Muccio and Glen P. Jackson; Isotope ratio mass spectrometry. First published as an Advance Article on the web 14th November 2008. DOI: 10.1039/b808232d.



    Ray H. Liu, Dennis V. Canfield, Sheng-Meng Wang;  Quantitation and Mass Spectrometric Data of Drugs and Isotopically Labeled Analogs. Published: August 5, 2009 by CRC Press.



    Karen A. Sap and Jeroen A. A. Demmers (2012); Labeling Methods in Mass Spectrometry Based Quantitative

    Proteomics, Integrative Proteomics, Dr. Hon-Chiu Le ung (Ed.), ISBN: 978-953-51-0070-6, InTech, Available

    from:http://www.intechopen.com/books/integrative-proteomics/labeling-methods-in-mass-spectrometry-bas

    ed-quantitative-proteomics


    Warscheid, Bettina (Ed.); Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods and Protocols.
    Methods in Molecular Biology, Vol. 1188, 2014, XIV, 372 p. 65 illus., 27 illus. in color. Humana Press.


    D. Rodriguez, G. Audi, J. Aystö, D. Beck, K. Blaum, et al.. Accurate mass measure
    ments on neutron-defcient krypton isotopes. Nuclear Physics A, Elsevier, 2006, 769, pp.1-15.10.1016/j.nuclphysa.2006.02.001.in2p3-00025070


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  • 12/12/14--00:00: Dual labeled probes
  •  Dual labeled probes

     

    Methods that use fluorescence are widely used in biological sciences including biochemistry, biophotonics, biophysics, cell biology, clinical chemistry, histochemistry, and molecular medicine. Molecules in electronically excited states emit light and fluoresce. Many biological molecules display intrinsic fluorescence, or can be labeled with molecules that exhibit fluorescence. Fluorescent molecules used as labels are also called fluorophores. Fluorescently labeled molecules are also called molecular probes. Typically, fluorescent molecules are aromatic compounds that display light absorption in the ultraviolet to visible regions of the electromagnetic spectrum. UV: 250 to 400 nm; Visible: 400 to 700 nm.


    Fluorescently labeled probes are useful for many biochemical assays for monitoring of specific molecular events such as binding, cleavage or conformational changes of oligonucleotides, proteins, and peptides. Dual labeled probes containing a fluorophore and a quencher molecule have many applications in genetic analysis.
    Dual labeled probes are often used for quantitative polymer chain reaction (qPCR). Dual labeled probes are single-stranded oligonuclotides labeled with two different dyes, a fluorophore and a quencher molecule. Dual labeled probes are hybridization probes that can be synthesized using standard automated oligonucleotide chemistry and are also referred to as hydrolysis probes. Dual labeled probes function by reporting the presence of specific nucleic acids in homogenous samples in solution. Usually, a reporter dye is located at the 5’-end and a quencher molecule located at the 3’-end. The quencher molecule inhibits or decreases the fluorescence intensity of a sample or fluorophore via fluorescence resonance energy transfer (FRET). When the primer is elongated by the polymerase during PCR the dual labeled probe can bind to the amplified specific DNA template. Hydrolysis releases the reporter molecule from the probe or target hybrid causing an increase in fluorescence and the measured fluorescence signal is directly proportional to the amount of target DNA.

    What is FRET?

    Resonance energy transfer or RET occurs when an energy quantum is transmitted from its site of absorption to the site of its utilization in a molecule, or system of molecules. This phenomenon occurs between chromophores over distances greater than interatomic, without conversion to thermal energy, and without kinetic collision of the donor and acceptor.

     

    The donor is the dye that initially absorbs the energy.

    The acceptor is the chromophore or dye to which the energy is transferred.

     

    Fluorescence resonance energy transfer or FRET is now widely used in many fluorescence based applications. In the past FRET has been widely used to measure dimensions and distances within or between molecules over distances of 10 to 100 Å. This distance range is well suited to probe structures of oligonucleotides, proteins and peptides. The range of applications includes medical diagnostics, DNA analysis as well as optical imaging and many others. Favorable distances for energy transfer are typically in the size of proteins, DNA or RNA or the thickness of a membrane. Furthermore, the extent of FRET is predictable from spectral properties of the selected fluorophors. These properties allow for the design of experiments based on the known sizes and structural features of the studied sample.

     

    FRET is an electromagnetic phenomenon that can be explained using the laws of classical physics. FRET occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state. Typically, the donor molecule emits at shorter wavelengths that overlap with the absorption spectrum of the acceptor molecule. The energy transfer occurs without the appearance of a photon as a result of long-range dipole-dipole interactions between donor and acceptor. The extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and the acceptor molecule determine the rate of energy transfer.  

     
    Figure 1: The classical Jablonski diagram, shown her in the left panel, illustrates electronic states of a molecule as well as photo induced processes related to absorption and emission of energy. A simplified energy-level diagram illustrating resonance energy transfer is shown on the right panel. An asterisks denotes an excited state.  

    What is fluorescence quenching?

    Fluorescence quenching is any process that decreases the fluorescence intensity of a sample. Many molecular interactions can result in quenching including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching. The efficiency of fluorescence quenching is distance dependent. If the reporter fluorophore and quencher are far apart, fluorescence occurs. However, if the reporter and quencher are close together in space fluorescence is suppressed and does not occur. In oligonucleotide probes, the reporter and quencher are typically placed such that a change in distance will produce a maximal change in fluorescence. The observed fluorescent signal monitors the event, for example, a hybridization or nuclease activity. In this case the oligonucleotide sequence acts as a flexible tether or link between the fluorescent reporter and quencher. Since, many dyes are known to aggregate, self-associate, form dimers, trimers, or polymers, the tendency for dyes to aggregate is the basis of the static quenching mechanism.

    In static quenching or contact quenching a reporter such as FAM and a quencher such as BHQ-1 label can bind together to form a new, nonfluorescent intramolecular dimer. Furthermore, the efficiency of static quenching is dependent on the affinity of the reporter and quencher for each other. Often the reporter and quencher are planar, hydrophobic molecules that stack together to avoid contact with water.

     

    Reference


    dos Remedios CG, Moens PD.; Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor.
     J Struct Biol. 1995 Sep-Oct;115(2):175-85. 

    Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers. Authors: Johansson, M.K. Book: Methods in Molecular Biology, v. 335; V.V. Didenko, Ed; Humana Press: Totowa, NJ,
    2006; pp 17-29. 

    Intramolecular Dimers: A New Design Strategy for Fluorescence-Quenched Probes.
      Authors: Johansson, M.K.; Cook, R.M. Journal: Chem.-Eur. J. 2003, 9, 3466. 

    Intramolecular Dimers: A
    New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes. Authors: Mary Katherine Johansson, Henk Fidder, Daren Dick and Ronald M. Cook. Journal: J. Am. Chem. Soc. 2002, 124, 6950.

    Joseph R. Lakowicz; Principles of Fluorescence Spectroscopy. Editors: ISBN: 978-0-387-31278-1 (Print) 978-0-387-46312-4 (Online)

    Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Authors: Salvatore A. E. Marras, Fred Russell Kramer and Sanjay Tyagi. Journal: Nucleic Acids Research, 2002, 30, e122.

    Peng L, Minbo H, Fang C, Xi L, Chaocan Z.;The interaction between cholesterol and human serum albumin. Protein Pept Lett. 2008;15(4):360-4.


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    Can cancer cells or their microenvironment be targeted selectively to treat tumors?

    Yes, is appears that this is possible.


    A number of peptides have been reported to specifically target tumor and tumor associated microenvironments, such as the tumor vasculature, after their systematic delivery. These peptides are known as “tumor-specific internalizing peptides” (TSIPs) or “tumor homing peptides” (THPs).

    Similar to anti-cancer peptides tumor-specific internalizing peptides are usually short peptides in sequence lengths of 3 to 15 amino acids that specifically recognize and bind to tumor cells or tumor vasculature. Since 1998 a number of these peptides have been identified using in vitro and in vivo phage display technology. Phage display is a molecular biology technology in which proteins or peptides are displayed on the surface of a phage as a fusion with one of the phage coated proteins. Phage display has been used intensively for the screening for protein-protein interactions. This screening method allowed for the identification of tumor-specific or tumor homing peptides that target specific tumor cells or tumor vasculature.

    According to the International Agency for Research on Cancer, an agency of the World Health Organization, cancer is now the world’s biggest killer. The “World Cancer Report” showed that there were 8.2 million deaths from cancer in 2012 and predicts that cancer cases worldwide will rise by 75 % over the next two decades. By then it is estimated that up to 25 million people may be suffering from cancer worldwide. Unfortunately, despite progress made in our understanding of the molecular basis of cancer and improvements made in treatment options, mortality rate is still high. This suggests that the availability of new types, more selective drugs that fight cancer would be of great benefit to humans.

    Tumor-specific internalizing peptides or tumor homing peptides have common sequence motifs like RGD, or NGR, which specifically bind to a surface molecule on tumor cells or tumor vasculature. The best known examples are the short peptides RGD and NGR. The RGD (Arg-Gly-Asp) peptide is known to bind α integrins and NGR (Asn-Gly-Arg) is known to bind to a receptor aminopeptidase N present on the surface of tumor endothelial cells, also called tumor angiogenic markers. It is no wonder that tumor-specific internalizing peptides are being used in cancer diagnosis and treatment. So far, many anti-cancer and imaging agents have been targeted to tumor sites in mice models by conjugation them to tumor-specific peptides. A database called “TumorHoPe” provides comprehensive information about experimentally validated tumor homing peptides and their target cells (http://crdd.osdd.net/raghava/tumorhope/). This is a manually curated database containing 744 entries of experimentally characterized tumor homing peptides that recognize tumor tissues and tumor associated micro environment, including tumor metastasis.


    A list of some tumor homing peptide motifs

     

    Motif

    Action

    NGR (Asn-Gly-Arg)

    Binds aminopeptidase N

    GSL (Gly-Ser-Leu)

    Inhibition of tumor homing

    RGD (Arg-Gly-Asp)

    Binds selectively to integrins which are overexpressed on endothelial cell surface in the cancer and facilitate cancer cell migration

    TSPLNIHGQKL

    Hn-1 appears to be HNSCC specific. Targeted drug delivery into solid tumors.

     

    Models of tumor homing peptides




    The specific internalization of peptides that target tumor cells has been evaluated for targeted siRNA delivery into human cancer cells. Un et al. in 2012 investigated the internalization of the HN-1TYR-anti-hRRM2 siRNAR peptide conjugate in human head and neck or breast cancer cells to establish its utility for targeted siRNA delivery into human cancer cells. The researchers used a FITC-HN-1TYR-anti-hRRM2 siRNAR construct to image its successful internalization into a human cancer cell line. For the synthesis of the fluorescent siRNA delivery vehicle, FITC-HN-1TYR-anti-hRRM2 siRNAR, a tyrosine and a FITC was added to the N-terminal end. Next, a synthetic anti-hRRM2 siRNA was synthesized with fluorine, incorporated at its 2’-OH position, to avoid degradation by RNases in vivo, and conjugated to the 5’-end of the antisense strand using a hexynyl phophoramidite linker. The selected HN1 peptide, a 12mer peptide that was isolated by peptide display library screening using a M13 phage library, contains the sequence TSPLNIHNGQKL. It has the ability to translocate drugs across the cell membrane into the cytosol, its uptake occurs in a tumor-specific manner, and it is capable of penetrating solid tumors. Ribonucleotide Reductase (RR), composed of the subunits hRRM1 and hRRM2, catalyses the conversion of ribonucleotides to their corresponding deoxy forms need for DNA replication. The researchers choose an anti-hRRM2 siRNA to allow for the degradation of hRRM2’s mRNA to suppress tumorgenesis.

    To conclude, tumor-specific internalizing peptides or tumor homing peptides appear to be future drug candidates for targeted siRNA delivery into human cancer cells that may enable a more selective treatment of tumors with less site effects.

    References

    Kapoor P, Singh H, Gautam A, Chaudhary K, Kumar R, et al. (2012); TumorHoPe: A Database of Tumor Homing Peptides. PLoS ONE 7(4): e35187. doi:10.1371/journal.pone.0035187.

    FRANK UN, BINGSEN ZHOU and YUN YEN; The Utility of Tumor-specifically Internalizing Peptides for Targeted siRNA Delivery into Human Solid Tumors. ANTICANCER RESEARCH 32: 4685-4690 (2012).

     


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    Control templates for molecular DNA/RNA diagnostics

    As the number and scope of disease-producing pathogens and their genetic variants that cause human disease have continued to increase, there has been a commensurate and rapid increase in the use of nucleic acid based tests for routine clinical diagnosis. Due to the complex nature of nucleic acids, these molecular tests must be fully controlled to accurately ascertain their specificity and sensitivity. However, the success of molecular diagnostics is often impeded by the availability of DNA- or RNA-based positive controls with the same or similar number of mutations as the organism being screened, for example, in the case of a pandemic or newly emerging disease, such as Ebola, where it can be difficult to acquire necessary positive controls.

    DNA or RNA standards allow a researcher to determine if an assay accurately represents the composition or quantities of known input as well as to derive standard calibration curves. This allows to relate read-out counts of analyte concentrations in the studied samples to accurate amounts or quantities. Furthermore, the use of control standards allows for direct measurement of error rates, coverage biases, and other veriables that can affect downstream analysis, such as the analysis of various isoforms.   

    Biosynthesis Inc.
     
     

    As Good Laboratory Practices, government agencies, and organizations that establish standards and control require diagnostic laboratories to use stringent quality controls (QCs) guidelines that include calibrating equipment against control samples and performing tests of patient samples in tandem with consistent references, it is critical that reference samples be used in a manner that provides comprehensive evaluation of every component in these highly complex procedures and reagent mixtures. The need for these controls and/or standards became particularly acute with the widespread use of high complexity and high volume DNA- or RNA-based real time testing platforms.

    Bio-Synthesis provides molecular assay services, focused on the design and development of nucleic acid-based, positive control templates (PCT) to monitor the molecular diagnostic testing process, including the extraction, amplification, and detection components of test systems used to measure disease producing organisms. We provide thousands of PCTs to genotype high value polymorphisms for various drug metabolism and transporter genes. These PCTs can be manufactured in our cutting edge molecular diagnostic facilities and significantly shorten your path from RESEARCH to RESULT by providing you with the full development process for control templates that may be used as standard references in the simultaneous detection of mutations in any genome. These laboratory-safe, synthetic or semi-synthetic DNA/RNA Positive Controls can be a relatively cost effective, simple and efficient alternative to difficult-to-acquire controls from infectious samples.


    Our contract services are confidential, fast, efficient and well-documented, with objective to support the improvement of analysis and control of human infectious diseases by providing high quality evaluation materials to aid in the advancement of nucleic acid technologies.

    Advantages


    Select Platform

     DNA/RNA control templates, length >1000 bp

    Technology Friendly

     Suitable for Real-Time PCR, qPCR, microarray...

     Laboratory-Safe

     Non-infectious, laboratory-safe synthetic controls

     Accurate and Reliable

     Reproducible results - known input copy number

     Well-Documented

     Well-characterized sequence to assure maximum fidelity

     Customized Solutions

    Optimized preparation for specific applicationsSelect Platform: DNA/RNA control templates, length >1000 bp

     

    Biosynthesis Inc.



    BSI's On-demand HPV and HLA controls can be used as positive controls in nucleic acid amplification reactions.

    These quantitative controls can also be used to generate standard curves for qPCR assays.

     Biosynthesis Inc.


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    Crotamine is a cell-penetrating peptide that accumulates in the nucleus and can be used to transport DNA into replicating cells.

    In addition, it is thought that crotamine has the potential to transport drugs into mammalian cells without the need for specific receptors.

     

    Crotamine is a highly basic polypeptide of 42 amino acids that has an isoelectric point (pI) of 10.3 and a molecular mass of 4.8 kDa. Crotamine is found in the venom of the Brazilian rattlesnake Crotalus durissus terrificus. Crotamine was first isolated in 1947 from the venom of this snake. Crotamine selectively inhibits and interferes with the functioning of Kv1.3 channels and promotes the permeability of bacterial membranes. Furthermore, crotamine has been reported recently to possess both antitumoral and antibacterial activities.

    Amino Acid Sequence 


    ykqchkkgghcfpkekiclppssdfgkmdcrwrwkcckkgsg



    Molecular weight


    4890.01 g/mol (reduced), 4884.01 (oxidized).

    Amino Acid Composition

    C = 6 (14.29),  D = 2 ( 4.76),  E = 1 ( 2.38),  F = 2 ( 4.76), G = 5 (11.90),  H = 2 ( 4.76),  I = 1 ( 2.38),  K = 9 (21.43), L = 1 ( 2.38),  M = 1 ( 2.38),  P = 3 ( 7.14),  Q = 1 ( 2.38), R = 2 ( 4.76),  S = 3 ( 7.14),  W = 2 ( 4.76),  Y = 1 ( 2.38).


    Crotamine contains three disulfide bridges and several isoforms have been characterized. The overall peptide fold is homologous to antimicrobial peptides (AMPs) belonging to the α-defensin, β-defensin and insect defensin families. Crotamine has the same structural scaffold as mammalian α-defensins and β-defensins, a three-stranded β-sheet core and a framework of loops stabilized by six disulfide-linked cysteines.



    Figure 1: Molecular models of the cell-penetrating polypeptide Crotamine from the venom ofCrotalus durissus terrificus as reported by Nicastro et al in 2003.
     
    Nicastro G, Franzoni L, de Chiara C, Mancin AC, Giglio JR, Spisni A.;
     Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom.Eur J Biochem. 2003 May;270(9):1969-79.

    Abstract

    Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 micro g per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal alpha-helix and a small antiparallel triple-stranded beta-sheet arranged in an alphabeta1beta2beta3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an alpha/beta fold, though with a beta1alphabeta2beta3 topology. In addition, as the antibacterial beta-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human beta-defensins shows a similar fold and a comparable net positive potential surface. To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel. PMID: 12709056 [PubMed - indexed for MEDLINE]”

    Biosynthesis Inc.

    Figure 2: Molecular models of the cell-penetrating polypeptide Crotamine from the venom ofCrotalus durissus terrificus as reported by Coronado et al in 2013.



    Figure 3: Disulfide bond connectivity of crotamine.

    Coronado MA, Gabdulkhakov A, Georgieva D, Sankaran B, Murakami MT, Arni RK, Betzel C.; Structure of the polypeptide crotamine from the Brazilian rattlesnake Crotalus durissus terrificus.Acta Crystallogr D Biol Crystallogr. 2013 Oct;69(Pt 10):1958-64. doi: 10.1107/S0907444913018003. Epub 2013 Sep 20.

    “Abstract

    The crystal structure of the myotoxic, cell-penetrating, basic polypeptide crotamine isolated from the venom of Crotalus durissus terrificus has been determined by single-wavelength anomalous dispersion techniques and refined at 1.7Å resolution. The structure reveals distinct cationic and hydrophobic surface regions that are located on opposite sides of the molecule. This surface-charge distribution indicates its possible mode of interaction with negatively charged phospholipids and other molecular targets to account for its diverse pharmacological activities. Although the sequence identity between crotamine and human β-defensins is low, the three-dimensional structures of these functionally related peptides are similar. Since crotamine is a leading member of a large family of myotoxic peptides, its structure will provide a basis for the design of novel cell-penetrating molecules.”

     


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  • 01/16/15--00:00: ZINC FINGER PEPTIDES
  •  Zinc finger peptides

     

    Zinc fingers are polypeptides that are part of small protein domains in which the zinc complex structure contributes to the overall stability of the domain. However, zinc fingers are structurally diverse polypeptides and a variety of zinc finger peptide motifs or zinc finger structural motifs have now been identified and classified. Since the discovery of the first zinc finger polypeptides or proteins intensive research has revealed over the years that zinc fingers are involved in a broad range of cellular processes. For example, zinc fingers are a part of nuclear receptor proteins including receptors for steroid hormones, thyroid hormone, and retinoic acid, and many others. In summary zinc binding polypeptide motifs are ubiquitous structures found in a multitude of proteins.

     

    In a typical zinc finger, as present in natural transcription factors, a zinc ion is held by a pair of histidines and a pair of cysteines. This zinc ion amino acid complex stabilizes the packing of an anti-parallel β-sheet against an α-helix. The α-helix is also called the “recognition helix”. This helix interacts with a triplet of bases in the DNA indicating that the DNA recognition is a one to one interaction between the three amino acids in the protein helix and the nucleotide bases. Apparently zinc fingers can function as independent modules. In recent years the engineering of unique non-natural zinc fingers has led to the development of the zinc finger technology.

     

    A comprehensive classification of zinc finger spatial structures was published by Krishna et al. in 2003. According to this classification scheme each available zinc finger structure can be placed into one of eight fold groups that are based on the structural properties in the vicinity of the zinc-binding site. The majority of zinc fingers are found in the three fold groups, C2H2-like finger, treble clef finger and the zinc ribbon.




    A whole family of synthetic peptides that interact with zinc ions have been modeled and designed using zinc figure domains. This has led to the production of fluorescent peptide-based sensor molecules for divalent zinc. The design for the chemosensor relies on a synthetic peptide sequence template covalently attached to a fluorescence reporter which is sensitive to metal induced changes in the conformation of the polypeptide construct.

     

    Zinc is a metallic element with the atomic number 30 and a molecular weight of 65.37. Zinc is an important and essential element in many organisms including human. A number of zinc salts are used in medicine and many proteins use zinc as a cofactor.

     

    Zinc-finger Proteins

     

    The transcription factor IIIA (TFIIIA) was the first well-characterized eukaryotic positive-acting regulator protein. The cloning and sequencing of the gene encoding for this transcription factor, TFIIIA, led to the discovery of a recurring cysteine and/or histidine amino acid residue motif spaced at regular intervals along the amino acid sequence of this protein. The protein was found to have nine repeated domains that contain cysteines and histidines spaced at regular intervals. These amino acids can bind zinc ions. In addition, it was found that the purified native protein contained an amount of zinc equal to the cystine-histidine clusters present in the transcription factor. These findings led to the proposal that TFIIIA contains ‘zinc finger” folds that protrude from the surface of the protein and bind to DNA. In the proposed zinc-finger folded structure of TFIIIA the repeated domains form loops in such as way that zinc ions are bound between a pair of cysteines and a pair of histidines. Furthermore, a phenylalanine or tyrosine residue and a leucine residue occur at nearly constant positions in the peptide loops. Mutational studies showed that these amino acid residues are required for DNA binding. Many regulatory proteins have now been found to contain zinc fingers, and because of the rapid growth of structural information on proteins and their folding a multitude of structures are now available for study.  


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  • 01/22/15--00:00: Telomere repeat sequences
  • Telomere repeat sequences


    Telomeres are DNA structures at the ends of eukaryotic chromosomes that protect them from degradation and DNA repair activities. Initially, telomeres were defined functionally as the natural ends of eukaryotic chromosomes. Without telomeres, a chromosome is unstable. Telomeric DNA consists of simple sequences repeated in tandem. These sequence stretchers have been found to be a conserved feature in throughout eukaryotes. The terminal DNA stretch can range in total length from under fifty base pairs in some protozoans, through a few hundred base pairs in yeasts and several other eukaryotes, to thousands of base pairs in mammalian cells. These sequence repeats appear to maintain a stable telomere. For this reason, telomeres can be functionally defined as regions of DNA at the end of linear chromosomes. Apparently these sequence repeat regions are needed for replication and stability of chromosomes. All known eukaryotic telomeres contain simple repeated sequences of G- and C-rich complementary strands. These telomere repeat sequences have the general structure (T or A)m(G)n. In Tetrahymena and Oxytricha the G-rich DNA strand, oriented in a 5' - 3' direction toward the end of the chromosome, is synthesized by an RNA-dependent "telomerase" activity.


    Protein telomere with telomere repeat sequence complex in Sterkiella nova

    In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. The association of telomere protein subunits is DNA-dependent. The alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, Buczek and Horvath in 2006 characterized several possible stepwise assembly pathways using isothermal titration calorimetry.

    The enzyme telomerase is a reverse transcriptase that elongates telomeres in the cells it is expressed in. Telomerase was found to be active in germ cells and stem cell populations but not in adult tissue cells. In normal adult tissue cells or somatic cells the telomere activity levels are not sufficient to prevent telomere shortening associated with cell division. During the lifetime of an organism including humans the number of times a normal human cell population will divide until cell division stops appear to be finite. However, cancer cell lines can almost live on forever and are considered to be immortal.

    Chromosomes can be stained with fluorescently or otherwise labeled oligonucleotide probes directed against highly conserved telomere repeat sequences.

    In general, to stain chromosomes during in-situ hybridization experiments labeled oligonucleotide probes directed against highly conserved mammalian telomere repeat sequences and against major satellite repeats are used. Popular probes of this type contain the fluorogenic dyes Cy3 or FITC at the 5’ or 3’ end.  Artificial nucleotides, such as bridged nucleic acids or BNAs, can be used to enhance the specificity of the probes.

    For example molecular probes that target the (TTAGG)4 repeat are often used for the detection and staining of  highly conserved sequence repeats consisting of (TTAGGG)n and (CCCTTA)n sequences in human chromosomes. Human chromosomes contain stretches of up to 30,000 C and G bases repeating over and over. These sequence stretches often occur adjacent to gene-rich areas and form a barrier between the genes and the non-coding DNA. In general many FISH probes are targeted towards repetitive sequences found in the chromosomes. Scientists now believe that CpG islands are involved in regulating gene activities and more evidence has been accumulated recently to support the functional significance of satellite DNA sequences.

    Structure of probes used for the staining of chromosomes


    A typical non-radioactive probe consists of a fluorophore that is conjugated to the 5’- or 3’-end of the oligonucleotide used.


    Telomer Probe:  Fluorophore-5’-CCC TAA CCC TAA CCC TAA-3’


    Centromere Probe:  Fluorophore-5’-TCG CCA TAT TCC AGG TC-3’

    Target:  Mouse major satellite repeats.

    Structure of the human telomere in K+ solution

    An intramolecular (3 + 1) G-quadruplex scaffold.



    Ref.: Luu, K.N.,  Phan, A.T.,  Kuryavyi, V.V.,  Lacroix, L.,  Patel, D.J.; (2006) J.Am.Chem.Soc.128: 9963-9970.

    Abstract

    We present the intramolecular G-quadruplex structure of human telomeric DNA in physiologically relevant K(+) solution. This G-quadruplex, whose (3 + 1) topology differs from folds reported previously in Na(+) solution and in a K(+)-containing crystal, involves the following: one anti.syn.syn.syn and two syn.anti.anti.anti G-tetrads; one double-chain reversal and two edgewise loops; three G-tracts oriented in one direction and the fourth in the opposite direction. The topological characteristics of this (3 + 1) G-quadruplex scaffold should provide a unique platform for structure-based anticancer drug design targeted to human telomeric DNA.
     

    Telomeric repeat sequences in eukaryotes


    Organism

    Telomeric repeat

    (5' to 3' toward the end)

    Reference

    Vertebrates, human

    TTAGGG n repeats

    Meyne et al. 1989

    Giardia lamblia

    TAGGG

    Adam et al. 1991

    Human, mouse, Xenopus, Neurospora crassa, Trypanosoma brucci, Aspergillus nidulans

    TTAGGG

    Bhattacharyya and Blackburn 1997b

    Neurospora crassa

    Cryptococcus

    TTAGGG

    TTAGGGGG

     

    Edman 1992

    Physarum, Didymium,

    TTAGGG

     

    Dictyostelium

    AG(1-8)

     

    Trypanosoma, Crithidia

    TTAGGG

     

    Tetrahymena, Glaucoma

    TTGGGG

     

    Paramecium

    TTGGG(T/G)

     

    Oxytricha, Stylonychia, Euplotes

    TTTTGGGG

     

    Plasmodium

    TTAGGG(T/C)

    TT(T/C)AGGG

     

    Arabidopsis thaliana

    TTTAGGG

     

    Chlamydomonas

    TTTTAGGG

     

    Bombyx mori

    TTAGG

     

    Ascaris lumbricoides

    (C)TTAGG(C)

    Muller et al. 1991

    Schizosaccharomyces pombe

    TTAC(A)(C)G(1-8)

    TTAC(A)AG2-7

     

    Saccharomyces cerevisiae

    TGTGGGTGTGGTG

    (from RNA template)
    or G(2-3)(TG)(1-6)T

    (consensus)

    TG2-3(TG)1-3

     

    Saccharomyces castellii

    TCTGGGTG

     

    Candida glabrata

    GGGGTCTGGGTGCTG

     

    Candida albicans

    GGTGTACGGATGTCTAACTTCTT

     

    Candida tropicalis

    GGTGTA[C/A]GGATGTCACGATCATT

     

    Candida maltose

    GGTGTACGGATGCAGACTCGCTT

     

    Candida guillermondii

    GGTGTAC

     

    Candida pseudotropicalis

    GGTGTACGGATTTGATTAGTTATGT

     

    Kluyveromyces lactis

    GGTGTACGGATTTGATTAGGTATGT

    McEachern and Blackburn 1996

     

    Reference

    Adam R.D., Nash T.E., and Wellems T.E., 1991. Telomeric location of Giardia rDNA genes. Mol. Cell. Biol. 11: 3326-3330.

    Blackburn, E. H. & Szostak, J. W. (1984) Annu. Rev. Biochem. 53, 163-194.

    Buczek P, Horvath MP; Structural reorganization and the cooperative binding of single-stranded telomere DNA in sterkiella nova.J.Biol.Chem. (2006) 281 p.40124.



    The RNA World, Second edition. Gesteland, Chech, Atkins, CSHL Press.

     

    Links to data bases

    http://telomerase.asu.edu/

    http://labs.fhcrc.org/bedalov/telomeredatabase.html

    http://www.biophysica.com/telomere.html

    http://www.rcsb.org/pdb/results/results.do?outformat=&qrid=31238D90&tabtoshow=Current

     

     


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    PEGylation of Therapeutic Proteins


    Therapeutic proteins such as 
    monoclonal antibodies and cytokines, after delivery to the body, are eliminated by proteolysis and clearance in the kidney or liver, a process that limits their usefulness as therapeutic agents. In addition, many of these proteins can also trigger an antibody immune response that would block their beneficial effects. Hence, significant efforts have been dedicated developing methods that while extending these proteins’ circulating life and decreasing their immunogenicity do not interfere with their specific biological functions. Between the different methods explored,
    pegylation or the covalent addition of polyethylene glycol (PEG) chains to a protein, has been the most successful. Pegylation is used in several types of  therapeutic proteins. Apparently it results in the increase of the hydrodynamic volume of a protein. However, addition of PEG can also have beneficial effects on nucleic acids by increasing their stability against nucleases.

    PEGylation, or pegylation, refers to the covalent attachment of polyethylene glycol polymer chains to molecules such as proteins, peptides, oligonucleotides or other biomolecules.

    The first FDA-approved pegylated therapeutic protein appeared on the market in 1990, which was Adagen®, a PEGylated form of adenosine deaminase. Following that, seven successful pegylated therapeutic proteins have been approved by the FDA, including Oncaspar®(Pegaspargase), PEGIntron ® (PEGylated IFN-α2b), Pegasys® (PEGylated IFN-α2a), Neulasta® (pegfilgrastm), Somavert® (Pegvisomant), Mircera® (PEGylatedepoetin-β), and Cimzia®(Certolizumabpegol). In addition to these already approved PEGylated biopharmaceuticals, many new products are currently under investigation and in different stages of clinical trials. Still, when considering pegylation as a strategy to extend the circulating life of a protein, several factors need to be considered, which will be discussed here.

    • Location of the PEG chains:

    As a rule, PEG chains should be covalently linked at a site distant from the binding site of the protein, i.e. that region of the protein that is involved in binding to a cell receptor or an antigen; this way, the blocking of specific groups needed for binding and/or the steric hindrance of the binding process caused by the PEG are avoided. That means that at a minimum we should have amino acid sequence information about the protein as well as its binding site, post-translational modifications, such as glycosylation may be also helpful as they provide additional potential sites for the covalent binding of the PEG chains. Information about the tertiary structure of the protein would help in fine tuning the choice for a pegylation site. Indeed, a new approach is to modify the gene coding for a protein to add a new reactive group, like a cysteine or lysine, which is distal from the binding site, to allow the covalent binding of the PEG chain(s). 


    One strategy commonly used is to add the PEG chain at the N-terminal group of a protein, as usually this amino acid residue is not part of the binding site and it is located far from that site. Specificity for the terminal amino group is based on the differences in the pKa values for the lysine α and ε amino groups, and that the N-terminal group is always α. Another strategy used with glycosylated proteins is to oxidize the sugar moiety with periodate and to link the PEG to the aldehyde groups; because glycosylation sites are usually distant from the binding site, this approach minimizes any potential steric hindrance due to the added PEG.

    • Size and number of PEG chains:

    In general, the larger the PEG chain the lower number of chains needed per mole of protein; yet, the length of the chain would be limited by its effects on stability and interference with binding. An advantage of longer PEGs is that usually a single chain will do, while smaller PEGs would require more chains added, which may interfere with the binding of the proteins. The size of the PEG is usually found by experimentation, in the case of monoclonal antibodies and their Fab fragments, PEGs of 40 kDaltons have been used successfully. However, while increase of the PEG size may result in a longer life, the biological activity of the protein may decrease as a function of the PEG size. Thus, the reason for the need to asses experimentally each therapeutic protein performance after pegylation. A new development in pegylation of therapeutic proteins is the use of branched lower molecular weight PEGs; these PEGs having usually two or three chains linked to a single linker carrying also a reactive group for binding to a protein, apparently has lower side-effects while maintaining a good stability. Also a higher circulating life of a pegylated protein may result in the accumulation of breakdown products that can damage the kidney; hence, as indicated above, pegylation effects need to be evaluated case by case.

    • Chemistry of the linking process:

    A number of PEGs are available that have a single terminal reactive group capable of forming a covalent bond with some of the functional groups from proteins. For instance, PEGs having an aldehyde group can be used to preferentially add a PEG chain at the protein N-terminal group. This goal can be achieved by adjusting the pH of the reaction to have most of α amino groups deprotonated and thus able to react with the aldehyde. Other reactive groups are carboxyl, amine, maleimide, succimidyl and triazinyl, which are able to react with many of the functional groups available in proteins. To prevent damage to the protein, the reactions are carried under aqueous conditions, avoiding extreme pH conditions and using a concentration of activated PEG closer to the one needed to deliver the desired degree of pegylation. Once the pegylation of therapeutic proteins has taken place the different conjugates showing different degrees of substitution can be separated by ion exchange chromatography using a salt gradient to remove the excess of activated PEG and resolve the proteins with different degrees of pegylation. The pegylated therapeutic proteins are then concentrated, filter sterilized, and stored in PBS at 4oC. It may be possible to lyophilize the pegylated proteins in the presence of an excipient like mannitol or sorbitol.

    • Analysis for the Pegylation of therapeutic proteins:

    Several methods can be used to assess the quality of the final product, such as SDS-PAGE, hydrophopic interaction chromatography (HIC), size exclusion chromatography (SEC), and matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). 

    SDS-PAGE:


    This method delivers information about the different conjugates prepared during the process. However, the molecular weight information is usually not related to the actual molecular weight.


        

    Hydrophobic Interaction Chromatography:


    This method that allows resolving the different species according to their lipophilicity can be done using a butyl column, where the different conjugates are bound to the matrix using a high salt concentration and eluted in order of increasing lipophilicity by using a decreasing linear gradient of a salt, such as ammonium sulfate.



    Size Exclusion Chromatography:


    This method uses matrices like cross linked agarose gels, and the different conjugates are separated according to their hydrodynamic volume that is a result of the volume occupied by the protein plus the volume added by the PEG chain(s). This parameter is somewhat correlated with mol. wt. but it is not reliable to deliver the actual mol. wts.


    MALDI-Mass spectrometry:


    Under optimal conditions, this method delivers the actual molecular weight of the conjugate and it is quite useful to establish the actual molecular weight of the pegylated proteins thereby indicating the presence of the correct pegylated protein conjugate.

    Storage:


    Pegylated proteins are best stored under sterile conditions at neutral pH and at 4oC. The buffer can be the common phosphate buffered saline solution at pH 7.2.        

     

    Reference

    Ginn C, Khalili H, Lever R, Brocchini S.; PEGylation and its impact on the design of new protein-based medicines.Future Med Chem. 2014;6(16):1829-46. doi: 10.4155/fmc.14.125.

    Jevsevar S, Kunstelj M, Porekar VG.; PEGylation of therapeutic proteins. Biotechnol J. 2010 Jan;5(1):113-28. doi: 10.1002/biot.200900218.

    Veronese FM; Peptide and protein PEGylation: a review of problems and solutions. Biomaterials. 2001 Mar;22(5):405-17.

    Yang T (2013) PEGylation – Successful Approach for Therapeutic Protein Conjugation. Mod Chem appl 1:e112. doi: 10.4172/2329-6798.1000e112

     


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     What are Circular Oligonucleotides?


    Circular oligonucleotides are covalently closed singular DNA or RNA strands that are resistant to degradation by many cellular DNA and RNA decay machineries.


     


    Models of circular DNA and RNA


    [Figures were adapted from: Modelling DNA Phil. Trans. R. Soc. A-2006-Harris-3319-34; David Goodsell at http://www.rcsb.org/pdb/101/motm.do?momID=65: The self-splicing RNA in PDB entry 1u6b caught in the middle of its splicing reaction is illustrated].

    Circular DNA


    Modern biology focuses on the study of molecules within cells as well as the interaction between cells to understand and describe multi-cellular organisms most of which are visible to a human’s eye. Molecular cell biology concentrates on the study of macromolecules, such as carbohydrates, DNA, polypeptides, proteins, RNA, lipids as well as others, their biochemistry and processes that regulate metabolic pathways in these organisms.

    Mitochondria and chloroplasts found in today’s eukaryotes contain circular DNAs encoding proteins. These proteins are essential for the function of organelles and the ribosomal and transfer RNAs required for their translation. 

    Eukaryotic cells have multiple genetic systems: 

    (1) a nuclear system, and 

    (2) systems with their own DNA present in mitochondria and chloroplasts. 


    Human mitochondrial DNA, mtDNA, is a circular DNA molecule which sequence is now completely known. This human mtDNA is among the smallest known mtDNAs. The circular mtDNA molecule is a compact, double-stranded circular genome contains 16,569 base pairs that encode two ribosomal RNAs (rRNAs). These are present in mitochondrial ribosomes and 22 transfer RNAs (tRNAs) needed to translate mitochondrial messenger RNAs (mRNAs). In addition, human mtDNA contains 13 sequences that begin with the ATG (methionine) codon and end with a stop codon. These DNA sequence stretches are long enough to encode polypeptides of more than 50 amino acids. All of the possible proteins encoded by these open reading frames have now been identified. Mammalian mtDNA has no introns and contains no long noncoding sequences. A single cell can have several hundred to thousands of copies of the circular mitochondrial genome. 

    Since the 1970s scientists have known that, bacteriophages contain circular DNA. For example, Arnberg et al. in 1976 reported that a DNA-protein complex contains circular DNA in the bacillus bacteriophage GA-1. In addition, the researchers found that proteins are bound to DNA purified from bacillus bacteriophage GA-1. The removal of DNA-associated proteins by treatment with proteolytic enzymes resulted in 

    (i)    a loss of transfecting activity of GA-1 DNA on competent Bacillus subtilis cells and 

    (ii)    an increase (0.004 g/cm3) in the buoyant density of the DNA. 


    GA-1 DNA, when analyzed by electron microscopy appeared as 6.4-µm-long circular and linear molecules. The analysis of 170 DNA molecules in the untreated GA-1 DNA preparation found 30 (18%) to be circular oligonucleotides. However, no circular molecules among 500 molecules analyzed were observed in trypsin-treated DNA. The researchers concluded that proteins are responsible for the circularization of GA-1 DNA.

    Since the 1990’s researchers have set out to synthesize and study circular DNA constructs. For example, Prakash and Kool reported in 1992 the synthesis of circular DNA oligomers ranging in sizes from 24 to 46 nucleotides. In addition, the scientist reported that oligomers containing four, eight, twelve, and eighteen nucleotides can form strong complexes with these circular DNA. The melting temperatures (Tm) of these complexes were observed to be 17 to >33 °C higher than the ones for the corresponding Watson-Crick duplexes of the same length. In addition, melting temperature studies confirmed that the circular DNAs can bind to complementary sites within longer oligonucleotides. These data indicated that circular DNA molecules can act as hosts for molecular recognition and form a triple helix with single-stranded nucleic acids.   

    Chen and Ruffner in 1998 reported that an in vitro procedure called ligation-during-amplification (LDA) can be used for the selective amplification of closed circular DNA using sequence-specific primers. Their report showed that LDA is a useful technique for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and the production of circular DNA. Scientists now know that triple helix formation can be used for the recognition of single-stranded DNA or RNA. Oligonucleotides that were designed to form intermolecular parallel triple helixes at a polypyrimidine RNA sequence stretch were shown to arrest protein synthesis from that RNA template in a cell-free system. Other types of circular oligonucleotides were synthesized in recent years either using  chemical or biological ligation. These circular oligonucleotides can also form a triple helix with single-stranded nucleic acid displaying higher mismatch discrimination for its complement as compared to normal DNA duplexes. Detailed kinetic studies showed that the rates of triple helix formation by circular oligonucleotides were approximately 100 times faster than that for normal triple helix formation.


    Circular RNA


    Circular ribonucleic acids (RNAs) are part of a newly revealed previously unexplored hidden world.


    Over the last 20 years may forms of RNAs have been discovered, some of them were unexpected. What made these discoveries possible? Most RNA sequencing methods detect molecules that contain tails. The old sequencing methods did not allow detecting and analyzing circular RNA molecules. However, newer sequencing methods now are revealing the presence of these molecules in many organisms such as humans, mice and worms.Furthermore, recent research indicates that circRNA molecules may be more abundant than what we previously thought. For example, the RNA genome of the hepatitis D virus (HDV) is a single-stranded, negative sense, small circular RNA molecule containing approximately 1700 nucleotides. This circular, covalently closed RNA strand is rod-shaped because of extensive base pairing. The HDV genome is surrounded by the d antigen core encoded by HDV that is subsequently encased in an envelope embedded with Hepatitis B antigens (HBsAg) or virus envelope proteins. Circular transcripts were observed in rodents 20 years ago. For example, the mouse SRY gene consists of a single exon. This gene determines the sex in male rodents. During the development of the animal, the RNA exists as a linear transcript that is translated into protein. However, in the adult testes the RNA exists primarily as a circular product predominantly localized to the cytoplasm and apparently not translated. It has been found that inverted repeats in the genomic sequence flanking the SRY exon direct transcript circularization.   

    Recently a new class of circRNAs has been reported to be abundant in mammalian cells. These circular RNAs are an enigmatic class of RNA with unknown function. However, emerging data indicate that these RNAs appear to regulate microRNAs (miRNAs). In recent years high-throughput sequencing has identified a large number of distinct RNAs generated from non-protein-coding regions of the genome. These noncoding RNAs can vary in length and like protein-coding RNAs appear to be linear molecules containing 5’ and 3’ termini. 

    Wang and Kool in 1994 reported the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides. These RNA molecules were designed to bind single-stranded nucleic acids by pyrimidine.purine.pyrimidine-type (pyr.pur.pyr-type) triple helix formation. The circular RNAs containing 34 nucleotides were cyclized employing a template-directed nonenzymatic ligation method. One nucleotide at the ligation site was a 2'-deoxyribose. These nucleotides was selected to ensure isomeric 3'-5' purity during the ligation reaction. One circular molecule was complementary to the sequence 5'-A12, and the second was complementary to the sequence 5'-AAGAAAGAAAAG. The researchers performed thermal denaturing experiments to show that both circular RNA molecules bind to complementary single-stranded DNA or RNA substrates by triple helix formation. According to the reported results two domains in a pyrimidine-rich circular RNA molecule sandwich a central purine-rich substrate. In addition, the measured affinities of these circular RNAs with their purine complements were much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. The comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence showed that they were similar in binding to DNA, but very different in the binding behavior towards RNA. The relative order of thermodynamic stability for the four types of triplex studied were found to be DDD >> RRR > RDR >> DRD. The researchers argued that triplex-forming circular RNAs represent a novel and potentially useful strategy for high-affinity binding of RNA.

    Wilusz and Sharp in 2013 (Science 26 April 2013) report that circular RNAs have covalently linked ends and are found in pathogens such as viroids, circular satellite viruses, and hepatitis B. 

    Viroids are small, circular, single-stranded RNAs that cause several infectious plant diseases. The RNA genome of the viroids contains functional motifs that allowing them to spread in the plant by recruiting host proteins. Many of these circular RNAs are thought to replicate by a rolling-circle-based mechanism. 

    In addition, a few circular RNAs generated from eukaryotic genomes have also been identified. However, until recently their exact role in the cells was unclear. The scientists report in this paper that a new method that combines high-throughput sequencing data with a new computational algorithms has recently revealed thousands of circular RNAs in a wide range of species ranging from humans to archaea. In addition, it was observed that human fibroblasts alone have more than 25,000 circular RNAs. These circular RNAs are derived from approximately 15% of actively transcribed genes, apparently mostly from exonic sequences. Large numbers of these RNAs accumulate in the cytoplasm of cells and sometimes exceed the abundance of associated linear mRNA by a factor of 10.

    Fibroblasts are cells that synthesize the extracellular matrix and collagen as part of the structural framework of animal tissues. In addition, these cells play a critical role in wound healing and are the most common cells of connective tissue in animals.

    Circular RNAs are resistant to degradation by many cellular RNA decay machineries since these recognize the ends of linear RNAs to function. The identification of a subset of these circular RNAs in humans and mice indicates that the circularization signals are evolutionarily conserved. It is hypothesized that the splicing machinery is involved in their biogenesis. However, the exact mechanism, how this works, will need to be elucidated in the future. 

    Salzman et al. in 2012 report their deep-sequencing results of normal and malignant cells that showed that RNA transcripts from exons in many human genes were arranged in a non-canonical order. The analysis of these transcripts with statistical and biochemical assays provided strong evidence that different modes of RNA splicing resulted in circular RNAs. Their results suggest that many eukaryotic RNAs exist in a circular form that may be part of a general feature of the gene expression program. The researchers proposed models to explain how these circular RNAs are formed. One model attempts to explain the mechanism of exon scrambling and the others to explain circle splicing.

    Memczak et al. recently sequenced and computationally analyzed human, mouse and nematode RNA. The research group detected thousands of well-expressed, stable circRNAs. Their sequence data and analysis indicated that the circRNAs showed tissue-developmental-stage-specific expression and that these RNAs may have important regulatory functions. Furthermore, the researchers report that a human circRNA that is antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as) is densely bound by microRNA (miRNA) effector complexes and contains 63 conserved binding sites for the ancient miRNA miR-7. In summary, the researchers data provided evidence that circRNas form a large class of post-transcriptional regulators and that numerous circRNA form by head-to-tail splicing of exons. The researcher argue that their data support the notion that animal genomes express thousands of circRNAs from diverse genomic locations from coding and non-coding exons, intergenic regions or transcripts antisense to 5’ and 3’ untranslated regions (UTRs). In addition to binding miRNAs circRNAs could function to store, sort, or localize ribonucleotide biding proteins (RBPs).

    Eric T. Kool in 1996, 17 year ago, reported in the journal “Annual Reviews in Biophysics and Biomolecular Structure” the synthesis of both circular DNA and RNA oligonucleotides as useful biological tools and potential therapeutics. The paper discusses synthetic methods to prepare circular oligonucleotides as well as the effect RNA versus DNA on the backbone of the formed helices and the stability and half-live of these molecules in human serum. Furthermore, the paper reported that small circular oligonucleotides can encode information by serving as a template for polymerase enzymes and that rolling circle synthesis can be used to form long DNA and RNA multimer strands using these circular molecules as catalytic templates.

    Seidl and Ryan in 2011 published a paper in which they showed how to use circular single-stranded DNA templates as synthetic DNA delivery Vectors for the production of miRNAs in human cells that express the proper RNApolymerases.


    Proposed functions of circRNAs


     Circular RNA appears to have the following functions:

    • Bind miRNAs
    • Store miRNAs as well as ribonucleotide biding proteins (RBPs)
    • Sort miRNAs as well as RBPs 
    • Localize miRNA as well as RBPs

     

    Whatever their function, more research in the future will hopefully more clearly define how these RNAs function. However, in recent years it has become increasingly clear that the cell can use a myriad of ways to process and stabilize RNA molecules.


    Caged circular antisense oligonucleotides can be used to modulate RNA digestion and gene expression in cells


    Wu et al. in 2012 report the chemical synthesis of 20mer caged circular antisense oligonucleotides. The researchers used the circular oligonucleotides to test their usefulness to perform photomodulated gene expression using ribonuclease H and non-enzyme antisense strategies. A photomodulated expression of green fluorescent protein (GFP) in HeLa cells to test the efficacy of the approach was used for this. Their results showed that the three synthetic caged circular antisense oligonucleotides containing 2’OMe modified RNA and phosphorothioate modifications were capable of photoregulating GFP expression in cells. 



    The oligonucleotides were synthesized using two 10mer oligonucleotides. A photocleavable linker and an amide bond linker were inserted between the two nucleotides to form the circular construct. The 3’-end of the linear oligonucleotide was modified with an amine group and the 5’-end contained a carboxylic acid group. After cyclization by forming an amide bond the caged circular oligonucleotide can be photo-cleaved in the middle of the previous linear oligonucleotide. The key feature is the resulting linear oligonucleotide strand that can hybridize with the complementary target RNA with the flexible linker in the middle.

    The scientists argue that this strategy can overcome the problems observed for cleaved caged moieties or inhibitor strands used in earlier studies. One interesting finding reported was that the size of the ring of the caged antisense oligonucleotide is the most important factor to enable photomodulation of target RNA digestion by ribonuclease H. Furthermore, the research group proposed that this design may help to minimize the binding of caged circular oligonucleotides with target sequences due to the non-base linkers present in the circular oligonucleotides. However, upon light activation, the binding ability can be restored. 

    The same research group used caged circular morpholino oligomers to photo modulate β–catenin-2 and no tail expression in zebrafish embryos.

    Morpholino antisense oligonucleotides have been used in the past, however, it has been noted that their use can generate misleading results. Eisen and Smith in 2008 discussed in their review published in the same year year how the use of morpholinos can lead to misleading results, including off-target effects.

    The main observed difficulties encountered in interpreting experiments using morpholino oligonucleotides are as follows: 

    • The effectively of the knock-down is hard to determine;
    • The possibility of “off-target” effects has to be addressed as well;
    • it can be difficult to inject precise and reproducible volumes of morpholino oligonucleotides.   

    It is expected that the use of caged circular antisense oligonucleotides will help to block protein translation through a non-enzymatic antisense strategy. The researchers further suggested that the circular oligonucleotides could be made more functional by using other artificial nucleotides, such as bridged nucleic acids (BNAs).

    Reference


    BSI Blog: http://blog-biosyn.com/2013/09/11/what-are-circular-oligonucleotides/ 

    Judith S. Eisen and James C. Smith; Controlling morpholino experiments: don't stop making antisense. May 15, 2008 Development 135, 1735-1743. Review.


    Thomas B. Hansen, Trine I. Jensen, Bettina H. Clausen, Jesper B. Bramsen, Bente Finsen, Christian K. Damgaard & Jørgen Kjems; Natural RNA circles function as efficient microRNA sponges. Nature 495, 384–388 (21 March 2013) doi:10.1038/nature11993.

    Kool ET.; Circular oligonucleotides: new concepts in oligonucleotide design. Annu Rev Biophys Biomol Struct. 1996;25:1-28.


    Sebastian Memczak, Marvin Jens, Antigoni Elefsinioti, Francesca Torti, Janna Krueger, Agnieszka Rybak, Luisa Maier, Sebastian D. Mackowiak, Lea H. Gregersen, Mathias Munschauer, Alexander Loewer, Ulrike Ziebold, Markus Landthaler, Christine Kocks, Ferdinand le Noble & Nikolaus Rajewsky; Circular RNAs are a large class of animal RNAs with regulatory potency.
    Nature 495, 333–338 (21 March 2013).


    Enrique Pedroso, Nuria Escaja, Miriam Frieden, Anna Grandas; Solid-Phase Synthesis of Circular Oligonucleotides.
    Methods in Molecular Biology Volume 288, 2005, pp 101-125.


    Martin Tabler and Mina Tsagris. Viroids: petite RNA pathogens with distinguished talents. TRENDS in Plant Science Vol.9 No.7 July 2004.


    Seidl CI, Ryan K; Department of Chemistry, City College of New York, New York, New York, United States of America.
    PloS onedoi:10.1371/journal.pone.0016925.g001.


    Yuan Wang, Li Wu, Peng Wang, Cong Lv, Zhenjun Yang and Xinjing Tang; Manipulation of gene expression in zebrafish using caged circular morpholino oligomers. Nucleic Acids Research, 2012, Vol. 40, No. 21 11155–11162 
    doi:10.1093/nar/gks840.  http://dev.biologists.org/content/135/10/1735.full


    Wu L, Wang Y, Wu J, Lv C, Wang J, Tang X.; Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells. Nucleic Acids Res. 2013 Jan 7;41(1):677-86. doi: 10.1093/nar/gks996. Epub 2012 Oct 26.

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