Are you the publisher? Claim or contact us about this channel


Embed this content in your HTML

Search

Report adult content:

click to rate:

Account: (login)

More Channels


Showcase


Channel Catalog


Channel Description:

Latest Articles of Bio-Synthesis Inc.

older | 1 | .... | 3 | 4 | (Page 5) | 6 | 7 | .... | 16 | newer

    0 0

    Klaus D. Linse

    A review of the scientific biomedical literature reveals that many peptides have anti-cancer activities or properties. The following table contains a list of peptides that are reported to have anti-cancer activities against solid and hematological tumors. Their oncolytic properties, or their ability to destroy tumor cells, are shown here as well. [Gaspar et al. 2013].

    Peptides with Anti-Cancer Properties
    Peptide
    Amino acid sequence
    Anticancer Activity
    Cancer Cell
    D-peptide A
    D-peptide B
    D-peptide C
    D-peptide D
    RLYLRIGRR
    RLRLRIGRR
    ALYLAIRRR
    RLLLRIGRR
    Cell membrane disruption
    Human cervix, glioma, lung, mouse myeloma, African green monkey kidney
    D-K6L9
    LKLLKKLLKK LLKLL
    Necrosis via membrane depolarization
    Human prostate
    NRC-03
    GRRKRKWLRR IGKGVKIIGG AALDHL
    Cell membrane lysis
    Human breast
    NRC-07
    RWGKWFKKAT HVGKHVGKAA LTAYL
    Cell membrane lysis
    Human breast
    Gomesin
    ZCRRLCYKQR CVTYCRGR
    Unclear
    Murine melanoma, human breast, colon and cervix adenocarcinoma
    Hepcidin TH2-3
    QSHLSLCRWC CNCCRSNKGC
    Cell membrane lysis
    Human cervix, hepatocellular carcinoma, fibrosarcoma
    Dermaseptin B2
    GLWSKIKEVG KEAAKAAAK AAGKAALGAV SEAV
    Necrosis
    Human prostate and breast
    PTP7
    FLGALFKALS KLL
    Apoptosis induction
    Human lung, breast and hepatocellular carcinoma
    MGA2
    GIGKFLHSAK KFGKAFVGEI MNSGGKKWKM RRNQF–WVKVQRG
    Necrosis and apoptosis
    Cervix and lung, melanoma, rat glioma
    HNP-1
    ACYCRIPACI AGERRYGTCI YQGRLWAFCC
    Mediation of antitumor immunity
    Mouse colon and brest
    Tachyplesin
    KWCFRVCYRG ICYRRCR
    Activation of the classic complement pathway
    Human prostate
    Temporin-1CEa
    FVDLKKIANI INSIF
    Membrane disruption, calcium release, ROS production
    Human breast
    NK-2
    KILRGVCKKI MRTFLRRISK DILTGKK
    Necrotic death after peptide intercalation into phospholipid phosphatidylserine -containing membranes.
    Human chronic myelogenous leukemia, histiocytic lymphoma, acute T cell leukemia, acute lymphoblastic leukemia, neuroblastoma, colorectal adenocarcinoma
    Bovine lactoferricin B6 (LbcinB6)
    RRWQWR
    Apoptosis, disruption of mitochondrial membrane
    Human acute lymphoblastic T leukemia, acute T cell leukemia
    Cecropin CB1
    KWKVFKKIEK MGRNIRNGIV KAGPKWKVFK KIEK
    Unclear
    Human acute lymphoblastic T leukemia, lung carcinoma, stomach carcinoma
     
     
    Reference
     
    Diana Gaspar, A. Salomé Veiga and Miguel A. R. B. Castanho; From antimicrobial to anticancer peptides. A review. Front. Microbiol., 01 October 2013 | doi: 10.3389/fmicb.2013.00294.

    0 0

    Luteinizing hormone-releasing hormone (LH-RH or LHRH) agonists and gonadotropin-releasing hormone (GnRH) agonists are peptides or drugs that are used for hormone therapy. These drugs lower the production of testosterone in a human's body. The drop in testosterone levels usually slows or stops the growth of prostate cancer for some time. These agonist drugs cause the pituitary gland to release the hormones that tell the testicles and adrenal glands to produce testosterone. These drugs are usually given by injection or implanted under the skin or may be given once a month, once every 3 to 4 months, or once a year.
     
     
    Peptide
    Sequence
    Indications
    Agonists
     
     
    Buserelin
     
    Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt
    Prostate cancer
    Gonadorelin
     
    Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
    Cystic ovarian disease, agent for evaluating
    hypothalamic-pituitary gonadotropic function
     
    Goserelin
    Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2
    Prostate cancer; breast cancer
    Histrelin
     
    Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu-Arg-Pro-NHEt
    Prostate cancer; breast cancer
    Leuprolide
     
    Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt
    Prostate cancer; breast cancer
    Nafarelin
     
    Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2
    Treat symptoms of endometriosis, central precocious
    puberty
    Triptorelin
     
    Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2
    Prostate cancer; breast cancer
    Antagonists
     
     
    Abarelix
    Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-NH2
    Prostate cancer
     
    Cetrorelix
    Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2
    Prostate cancer; breast cancer
     
    Degarelix
     
    Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)- D-4-aminoPhe(carbamoyl)-Leu-isopropylLys-Pro-D-Ala-NH2
    Prostate cancer
     
    Ganirelix
     
    Ac-D-2Nal-D-4-chloroPhe-D-3-(3’-pyridyl)
    Ala-Ser-Tyr-D-(N9, N10-diethyl)-homoArg-Leu-(N9,
    N10-diethyl)-homoArg-Pro-D-Ala-NH2
    Fertility treatment
     
     

     
    Reference
     
    Thundimadathil J.; Cancer treatment using peptides: current therapies and future prospects. J Amino Acids. 2012;2012:967347. doi: 10.1155/2012/967347. Epub 2012 Dec 20.
     

    0 0

     Peptide receptors are important for cancer therapies
     
     
    By Klaus D. Linse
     
    Our understanding of estrogen signaling has increased significantly in recent decades. Research findings indicate that estrogen signaling is a balance between two opposing forces. The specific estrogen receptors (ERα and ERβ) and their splice variants were identified as the two major players in this receptor force-field. These receptors act as transcription factors and it was found that the two pathways involved can be selectively stimulated or inhibited using selective drugs. This opened up new promising therapeutic opportunities in clinical research areas as diverse as hormone replacement, autoimmune diseases, prostate and breast cancer, and depression. This invaluable information from molecular, biological, biochemical, and structural studies allows for the development of more selective and effective estrogen receptor (ER) ligands needed for the development of more effective drugs to modulate and regulate the action of these receptors. For example, anti-estrogens and aromatase inhibitors are now routinely used clinically to arrest the estrogen-dependent growth of breast cancer.
     
    In addition, it is now also well known that ERs are over-expressed in approximately 70% of breast cancer cases. These types of cancers are referred to as "ER-positive" and a technique called immune-histochemistry can be used to reveal the over-expression of the receptors in tissue cells. Estrogen receptor proteins are found inside cells and are activated by the hormone estrogen or 17β-estradiol. In women the three major naturally occurring estrogens are estrone (E1), estradiol (E2), and estriol (E3). Estradiol is the predominant estrogen during the reproductive years. However, two classes of estrogen receptor have been found to exist; The first is ER which is a member of the nuclear hormone family of intracellular receptors. The other is GPR30, a member of the rhodopsin-like family of G protein-coupled receptors. It is now known that estrogens play key roles in the development and maintenance of normal sexual and reproductive functions.
     
    Selective estrogen receptor modulators (SERMs) that act on the estrogen receptor, in addition to pure receptor agonists and antagonists are also investigated for their ability to selectively inhibit or stimulate estrogen-like action in various tissues. Also, enzyme inhibitors such as aromatase inhibitors which represent a newer drug type, are sometime, also used to treat breast cancer or to help keep breast cancer from reemerging after surgery.
     
    The goal of these cancer drugs is to lower estrogen levels in the body so as to inhibit the growth of estrogen receptor-positive breast cancers.  Another type of inhibitors called aromatase inhibitors, block estradiol synthesis and/or anti-estrogens that compete with hormone binding to the receptors. These drugs are now routinely prescribed. Unfortunately, the emergence of tumor resistance creates a need to develop new drug types. One promising approach is to design and synthesize peptides that can specifically inhibit intra- or inter-molecular interactions between proteins or peptide receptors and their target ligands or interfaces. Protein-protein interaction studies have allowed the identification of functional protein or peptide sequence motifs that are potentially suitable for the design of such antagonists. In addition, peptide and non-peptide mimics of these motifs are good candidates for this design or synthesis approach.
     
    The following table shows a list of peptide receptors that are potential targets for new types of cancer therapies using peptide mimics.

     
    Peptide receptors which have potential in cancer therapy
     
    Peptide receptors
    Receptor subtypes
    Expressing tumor type
    Targeting agents
     
    Somatostatin
    sst1, sst2, sst3, sst4, and sst5
     
    GH-producing pituitary adenoma, paraganglioma, nonfunctioning pituitary
    adenoma,  heochromocytomas
    Radioisotopes, AN-201 (a potent cytotoxic radical 2-pyrrolino-doxorubicin), doxorubicin
    Pituitary adenylate cyclase activating peptide (PACAP)
    PAC1
     
    Pheochromocytomas
    and paragangliomas
     
    Radioisotopes, doxorubicin
     
    Vasoactive intestinal
    peptide (VIP/PACAP)
    VPAC1, VPAC2
     
    Cancers of lung stomach, colon, rectum, breast, prostate, pancreatic ducts,
    liver, and urinary bladder
    Radioisotopes,
    camptothecin
     
    Cholecystokinin (CCK)
     
    CCK1 (formerly CCK-A)
    and CCK2
     
    Small cell lung cancers, medullary thyroid carcinomas, astrocytomas, and ovarian cancers
    Radioisotopes, cisplatin
     
    Bombesin/gastrin-releasing
    peptide (GRP)
    BB1, GRP receptor subtype (BB2), the BB3 and BB4
    Renal cell, breast, and
    prostate carcinomas
     
    Doxorubicin,
    2-pyrrolinodoxorubicin
     
    Neurotensin
    NTR1, NTR2, NTR3
    Small cell lung cancer, neuroblastoma,
    pancreatic and colonic cancer
    Radioisotopes
     
    Substance P
    NK1 receptor
    Glial tumors
    Radioisotopes
    Neuropeptide Y
    Y1–Y6
    Breast carcinomas
    Radioisotopes
     
     
    Reference
     
    Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague M, Ström A, Treuter E, Warner M, Gustafsson JA.; Estrogen receptors: how do they signal and what are their targets. Physiol Rev. 2007 Jul;87(3):905-31
     
    Leclercq G, Gallo D, Cossy J, Laïos I, Larsimont D, Laurent G, Jacquot Y.; Peptides targeting estrogen receptor alpha-potential applications for breast cancer treatment.Curr Pharm Des. 2011;17(25):2632-53.
     
    Renoir JM, Marsaud V, Lazennec G.; Estrogen receptor signaling as a target for novel breast cancer therapeutics. Biochem Pharmacol. 2013 Feb 15;85(4):449-65. doi: 10.1016/j.bcp.2012.10.018. Epub 2012 Oct 24.
     
    Thundimadathil J.; Cancer treatment using peptides: current therapies and future prospects. J Amino Acids. 2012;2012:967347. doi: 10.1155/2012/967347. Epub 2012 Dec 20.
     
     
     
     

    0 0

    Molecular probes and their applications in real-time quantitative PCR (qPCR)

    By Andrei Laikhter
     
    The past decades have seen a remarkable growth in the use of modified oligonuleotides as tools for the study of biological phenomena such as gene regulation, siRNA as well as for drug discovery. In particular, modified oligonucleotides have been used in a variety of applications such as antisense gene regulation and as reporter molecules in hybridization-based assays. A large arsenal of modified synthetic oligonucleotides conjugated with reporter molecules have also been employed in hybridization-based assays. The use of these methods proved to have broad applications in basic molecular biology, biochemistry research, in clinical diagnostics, and in physiology and molecular medicine. The reason for all this is the characteristic high specificity to a target nucleic acid sequence of these modified oligonucleotides. The more recently developed process of fluorescence resonance energy transfer, or FRET, has improved the performance and utility of hybridization-based assays in molecular biology and medical diagnostics. Fluorescence typically occurs from aromatic molecules, and fluorescence spectral data are generally presented as emission spectra. Furthermore, the conjugation of fluorescent substances to non-fluorescenting molecules such as oligonucleotides allows for high sensitivity detection of these molecules in vivo and in vitro. A fluorescent substance, called fluorophore, has a fluorescence lifetime and a quantum yield, which is the number of emitted photons relative to the number of absorbed photons. The characteristics of fluorophores have been outlined in the Jablonski diagram. In addition, the intensity of fluorescence can be decreased by a wide variety of processes. This decrease of fluorescence intensities is called quenching which can occur by different mechanisms. Another process of importance that occurs in the excited state is called resonance energy transfer, or RET. Whenever the emission spectrum of a fluorophore, called the donor, overlaps with the absorption spectrum of another molecule, called the acceptor, this process will occur. However, the acceptor does not need to be fluorescent. The donor and acceptor are coupled by a dipole-dipole interaction. The terms RET and FRET are commonly used to describe this process. FRET can be used to measure the distances between a donor and acceptor and Foerster distances are typically in the range of 15 to 60 Å. This distance is comparable to the diameter of many proteins and the thickness of membranes. However, the use of this methodology requires a fluorescent reporter/donor, and a chromophore or flurophore acceptor conjugated to the target molecule that may be used as a probe. The conjugate can be either another fluorophore or a quencher molecule. When one acceptor molecule is a quencher, and two chromophores are in close proximity, the energy transfer will result in relatively low levels of heat. When the distance between chromophores is considerably higher than the Forster distance (Ro), the fluorescence from a fluorophore reporter molecule will result in a much higher intensity. Therefore, assays based on FRET are typically designed in such a way that the fluorophore and quencher molecules are in close proximity. When used in the assay the chromophores become separated and fluorescence of the fluorophore can be easily registered.

    FRET Probes

    Figure 1.  Mode of action of FRET probes during the qPCR process



    Real-time PCR is one of the most powerful hybridization-based diagnostic assay in use today (1,2). Real-time PCR allows a researcher to detect and measure the progress of PCR as reactions occur when using spectrofluorometric thermo-cyclers. This assay typically uses DNA probes, which fluoresce during the DNA amplification progresses.  During the PCR process the 5’-nuclease activity of the DNA polymerase causes cleavage of the fluorogenic probe, effectively separating the reporter molecule from the quencher dye, resulting in an increase in reporter-associated fluorescence. With this system, the monitoring of the increase in fluorescence allows quantitation of the amount of PCR product amplified in a simple manner that does not require gel electrophoresis. 

    qPCR Amplification Plot

    qPCR Amplification Plot

    Figure 2.  An amplification plot showing typical qPCR fluorescent profiles at various template concentrations is illustrated here. This type of detection methods is based on changes in fluorescence proportional to the increase of the target. The monitoring of the increasing fluorescence during each PCR cycle allows the user to follow the reaction in real time.   
     
     

    Molecular beacons are another class of DNA probes that have been successfully used for real-time PCR (3). This approach is based on fluorescence resonance energy transfer (FRET).  Oligonucleotides designed in this way usually carry a fluorescein derivative (FAM) at the 5’ end as a common fluorescent donor and fluorescein or rhodamine derivatives (FAM, JOE, TAMARA, ROX, Cy3, Cy5, and others) attached to a modified thymidine within the primer sequence as a fluorescence quencher.  Oligonucleotides labeled in this way make them valuable in areas where high sensitivity and spectroscopic discrimination of multiple fluorescent labels are important. Examples are automated DNA sequencing from very low amounts of template (reducing the need for cycle sequencing), DNA sequencing by hybridization in conjunction with microchip technology, in situ  hybridization, and short tandem repeat detection, to name a few.  These probes have a hairpin DNA structure where the fluorophore and the quencher is in close proximity to each other. When the probe hybridizes to its target complementary DNA template, both chromophores are forced away from each other and are thus generating a fluorescent signal.

    qPCR Amplification Plot

    Figure 3.  Mode of action of molecular beacon probes during the hybridization process


     

    A similar class of fluorogenic hairpin probes, commonly referred as “ampliflours” and “scorpions”, are also utilized as a primer in PCR reactions (4).

    Ampliflour Probes

    Figure 4.  Mode of action for “ampliflour” type probes during the qPCR process




    Scorpion Probes

     
    Figure 5.  Mode of action for “scorpion” type probes or “scorpion probes” during the qPCR process
     


    Yet another unique type of real-time PCR probes that was developed in collaboration with Steve Benner lab at the University of Florida is Plexor that utilizes non-natural iso-dG and iso-dC base-pairing (5,6). During the reverse polymerase reaction the dabcyl labeled iso-dGTP will be incorporated in close proximity to the fluorescently labeled iso-dC nucleo-base and subsequently will quench the fluorescence. The signal will be detected by diminishing corresponding fluorescence resulting from the incorporated forward primer. 

    Plexor Probes


    Figure 6.  Mode of action of “Plexor” type probes in the qPCR process



    Several new, efficient azo-quenchers for use in DNA based probes have been reported recently. However, each of those dyes has a narrow and limited range of quenching that is predetermined by their narrow absorbance spectra. Therefore, each of those quencher dyes requires a fluorophore within a certain range of the fluorescence emission spectrum in order to have an efficient energy transfer between the two dyes.  The broad absorbance spectra of our new generation of the quencher dyes such as Instant Quencher dyes (IQ4) (7) makes these probes suitable for multiplexing. The use of novel quencher dyes significantly improved sensitivity and the Ct value number of the corresponding linear probes.  The Ct or threshold cycle number is found at the intersection between the amplification curve and a threshold line which is a relative measure of the concentration of target in the PCR reaction.

    Decamer UV Spectra


    Figure 7.  UV Spectra of standard mono-labeled decamers labeled with the leading quencher dyes
     


    References

    1.  Landgraf, A.; Reckmann, B.; Pingoud, A., Anal. Biochem., 1991, 193, 231.

    2.  Lee, L. G.; Connell, C. R. and Bloch, W. Nucleic Acids Res., 1993, 21,  3761.

    3.  Tyagi, S.; Kramer, F. R., Nature Biotechnology, 1996, 14, 303.

    4.  Nazarenko I. A.; Bhatnagar S. K. and Hohman R. J. Nucleic Acids Res., 1997, 25,  2516.

    5.  Sherrill, C.B. et al. J. Am. Chem. Soc. 2004, 126, 4550–6.

    6.  Frackman, S. et al. Plexor® technology: A new chemistry for real-time PCR Promega Notes 2005, 90, 2–4.

    7.  Laikhter A. et al. US patent 7,956,169

      

    0 0

    Primary alkyamino modified oligonucleotides are commonly used in oligonucleotide labeling and conjugations towards such electrophilic moieties as active esters, halogenated aliphatic and aromatic derivatives.1 In general, labeling oligonucleotides with different reporter molecules requires post synthetic solution phase process, where both amino labeled oligonucleotide and corresponding reporter NHS ester dissolved in aqueous organic solvent system. It is important that both reacting synthons are completely soluble in the reaction mixture. Second important requirement for the successful process that the reaction mixture is basic enough to drive coupling reaction to the completion and active ester is stable at least for duration of the reaction.

    3’-Amino modified oligonucleotides demonstrated resistance to 3’-exonuclease activity2 and also used as efficient polymerase chain terminator or 3’-blocking group.

    Bio-synthesis offers not only wide varieties of 3’-, 5’- and internally amino modified oligonucleotides and also their conjugates with peptides, proteins and antibodies.1

    5' Amino Modifications
    • 5' C3 Amino Linker
    • 5' C6 Amino Linker
    • 5' C12 Amino Linker
    • 5' Amino Modifier 5
    • 5' Amino TEG (triethylene glycol 12 atoms)
    • 5' PC Amino Linker
    3' Amino Modifications
    • 3' C3 Amino Linker
    • 3' C6 Amino Linker
    • 3' Amino C6 dC
    • 3' Amino C6 dT
    • 3' C7 Amino Linker
    Internal Amino Modifications
    • 2'-Deoxyadenosine-8-C6 Amino Linker (Amino C6 dA)
    • 2'-Deoxycytidine-5-C6 Amino Linker (Amino C6 dC)
    • 2'-Deoxyguanosine-8-C6 Amino Linker (Amino C6 dG)
    • C7 Internal Amino Linker
    • Thymidine-5-C2 Amino Linker (Amino C2 dT)
    • Thymidine-5-C6 Amino Linker (Amino C6 dT)
     

     Reference:
     

    1. http://www.biosyn.com/Bioconjugation.aspx
    2. Zendegui, J. G.; Vasquez, K. M.; Tinsley, J. H.; Kessler, D. J.; Hogan, M. E., Nucleic Acids Res., 1992, 20, 307  

    0 0

    Biosynthesis offers several different Oligonucleotide biotinylation synthesis options. Avidin, streptavidin and other biotin-binding proteins have the ability to form an intense association with biotin-containing molecules. This association has been used for many years to develop systems designed to capture biotinylated biomolecules. A variety of applications are in routine use to exploit the extraordinary affinity of these biotin-binding proteins for biotinylated molecules. However, the biggest bottleneck of biotin-binding proteins for biotin is that the association is essentially irreversible. Extremely low pH or highly concentrated chaotropic reagents are required to break teh association and these conditions are not entirely compatible with oligonucleotides. Biotin analogue such as desthiobiotin exhibits lower binding to biotin-binding porteins such as streptavidin. This biotin analogue is lacking the sulfur group from the molecule and has a dissociation constnat (Kd) several orders of magnitude less than biotin/streptaviding. As a result, biomolecules containing desthiobiotin are dissociated from streptavidin simply in the presence of buffered solutions of biotin.

    Biotinylated Oligonucleotide has been used in numerous applications, primarily, centering around the use of the biotin moiety as means to effect the avidin-mediated separation of a biotinylated oligonucleotide which has undergone hybridization with a target sequence. Biotin is also used as a way to attach avidin-conjugated enzymes used in chemiluminescent and colormetric1 detection protocols. Biotinylated oligonucleotides have been used in solid-phase differential display protocols as well as solid phase genomic and plasmid sequencing protocols such as solid phase capture by Streptavidin coated magnetic beads for use in restriction mapping,2 PCR based genomic walking,3 differential display detection of unique mRNA species,4 and DNA sequencing.5 5’- And 3’-biotinilated oligonucleotides as well as multi biotinilated oligonucleotides can be used for those applications.

    Oligonucleotide biotin modification includes:

    • 5' Biotin
    • Biotin dT
    • Photocleavable PC Biotin
    • Desthiobiotin
    • 3' Biotin
    Biotin dT can replace dT resiudes within the oligonucleotide sequence. 5' Biotin can be addedd only once to the 5'-terminus of an oligonucleotide, PC Biotin is a photocleavable 5'-biotin. Biotin TEG contain a triethyleneglycol long chain spacer.

    Contact us for Biotinlyated Oligonucleotide Synthesis Services.

    References:
    • Misra, R. R.; Chiang, S. Y.; Swenberg, J. A. Carcinogenesis, 1994, 15, 1647.
    • Conrad, F.; Krupp, G., Nucleic Acids Res., 1992, 20, 6423.
    • Rosenthal, A.; Jones, D. S. C., Nucleic Acids Res., 1990, 18, 3095.
    • Rostok, O.; Odeberg, J.; Rode, M.; Stokke, T.; Fundruck, S.; Smeland, E.; Lunderberg, J., Biotechniques, 1996, 21, 114.
    • Huitman, T.; Stahl, S.; Hornes, E.; Uhlen, M., Nucleic Acids Res., 1989, 17, 4937.

    0 0

    Real time detection of influence virus strain H7N9

    The development of the polymer chain reaction (PCR), for which Kary B. Mullis received a Nobel Prize in 1993 together with Michael Smith, has completely revolutionized the detection of RNA and DNA. PCR based methods now allow the detection of the amplified product at the end-point of the reaction or while the reaction is occurring. The real-time polymerase reaction (RT-PCR), sometimes also called the quantitative chain reaction (qPCR), is a technique based on the polymerase chain reaction (PCR), which can be used to amplify and simultaneously quantify specific DNA molecules.

    PCR enables both, detection and quantification of one or more specific DNA sequences in a sample. Detected quantities are reported either as absolute copy numbers or as relative amounts when normalized to DNA input or additional normalizing genes. Real-Time chemistries allow the detection of the PCR amplification products during the early phases of the reaction. The measurement of the reaction kinetics in the early phases of the PCR reaction provides a distinct advantage over traditional PCR detection methods. Usually, traditional methods use agarose gels for detection of PCR amplification products at the final phase or end-point of the PCR reaction. RT-qPCR or RT-PCR can be used to detect viral DNA or RNA.

    Classically respiratory viruses have been identified using viral cultures in a variety of permissive cell lines. However, viral cultures are hard to maintain because of the need to rapidly inoculate clinical samples into multiple cell lines to achieve optimal sensitivity. In addition, many rapid immune-enzymatic assays have been developed by many companies to allow detection of influenza virus A and B antigens and HRSV antigens. Unfortunately, the accuracy of these tests depends on many variables, and their sensitivity has generally been lower than that of viral cultures. Therefore PCR assays have now been developed for many respiratory viruses, allowing their detection of small amounts of viral nucleic acid.

    Influenza (Flu) viruses occur in two main types: Types A and B. The influenza A and B viruses that routinely spread in people, the human influenza viruses, are responsible for seasonal flu epidemics each year.

    Influenza A viruses can be classified into sub-types depending on the genes that code for the surface proteins of the virus strain. However, during a flu season, different types and subtypes of influenza can circulate and cause illness.

    According to the CDC human infections with a new avian influenza A (H7N9) virus were first reported in China in March 2013. Most of these infections are believed to result from exposure to infected poultry or contaminated environments, since H7N9 viruses have also been found in poultry in China. Most patients infected with human H7N9 have had severe respiratory illness, with about one-third resulting in death. The first case outside of China was in Malaysia and was reported on February 12, 2014. The case was detected in a traveler from an H7N9-affected area of China. According to the CDC the new H7N9 virus strain has not been detected in people or birds in the United States.
     

    The Influenza A Virus Genome

    The influenza virus belongs to the Orthomyxoviridae family composed of six different RNA viruses. This virus is responsible for the common flu in birds, mammals, and humans. The estimated death toll in the United States on average is 50,000 people annually. Symptoms usually include fever, headache, and nasal discharge which can develop into more obstructive pulmonary and heart problems including cardiac failure and bacterial pneumonia. The Influenza A virus genome is contained on eight single (non-paired) RNA strands that code for eleven proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The total genome size is 13,588 bases. The segmented nature of the genome allows for the exchange of entire genes between different viral strains during cellular cohabitation. Hemagglutinin (HA) determines the extent of the infection of the host organism. During infection influenza viruses bud from the apical surface of polarized epithelial cells (e.g. bronchial epithelial cells) into the lumen of lungs. The infection primarily occurs in the lungs since HA is cleaved by tryptase clara which is restricted to lungs. Clara cells are dome-shaped cells with short microvilli found in the small airways (bronchioles) of the lungs. However HAs of H5 and H7 pantropic avian virus subtypes can be cleaved by furin and subtilisin-type enzymes, allowing the virus to grow in other organs as well.


    (Source: Science Volume 340 12 April 2013 page 129 – 130 and Wikepidia.)

    The eight RNA segments of the viral genome

    1 HA or H Encodes hemagglutinin About 500 molecules of hemagglutinin are needed to make one virion
    2 NA or N Encodes neuraminidase About 100 molecules of neuraminidase are needed to make one virion
    3 NP Encodes nucleoprotein A protein that is structurally associated with nucleic acids
    4 M Encodes two matrix proteins (the M1 and the M2) by using different reading frames from the same RNA segment About 3000 matrix protein molecules are needed to make one virion
    5 NS Encodes two distinct non-structural proteins (NS1 and NEP) by using different reading frames from the same RNA segment NS1 of influenza A is a 26,000 Dalton protein that prevents polyadenlylation of cellular mRNAs to circumvent antiviral responses of the host
    6 PA Encodes an RNA polymerase An enzyme that produces RNA
    7 PB1 Encodes an RNA polymerase and PB1-F2 protein by using different reading frames from the same RNA segment Targets the inner mitochondrial membrane via a amphipathic helix and disrupts mitochondrial function
    8 PB2 Encodes an RNA polymerase PB2 PB2 subunit of the RNA Polymerase affects virulence by interacting with the mitochondrial antiviral Signaling Protein and inhibiting expression of beta interferon

    Real-time PCR (RT-PCR) based assays have been developed in many laboratories now and can be used to detect the different viral strains. The following tables contain lists of real-time detection assays for human H7N9 which has been made possible with the generous contributions of sequence data and viruses by the People's Republic of China, and through the contribution of protocols and validation data from OFFLU network laboratories.
     

    Detection of RNA segment H7 [Hemagglutinin]

    RT-PCR Assay Name H 7 H7N9 Detection $ Primer and Probes 5’ – 3’ Protocol & HA alignment GISAID sequences 10/15/13 99 HA sequences HA region
    CNIC H7(N9) RT qPCR VALIDATED Detecton of novel H7N9, very limited detection of other H7Nx viruses. Developed by China National Influenza Center, Beijing. Forward Primer:
    AGAAATGAAA TGGCTCCTGT CAA
    Reverse Primer:
    GGTTTTTTCT TGTATTTTTA TATGACTTAG
    Probe:
    FAM-AGATAATGCT GCATTCCCG CAGATG-BHQ1
    CNIC {http://www.cnic.org.cn/eng/} FOR primer: 3 nt changes to Shanghai/1; 1 nt change to homing pigeon / Jiangsu / SD184 and ck / Guangdong / SD641. REV primer: all 1 nt change but Shanghai/1) PROBE: 1 nt change to Shanghai/4, Hangzhou/2, chicken/Jiangsu/SC537, Guangdong/1 468‐550
    RT‐qPCR H7.2‐FLI & VALIDATED Detection of novel H7N9 and of other H7Nx viruses. Used as standard H7 RT‐qPCR at FLI. Validated with AgPAth reagents. Forward Primer:
    AYAGAATACA GATWGACCCA GT
    Reverse Primer:
    TAGTGCACYG CATGTTTCCA Probe:
    FAM-TGGTTTAGCT TCGGGGCATC ATG-BHQ
    FLI CODA-CERVA, Belgium
    FOR primer:
    0 Changes REV primer:
    0 Changes PROBE:
    0 changes
    1523-1672
    RT-qPCR CODA VALIDATED Validation used FLI probe with CODA primers. Detection of novel H7N9 and of other H7Nx viruses. Forward Primer:
    GYAGYGGYTA CAAAGATGTG
    Reverse Primer:
    GAAGACAAGG CCCATTGCAA
    Probe:
    FAM-TGGTTTAGCT TCGGGGC-BHQ
    FOR primer: 1 nt change to Wuxi/2, env/Shandong/SD039, env / Shandong / SD049 REV primer: all 1 nt change but: Shanghai/1, Shanghai/4655T and Shanghai / 4665T PROBE: 0 changes (FLI) 1553‐ 1638
    RT‐qPCR-IZSVe VALIDATED Monne et al., 2008 ‐ modified probe. Detection of novel H7N9. Forward Primer:
    TTTGGTTTAG CTTCGGG Reverse Primer:
    GAAGAMAAGG CYCATTG Probe:
    VIC-CATCATGTTT CRTACTTCT-MGB
    IZSVe
    FOR primer: 0 changes REV primer: all 1 nt change but: Shanghai/1, Shanghai/4655T and Shanghai/4665T;2 nt changes to ck/Hangzhou/50 PROBE: 0 changes
    1577- 1638
    Eurasian H7 HA2 RT-qPCR-AHVLA VALIDATED Detection of novel H7N9 and of other H7Nx viruses. Forward Primer:
    GGCCAGTATT AGAAACAACA CCTATGA
    Reverse Primer:
    GCCCCGAAGC TAAACCAAAG TAT
    Probe:
    FAM-CCGCTGCTTA GTTTGACTGG GTCAATCT- BHQ-1
    AHVLA, and IZSVe, AARIAH FOR primer: all 1nt change but ck/Jiangsu/SC035; 2 nt changes to Shanghai/1, Shanghai/4655T, Shanghai/4665T, env/Shanghai/S1438, Ck / Shanghai / S1358; REV primer: 0 changes PROBE: 0 changes 1464-1595
    Eurasian H7 RT‐qPCR assay 2 ‐ HKU yes Modified from J. Clin. Microbiol. 48: 4275‐4278. Forward Primer:
    ATAGATAGCA GGGCAGTTGG
    Reverse Primer:
    GATCWATTGC HGAYTGRGTG
    Probe:
    FAM-CCY TCY CCY TGT GCR TTY TG-3BHQ1
    HKU  
    Eurasian H7 RT-qPCR assay 1-HKU yes Eurasian H7 strains are positive with this test. From J. Clin. Microbiol. 48:4275--‐4278. Forward Primer:
    ATMAATAGCA GRGCARTRGG
    Reverse Primer:
    GATCWATTGC HGAYTGRGTG
    Probe:
    FAM-CCYTCYCCYT GTGCRTTYTG-3BHQ1
    HKU  
    H7CS RT-qPCR-AHVLA yes Published validation has shown this H7 CS RRT-PCR to be less sensitive than the H7 HA2 rRT-PCR (AHVLA). Advantage when molecular pathotyping may be required (amplicon sequencing). Forward Primer:
    CGTGCAAGTT TTCTGAGAGG
    Reverse Primer:
    GACCTTCCCA TCCATTTTCA
    Probe:
    FAM-AACCCGCTAT GGCACCAAATA GGCCTC- BHQ-1
    AHVLA  
    RT-qPCR (H7-TQM)-AAHL Low sensitivity Low sensitivity in detecting novel H7N9 ‐ not recommended. Highly sensitive for other H7 viruses. Forward Primer: GGATGGGAAG GTYTGGTTGA Reverse Primer: CCTCTCCTTG TGMATTTTGA TG Probe: FAM-TGAAACCATA-CCACCCA- BHQplus; antisense AAHL  
    RT-qPCR (H7-TQM)-USDA / CDC fail   USDA/CDC  


    Detection of RNA segment N9 [neuraminidase]

    RT-PCR Assay Name N 9 Prediction of novel H7N9 detection Primer and Probes 5’ – 3’ Protocol & HA alignment GISAID sequences 10/15/13 99 HA sequences HA region
    (H7) N9 RT-qPCR VALIDATED Sensitive detection of the N9 of novel H7N9, detection of other HxN9 viruses. Developed by China National Influenza Center, Beijing Forward Primer:
    TGGCAATGAC ACACACTAGT CAGT
    Reverse Primer:
    ATTACCTGGA TAAGGGTCGT TACACT
    Probe:
    FAM-AGACAATCCC CGACCGAATG ACCC- BHQ1
    CNIC FOR primer: all 1 nt change, 2 nt changes to Shanghai/1, ck/Shanghai/S1053, Hangzhou/3, Zheijiang/HZ1, Nanjing/1, env/Nanjing/2913, ck/Zheijiang/SD007, ck/Zheijiang/S1358, ck/Zheijiang/S1055, ck/Jiangsu/SC099, ck/Jiangsu/SC002, env/Wuxi/1, Jiangsu/1, Jiangsu/2 REV primer: all 1 nt change, 2 nt changes to Hangzhou/3, Zheijiang/HZ1, Nanjing/1, ck/Zheijiang/S1358, ck/Zheijiang/S1055, ck/Jiangsu/SC099, ck/Jiangsu/SC002, env/Wuxi/1, Jiangsu/1, Jiangsu/2 PROBE: 0 changes (Shanghai/Patient6‐3 nt changes or sequencing slip) 914-1020
    Eurasian N9 RT-qPCR-SEPRL VALIDATED Contains two quenchers: an internal quencher: ZEN and a 3'quencher IABkFQ. Sensitive detection of the N9 of novel H7N9 virus. Forward Primer:
    GCATTGACAG ATGATAGATC AAA
    Reverse Primer:
    AGCCCAATAG TCCATGAAAG A
    Probe:
    FAM- CTGACTGGA/ZEN/G TGGTTACAGTGG- IABkFQ
    USDA/SEPRL FOR primer: 1 nt change in Hangzhou/3, Shanghai/Patient5, Zheijiang/HZ1, ck/Zheijiang/SD007, ck/Jiangsu/SC002, env/Wuxi/1 REV primer: 0 changes PROBE: 1 nt change in Shangsha/1 1132-1230


    Detection of RNA segment M [matrix proteins]

    RT-PCR Assay Name M Prediction of novel H7N9 detection Primer and Probes 5’ – 3’ Protocol & HA alignment GISAID sequences 10/15/13 99 HA sequences HA region
    InfA RT-qPCR (by CDC / WHO VALIDATED Detection of influenza A including novel H7N9. Forward Primer:
    GACCRATCCT GTCACCTCTG AC
    Reverse Primer:
    AGGGCATTYT GGACAAAKCG TCTA
    Probe:
    FAM-TGCAGTCCTC GCTCACTGGG CACG- BHQ1
    CNIC/WHO/CDC FOR primer: 1 change in Shanghai/Patient14 REV primer: all 2 nt changes, 3 nt changes in ck/Zheijiang/SD007, Shanghai/Patient4, Shanghai/Patient14, env/Hangzhou/37, 4 nt changes in ck/Hangzhou/48 PROBE: 0 changes 146-251

    $ detection either predicted by none/few mismatches in primer/probes with template OR validated with H7N9 RNA.
    * of primers and probe sequences to novel H7N9 sequence.
    # other dyes and quenchers are also expected to work. Indicated dye/quencher combinations are used for these protocols in the contributing laboratories.
    & Geelong (AAHL) supplied reagents to ASEAN partners.


    Assay Protocol

    Real-time RT-PCR Protocol for the Detection of Avian Influenza A(H7N9) Virus 8 April 2013 Updated on 15 April 2013 Updated on 15 April 2013

    The WHO Collaborating Center for Reference and Research on Influenza at the Chinese National Influenza Center, Beijing, China, has made available attached real-time RT-PCR protocol for the detection of avian influenza A(H7N9) virus. For further information please contact us at:gisrs-whohq@who.int

    It is strongly recommended that all unsubtypeable influenza A specimens should be immediately sent to one of the six WHO Collaborating Centres for Influenza in the Global Influenza Surveillance and Response System (GISRS)1 for testing and analysis

    1http://www.who.int/influenza/gisrs_laboratory/collaborating_centres/list/en/
    The protocol was developed by and belongs to the WHO Collaborating Centre in Beijing. It is made available for emergency use as a service to the public health. It is not for commercial development or for profit. .
    1. Purpose

      To specifically detect avian influenza A(H7N9) virus using real-time RT-PCR with specific primers and probes targeting the matrix, H7 and N9 genes.

    2. Materials and equipments
      1. Real-time fluorescence quantitative PCR analysis system
      2. Bench top centrifuge for 1.5mL Eppendorf tubes
      3. 10, 200, 1000μL pipettors and plugged tips
      4. Vortex
      5. QIAGEN RNeasy Mini Kit
      6. AgPath one-step RT-PCR kit
      7. The specific primers and probes for the H7and N9 genes are summarized in the table below. In addition, the use of a primer and probe targeted M gene and house-keeping gene such a RNP is recommended for typing all influenza A virus and internal control in the tests.

        Table of PCR primers and probes

        ID Sequence Note
        H7
        CNIC-H7F 5'-AGAAATGAAATGGCTCCTGTCAA-3' Primer
        CNIC-H7R 5'-GGTTTTTTCTTGTATTTTTATATGACTTAG-3' Primer
        CNIC-H7P 5'FAM-AGATAATGCTGCATTCCCGCAGATG-BHQ1-3' Probe
        N9
        CNIC-N9 5’ –TAGCAATGACACACACTAGTCAAT-3’ Primer
        CNIC-N9R 5’ –ATTACCTGGATAAGGGTCATTACACT-3’ Primer
        CNIC-N9P 5’FAM- AGACAATCCCCGACCGAATGACCC -BHQ1-3’ Probe
        FluA
        InfA Forward 5’ GACCRATCCTGTCACCTCTGA C 3’ Primer
        InfA Reverse 5’ AGGGCATTYTGGACAAAKCGTCTA3’ Primer
        InfA Probe1 5’ FAM-TGC AGT CCT CGC TCA CTG GGC ACG-BHQ1-3’ Probe
        RnaseP
        RnaseP Forward 5’ AGATTTGGACCTGCGAGCG 3’ Primer
        RnaseP Reverse 5’ GAGCGGCTGTCTCCACAA GT3’ Primer
        RnaseP Probe1 5’FAM-TTCTGACCTGAA GGCTCTGCGCG-BHQ1-3’ Probe

        Note: FluA and RNase primer/probe sets were from published WHO protocol provided by CDC, Atlanta.

      8. Other materials: RNase-free 1.5mL eppendorf tubes, RNase-free 0.2mL PCR tubes, powder-free disposables latex glove, goggles, headgear, shoe cover, tips for pipettors, β- thioglycol, 70% alcohol.
    3. Biosafety

      The lysis of the specimen (500 μL lysis buffer with 200 μL clinical samples is recommended) should be to be carried out in a BSL-2 facility with BSL-3 level personal protection equipment. Subsequent procedures can be performed in a BSL-2 laboratory which has separate rooms including reagent preparation area, specimen preparation area and amplification/detection area. The DNA-free area is the clean area and the area of amplified DNA is the dirty area. The work flow is from clean to dirty areas.

    4. Procedures
      1. Nucleic acid extraction
        The procedure is performed in a BSL-2 biohazard hood in the specimen preparation area according the manufacturer. Elution of the RNA using a final volume of 50 μL H2O is recommended.
      2. Quality control parameters
        Negative control: Sterile water is extracted as a negative control at the same as the nuclear acid extraction of the other specimens. Reagent blank control: RNase free H2O.
        Positive control: RNA of the A(H7N9) virus provided. Internal positive control: ribonucleoprotein (RNP) is recommended.
      3. The reaction system preparation
        1. (1) Thaw the RT-PCR Master Mix, primers and probes at room temperature in the reagent preparation area of the BSL-2 facility.
        2. (2) Prepare reaction mixture. Different primer pairs and probes should be prepared in the different tubes respectively. For each reaction:
        Components volume(μL)
        2× RT-PCR Master Mix 12.5
        primer-forward(40μM) 0.5
        primer-reverse(40μM) 0.5
        Probe (20 μM) 0.5
        25xRT-pcr enzymes mix 1
        Template RNA 5.0
        RNase Free H2O 5
        Total 25
      4. Aliquot the reaction mixture into 0.2mL PCR tubes or a 96-well PCR plate as 20μL per tube and label clearly.
      5. Add five μL of the template RNA for the negative control, test specimens, or positive control into the separate tubes with the reaction mixture in a BSL-2 biohazard hood in the specimen preparation area.
      6. Load the tubes in the PCR cycler for Real-time RT-PCR detection and use the following programme for cycling:
        1. 45°C 10min
        2. 95°C 10min
        3. 95°C 15s
        4. 60°C 45s

        Return to the 3th step, and perform 40 cycles

      7. Result analysis:
        The results are determined if the quality controls work.
        1. The specimen is negative if the value of Ct is undetectable,
        2. The specimen is positive if Ct value is ≤38.0.
        3. It is suggested that specimens with a Ct higher than 38 are repeated. The specimen can be considered positive if the repeat results are the same as before i.e. Ct is higher than 38. If the repeat Ct is undetectable, the specimen is considered negative.
      8. Criteria for quality control:
        1. The result of the negative control should be negative.
        2. The Ct value of positive control should not be more than 28.0.
        3. Otherwise, the test is invalid.
    5. Troubleshooting
      1. False positives may be due to environmental contamination if there is amplification detected in the negative control and reagent blank control. The unidirectional work flow must be strictly obeyed. The following measures should be taken should there be false positives: ventilate the labs, wash and clean the workbench, autoclave centrifuge tubes and tips, and use fresh reagents.
      2. RNA degradation should be taken into consideration if the Ct value of the positive control is more than 30. All materials should be RNase-free.
    6. Cautions
      1. In order to avoiding nucleic acid cross-contamination, add the negative control to the reaction mixture first, then the specimen, followed by the positive control respectively.
      2. Dedicated equipment for each area including lab coats, pipettors, plugged tips and powder-free disposal latex glove are required.
      3. Follow the instructions for maintenance of the incubator, PCR cycler and pipettors. Calibration should be performed every 6 months.
    7. Protocol Use Limitations

      These protocols were optimized using the quantitative one-step probe RT-PCR (AgPath one-step RT-PCR kit ) that have been shown to produce comparable results on 96-well format thermocycler systems such as Stratagene QPCR instruments (MX3000®or MX3005®).


    0 0

    A novel bead-based suspension assay using BNA-NC probes to detect and quantify somatic mutations in leukemia

    A novel bead-based suspension assay using Bridged Nucleic Acid, BNA-NC, probes for the Luminex (USA) LabScan200 flow platform was develop and validated by Shivarov et al. in 2014. This assay allows quantitative detection of DNMT3A p.R882C/H/R/S mutations. The comparison with LNA based probes revealed the superior hybridization characteristics of the BNA based probes. The researchers demonstrated for the first time the benefit of BNA-NC probes coupled to fluorescently labeled beads for quantitative detection of DNMT3A R882 mutations. This type of assay adds to the list of molecular diagnostics techniques used for the analysis of biological markers in genomic and proteomic research. The specific diagnosis and monitoring of diseases enables detection and evaluation of disease risks for individual patients. By analyzing the specifics of a patient’s disease, molecular diagnostics offers the promise to enable and optimize personalized medicine.

    Leukemia is an often malignant blood disease that has the tendency to become progressively worse. This type of cancer of the blood or bone marrow is characterized by an abnormal increase of immature white blood cells. However, the term leukemia is also used to cover a whole spectrum of diseases affecting the blood, bone marrow, and lymphoid system, all known as hematological neoplasms. Leukemia is now considered to be a treatable disease. Several hematologic malignant tumors are characterized by genome instabilities. As identified by whole genome sequencing, these cancer types frequently have between 10,000 and 100,000 mutations in their entire genomes. Mutations in the human DNA methyl transferase 3A (DNMT3A) gene have now been identified in several blood diseases. The methyl-group transferring enzyme, DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for the regulation of gene expression and mutations. and deletions in this protein have been observed in acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL), myelodysplastic sydromes and myeloproliferative neoplasms. Myeloid cells represent a prominent part of local inflammatory infiltrates in the central nervous system (CNS) and appear to strongly contribute to the local inflammatory milieu and the pathological outcome of diseases involving these cells.

    Kim et al. in 2013 used PCR and direct sequencing to analyze mutations of DNMT3A amino acid residue R882 in 99 acute leukemia patients, including 57 AML patients, 41 ALL patients and a single biphenotypic acute leukemia (BAL) patient. The most common immunophenotype  in BAL patients is defined by the coexpression of B-lymphoid and myeloid markers and less frequently, T-lymphoid and myeloid markers. BAL has a high incidence of clonal chromosomal abnormalities, the most common being the t(9;22) (q34;q11) (Ph chromosome) and structural abnormalities involving 11q23. Data are emerging that BAL has a negative prognosis in both children and adults and this may be related to the underlying chromosome abnormalities. The research group detected DNMT3A expression in mononuclear cells of the bone marrow in these patients and in normal individuals using real‑time quantitative polymerase chain reaction. Approximately 17.5% (10/57) of AML patients were found to exhibit DNMT3A mutations, and four missense mutations were observed in the DNMT3A‑mutated AML patients, including R882 mutations and a novel single nucleotide polymorphism resulting in a M880V amino acid substitution. It is now known that somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies. The majority (∼60%) of these affect a single amino acid, Arg882 (R882), located in the catalytic domain of the enzyme. In 2013, Kim et al. could show that exogenously expressed mouse Dnmt3a proteins that have the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells but are capable of interacting with wild-type Dnmt3a and Dnmt3b. The coexpression of the Dnmt3a R878H (histidine) mutant protein resulted in inhibition of the wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. In addition the expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induced hypomethylation which suggests that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects. The current literature suggests that the presence of DNMT3A mutations is an adverse prognosis biomarker in adult acute myeloid leukemia and that the rapid detection of DNMT3A R882 codon mutations allows for early identification of poor risk patients with acute myeloid leukemia.

    Shivarov et al. therefore set out to develop a novel bead-based suspension assay using BNA-NC probes for the LabScan200 flow platform from Luminex, (USA). The research group developed and validated a bead-based method to quantitatively detect DNMT3A p.R882C/H/R/S mutations using BNA-NC-modified probes. The comparison with probes that were modified with LNAs, a first generation bridged-nucleic acid, revealed the superior hybridization characteristics for the BNA based probes. The researchers demonstrated for the first time the applicability of BNA-NC probes coupled to fluorescently labeled beads for quantitative detection of DNMT3A R882 mutations.

    How does the assay work?

    1.      Primers are designed to allow for the amplification of a DNA sequence fragment that contains the mutated sequence codon. One of the primers is labeled with biotin.

    2.      First, human genomic DNA is extracted from blood.

    3.      The exon 23 of the human DNMT3A gene is amplified using the selected forward and reverse primer.

    4.      To determine the exact sequence the purified and amplified DNA can be sequenced using Big Dye terminator cycle-sequencing.

    5.      Next, the exon 23 DNMT3A fragments are amplified from either genomic or plasmic DNA samples using a 5’-biotinylated forward primer.

    6.      Genotyping is performed with the BNA-NC modified oligonucleotide probes connected to microsphere beads, specific for the wild type or the mutant alleles, by direct hybridization.

    7.      The captured DNA fragment containing biotin on its 5’-end is detected with the help of streptavidin-phycoerythrine (SAPE) in the hybridization buffer using the LabScan200 flow platform from Luminex (USA). For more detail review Shivarov et al. 2014.  

    The outline of the assay is illustrated as follows:


     

    Bead-based suspension assay using BNA-NC probes

    bead based suspension assay - 1

    Figure 1:  Bead-based suspension assay using BNA-NC probes to detect and quantify somatic mutations in leukemia. The amplified DNA fragment containing the mutations is captured by the BNA/DNA probes and quantitatively detected with the help of the SAPE complex allowing the analysis in a Luminex system.

    The following illustration explains how the assay works in the Luminex platform.

    Bead-based suspension assay using BNA-NC probes

    bead based suspension assay - 2

    bead based suspension assay - 2

    Figure 2:  Bead-based suspension assay using BNA-NC probes and the Luminex system to detect and quantify somatic mutations in leukemia.
    1. Primers and probes are designed specific for the mutated sequence codons.
    2. DNA is isolated from blood.
    3. Amplified by PCR.
    4. Biotinylated DNA fragments are captured with the capture probe connected to the beads.
    5. 
    Biotinylated DNA fragments are detected with a streptavidin-phycoerythrine (SAPE) complex.
    6. The sample is analyzed using a Luminex instrument. The level of SAPE fluorescence is proportional to the amount of the captured DNA fragment.


    To illustrate how primers and probes were designed, the location of primer and probe sequences along the DNMT3A reference gene sequence are illustrated in the following paragraph showing a partial sequence segment of the gene containing the mutated target codon.


    Sequence Fragment for the DNMT3A gene: gi|340523094|ref|NG_029465.1| Homo sapiens DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A), RefSeqGene on chromosome 2:

    ----

    TTTTTCGCCTTCCCTGCTCTTGATGGGGAGGATGCTAGGGCTACGTGTTCGTTACATGCAGACTCCAGGTGTACGTTGCC

    GAGCTAACCCTGCAGGAGAGGAGAACTAGAGAGTGTGTCCTGGAGGTATTTCTGAGGGATTGTTCAAAATTACTCTTATT

    TCTGCTGGGTTGTGAAACTCTAGGCAGTGATGACCTTACTACCTTTAAGGTCACAGAAACCAGCACAGTGCCTGGCACAT

     

    5’-3’

    GGTTGGTGATCTGAGTGCCGGGTTGTTTATAAAGGACAGAAGATTCGGCAGAACTAAGCAGGCGTCAGAGGAGTTGGTGG

     

                                                     Forward primer

    GTGTGAGTGCCCCTGTCCCTGCACTTCGGGTGGCTGCTGGTCCTCCGGGTCCTGCTGTGTGGTTAGACGGCTTCCGGGCA

                                                     |

                                                   Biotin

     

    GCCTGGTCTGGCCAGCACTCACCCTGCCCTCTCTGCCTTTTCTCCCCCAGGGTATTTGGTTTCCCAGTCCACTATACTGA

    CGTCTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGGTCATGGAGCGTGCCAGTCATCCGCCACCTCTTCG

        3’-TTGTACTCGGCGAACCGA-5’-BEAD

     

        5’-AACATGAGCTGCTTGGCG CGC -> TGC; R -> C

        3’-TTGTACTCGGCAAACCGA-5’-BEAD

     

           AACATGAGCCCCTTGGCG CGC -> CCC; R -> P

        3’-TTGTACTCGGGGAACCGA-5’-BEAD

     

           AACATGAGCAGCTTGGCG CGC -> AGC; R -> S

        3’-TTGTACTCGTCGAACCGA-5’-BEAD

     

           AACATGAGCCACTTGGCG CGC -> CAC; R -> H

        3’-TTGTACTCGGTGAACCGA-5’-BEAD

     

    CTCCGCTGAAGGAGTATTTTGCGTGTGTGTAAGGGACATGGGGGCAAACTGAGGTAGCGACACAAAGTTAAACAAACAAA

    CAAAAAACACAAAACATAATAAAACACCAAGAACATGAGGATGGAGAGAAGTATCAGCACCCAGAAGAGAAAAAGGAATT

    TAAAACAAAAACCACAGAGGCGGAAATACCGGAGGGCTTTGCCTTGCGAAAAGGGTTGGACATCATCTCCTGATTTTTCA

    ATGTTATTCTTCAGTCCTATTTAAAAACAAAACCAAGCTCCCTTCCCTTCCTCCCCCTTCCCTTTTTTTTCGGTCAGACC

    TTTTATTTTCTACTCTTTTCAGAGGGGTTTTCTGTTTGTTTGGGTTTTGTTTCTTGCTGTGACTGAAACAAGAAGGTTAT

               3’-GAAAAGTCTCCCCAAAAGACA-5’

                     Reverse Primer

     

    TGCAGCAAAAATCAGTAACAAAAAATAGTAACAATACCTTGCAGAGGAAAGGTGGGAGAGAGGAAAAAAGGAAATTCTAT

    AGAAATCTATATATTGGGTTGTTTTTTTTTTTGTTTTTTGTTTTTTTTTTTTGGGTTTTTTTTTTTACTATATATCTTTT

    TTTTGTTGTCTCTAGCCTGATCAGATAGGAGCACAAGCAGGGGACGGAAAGAGAGAGACACTCAGGCGGCAGCATTCCCT

    ----

     

    Probe Design for BNA/DNA Probes.

    This table shows the uniform Tm values for the BNA-NC probes, whereas the LNA probes had slightly lower Tm values. The positions of the modified nucleotide for the BNA-NC probes are shown as well. However, since the positions for the LNA probes are propriotary informatioin, they can not be shown here.

    probe design table

    (Source: Shivarov et al. 2014)

    The Luminex® 100 and 200™ Systems

    Luminex

    Luminex

    The Luminex® 100 and 200™ Systems are analyzers that can perform up to 100 or 200 assays simultaneously in a single well of a microtiter plate. This system is based on the principles of flow cytometry, integrated into the xMAPR technology (multi-analyte profiling beads) enabling the detection and quantitation of multiple RNA or protein targets simultaneously. The xMAP system combines a flow cytometer, fluorescent-dyed microspheres (beads), lasers and digital signal processing to allow multiplexing of up to 100 or 200 unique assays, depending on the instruments used within a single sample. [http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/immunoassays/luminex-instruments/luminex-200-system.html]

     

    References

    Soo Jin Kim, Hongbo Zhao, Swanand Hardikar, Anup Kumar Singh, Margaret A. Goodell, and Taiping Chen; A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells. December 12, 2013; Blood: 122 (25).

    Shivarov V, Ivanova M, Naumova E; Rapid Detection of DNMT3A R882 Mutations in Hematologic Malignancies Using a Novel Bead-Based Suspension Assay with BNA-NC(NC) Probes. PLoS One. 2014 Jun 10;9(6):e99769. doi: 10.1371/journal.pone.0099769. eCollection 2014.


    0 0

    The amino acid acivicin [(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; AT-125; NSC-163501] is a fermentation product of Streptomyces sviceus. Acivicin is a modified amino acid, a glutamine analog that irreversibly inhibits glutamine-dependent amidotransferases known to be a potent γ-glutamyl transpeptidase inhibitor that has been used to elucidate aspects of glutathione metabolism and has known anti-tumorigenic activity. This amino acid inhibits tumor growth in cell lines dependent on glutamine metabolism. Glutamine-dependent amidotransferases are enzymes that are involved in nucleotide and amino acid biosynthesis active in a variety of mouse tumor models including the L1210 and P388 leukemias, the M5076 ovarian carcinoma, and the MX-1 human breast tumor xenograft. It is thought that antitumor activity is mediated through the inhibition of enzymes that catalyze the transfer of an amido group from L-glutamine, especially the enzymes cytidine triphosphate (CTP) synthetase and xanthosine-5'-phosphate:ammonia ligase (XMP) aminase. CTP synthetase is an enzyme involved in pyrimidine biosynthesis that interconverts Uridine-5'-triphosphate (UTP) and Cytidine triphosphate (CTP). XMP aminase or GMP synthase (EC6.3.4.1, xanthosine-5'-phosphate-ammonia ligase, guanylate synthetase,  xanthosine 5'-monophosphate aminase) is an enzyme essential for the biosynthesis of nucleic acid guanine. Acivicin is known to have anticancer and antitumor activity in vivo and to prevent melanoma metastasis in vitro.

     

    Since this molecule is a modified non-typical amino acid it contains an amino group that allows the analysis with the help of standard amino acid analysis or pre- or post-column derivatization prior to the analysis using HPLC-UV or DAD detection. In addition, the non-derivatized amino acid me be detected using liquid-chromatography mass-spectrometry based methods as well.


    acivicin molecule

    Acivicin [(2S)-Amino[(5S)-3-chloro-4,5-dihydro-1,2-oxazol-5-yl]ethanoic acid]; molecular formula: C5H7ClN2O3; molar mass: 178.57 g mol−1.

     

    Williams et al. in 2009 solved the crystal structure of the acivicin-modified H.pyloriγ-glutamyltranspeptidase (HpGT). The structure of acivicin-modified HpGT suggests the nucleophilic attack of Thr 380 Oγ at the C3 of acivicin and the displacement of chloride. Furthermore, the researchers report that the integrity of the dihydroisoxazole ring is likely maintained during the reaction since C3 retains its sp2 hybridization. The observed unique C-terminal capping of the active site within the acivicin-modified HpGT structure, together with mutagenesis studies, demonstrated that Phe 567 contributes to the overall catalytic efficiency of the enzyme. The researcher examined the contributions of residues within the C-terminal domain of the 20 kDa subunit and found that local structural motifs are critical for optimal enzymatic activity and autoprocessing. These new insights into the HpGT structure and function relationships are proposed to help facilitate the design of future selective γGT inhibitors. Figure 1 depicts the structure model of the acivicin-inhibited γ-glutamyltranspeptidase (γ-GT).



    acivicin model structure

    Figure 1:  Structure model of the acivicin-inhibited γ-glutamyltranspeptidase (γ-GT). (Left) The domain structures are highlighted in different colors. (Middle) The secondary structures are highlighted in different colors. (Right) The structure of acivicin is shown and its location inside the protein binding pocket is indicated by the red arrow.

     

    References

    Poster DS, Bruno S, Penta J, Neil GL, McGovren JP.; Acivicin. An antitumor antibiotic. Cancer Clin Trials. 1981 Fall;4(3):327-30.

    Williams K, Cullati S, Sand A, Biterova EI, Barycki JJ.; Crystal structure of acivicin-inhibited gamma-glutamyltranspeptidase reveals critical roles for its C-terminus in autoprocessing and catalysis. Biochemistry. 2009 Mar 24;48(11):2459-67. doi: 10.1021/bi8014955.


    0 0
  • 06/25/14--00:00: Amino Acids
  • SYMBOL AMINO ACID STRUCTURE FORMULA MOL WT MW-H2
    βAla β-Alanine C3H7NO2 89.09 71.07
    Ala Alanine C3H7NO2 89.09 71.07
    Aad Aminoadipic acid C6H11NO4 161.16 143.14
    Abu 2-Aminobutyric acid C4H9NO2 103.12 85.10
    4-Abz 4-Aminobenzoic acid C7H7NO2 137.14 119.10
    γAbu 4-Aminobutyric acid C4H9NO2 103.12 85.10
    ЄAhx 6-Aminocaproic acid C6H13NO2 131.17 113.15
    Aib α-Aminoisobutyric acid C4H9NO2 103.12 85.10
    Ach 1-aminocyclohexane-1-carboxylic acid C7H13NO2 143.18 125.16
    Acpe 1-aminocycropentane-1-carboxylic acid C6H11NO2 129.16 111.14
    Acpe 1-aminocycropentane-1-carboxylic acid C6H11NO2 129.16 111.14
    Acpp 1-aminocycropropane-1-carboxylic acid C4H7NO2 101.10 83.08
    Phe(4-NH2) 4-Aminophenylalanine C9H12N2O2 180.20 162.18
    Asu α-Aminosuberic acid C8H15NO4 189.21 171.19
    Arg Arginine C6H12N4O2 174.20 156.18
    Asn Asparagine C4H8N2O3 132.12 114.10
    Asp Aspartic Acid C4H7NO4 133.10 115.08
    Bpa 4-Benzoylphenylalanine C16H15NO3 269.30 251.28
    Bip 4,4’-Biphenylalanine C15H15NO2 241.29 233.27
    tBuAla β-t-Butylalanine C7H15NO2 145.20 127.18
    Tle 2-t-Butylglycine C6H13NO2 131.17 113.15
    Phe(pCl) 4-Chlorophenylalanine C9H10CINO2 199.63 181.63
    Cit Citrulline C6H13N3O3 175.19 157.17
    Cha β-Cycrohexylalanine C9H17NO2 171.24 153.22
    Chg α-Cycrohexylglycine C8H15NO2 157.21 139.19
    Cpa β-Cycropentylalanine C8H15NO2 157.21 139.19
    Cpg Cyclopentylglycine C7H13NO2 143.18 125.16
    Cpr β-Cycropropylalanine C6H11NO2 129.16 111.14
    Cys Cysteine C3H7NO2S 121.16 103.14
    Δ-Pro 3,4-Dehydroproline C5H7NO2 113.11 95.09
    Dab α,r-Diaminobutyric acid C4H10N2O2 118.13 100.11
    Dap α, β-Diaminopropionic acid C3H8N2O2 104.11 86.09
    Deg Diethylglycine C6H13NO2 131.17 113.15
    Dmt 2’,6’-Dimethyltyrosine C11H15NO3 209.24 191.22
    Tyr(3,5-di-l) 3,5-Diiodotyrosine C9H9I2NO3 432.98 414.96
    Dpa 3,3-Diphenylalanine C15H15I2NO3 432.98 223.27
    Phe(4-Fl) 3,3-Diphenylalanine C9H10FNO3 183.18 223.27
    Glu Glutamic acid C5H9NO4 147.13 129.11
    Gln Glutamine C5H10N2O3 146.14 128.12
    Gly Glycine C2H5NO2 75.07 57.05
    His Histidine C6H9N3O2 155.15 137.13
    Hci Homocitrulline C7H15N3O3 189.21 171.19
    Hcy Homocysteine C4H9NO2S 135.19 117.17
    Hle Homoleucine C7H15NO2 145.20 127.18
    Hph Homophenylalanine C10H13NO2 179.22 161.20
    Hpr Homoproline C6H11NO2 129.16 111.14
    hSer Hmoserine C4H9NO3 119.12 101.10
    Hyp Hydroxyprorine C5H9NO3 131.13 113.11
    Trp(5-OH) 5-Hydroxytryptophan C11H12N2O3 220.22 202.20
    Igl Indanylglycine C11H13NO2 191.23 173.21
    allo-Ile Allo-Isoleucine C6H13NO2 131.17 113.15
    Ile Isoleucine C6H13NO2 131.17 113.15
    Leu Leucine C6H13NO2 131.17 113.15
    Lys Lysine C6H12N2O2 146.19 128.17
    Met Methionine C5H11NO2S 149.21 131.19
    α-Me-Leu α-Methylleucine C7H15NO2S 145.20 127.18
    Phe(4-Me) 4-Methylphenylalanine C10H13NO2 179.22 161.20
    α-Me-Phe α-Methylphenylalanine C10H13NO2 179.22 161.20
    Ala(2-naphthyl) 3-(2-Naphthyl)-alanine C13H13NO2 215.25 197.23
    Phe(4-NO2) 4-Nitrophenylalanine C9H10N4O2 210.19 192.17
    Nle Norleucine C6H13NO2 131.17 113.15
    Nva Norvaline C5H11NO2 117.15 99.13
    Oic Octahydroindole-2-carboxylic acid C9H15NO2 169.22 151.20
    Orn Ornithine C5H12N2O2 132.16 114.14
    Pen Penicillamine C5H11N2O2S 149.21 131.19
    Phe Phenylalanine C9H11N2O2 165.19 147.17
    Phg Phenylglycine C8H9NO2 151.16 133.14
    Pro Proline C5H9NO2 151.13 97.11
    Pra Propargylglycine C5H7NO2 113.11 95.09
    Ala(2’-pyridyl) 3-(2’-Pyridyl)-alanine C8H10N2O2 166.18 148.16
    Pyr, Glp Pyroglutamine C5H7NO3 129.11 111.09
    Ala(2’-quinoyl) 3-(2’-Quinoyl)-alanine C12H12N2O2 216.24 198.22
    Sar Sarcosine C3H7NO2 89.09 71.07
    Ser Serine C3H7NO3 105.09 87.07
    Sta Statine C8H17NO3 175.23 157.21
    Tic 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid C10H11NO2 177.20 159.18
    Tpi 1,2,3,4-Tetrahydronorhorman-3-carboxylic acid C12H12N2O2 216.24 198.22
    Thz Thiaproline C4H7NO2S 133.17 115.15
    Thi β-(2-Thienyl)alanine C7H9NO2S 171.22 153.20
    Thr Threonine C4H9NO2S 119.12 101.10
    Trp Tryptophan C11H12N2O2 204.23 186.21
    Tyr Tyrosine C9H11NO3 181.19 163.17
    Val Valine C5H11NO2 117.15 99.13

    0 0

    Melting Temperature (Tm) Calculation for BNA Oligonucleotides 
     
    By Klaus D. Linse
     
    Several mutation detection techniques rely on the chemical- or temperature-driven melting behavior of short oligonucleotides that are used for the hybridization of PCR-amplified DNA. Techniques include temperature gradient gel electrophoresis (TGGE) or denaturing gradient gel electrophoresis (DGGE), DNA blotting and hybridization probe assays among others. The design of synthetic probes that increase the stability of the dsDNA oligonucleotids makes the use of these techniques possible. The influence of mismatches on dsDNA stability is based on thermodynamic principles which underlying physico-chemical mechanisms have been investigated extensively in the last decade. The stability of a single base pair binding is influenced by the surrounding nearest neighbor base pairs. The thermodynamic parameters for every matched or mismatched base pair were experimentally defined and published and allow for the prediction of oligonucleotide duplex stability with or without mismatches.
     
    What is the melting or annealing temperature of a DNA or RNA molecule?
     
    The melting or annealing temperature (Tm) of a DNA or RNA molecule is the temperature at which a DNA, RNA or DNA/RNA double helix dissociates into single strands and where half the molecules are double stranded and half are single stranded.
     
    The Tm reflects the stability of DNA or RNA duplexes. More stable complexes will have higher melting temperatures. Experimental conditions such as salt and oligonucleotide concentrations will affect the hybridization process therefore the Tm needs to be measured under standard conditions.
     
    In general, longer strands have higher melting temperatures, as do sequences with higher G and C content. The best estimate of the oligonucleotide melting temperature is the use of thermodynamic parameters for the calculations. These estimates are useful for evidence based design of molecular biological experiments in which the melting behavior is important. Values for entropy and enthalpy of Watson-Crick pairs have been determined by many researchers in the past and can be found in the literature. SantaLucia in 1998 published enthalpy and entropy based unified values for the reliable prediction of Tm reflecting the DNA duplex stability. Since the Tm of the duplex is also influenced by the concentration of the oligonuleotides used in an experiment experimentally derived correction equations are also needed. For longer sequences alternative methods to estimate melting temperatures for duplex stability were developed as well. These methods are based on the length and GC content of the oligonucleotides.
     
    Reliable calculations should allow for the design of oligonucleotide probes allowing the discrimination of wild-type and mutant sequences by designing probes with the proper Tm.
     
    Good sequencing primers or hybridization probes should have the following characteristics:
     
    1.         They need to form stable duplexes with the target sequence under the experimental conditions used;
     
    2.         Be highly specific for the intended target sequence, and no form base-pairs to other regions within the template; and
     
    3.         Very importantly the sequences should not anneal to themselves.
     
    The formation of stable duplexes is especially important if the oligonucleotide probe is used for screening of complex DNA libraries. High specificity and not annealing to itself are important c parameters needed for both screening and sequencing.
     
    Computerized methods have made the search for an optimal oligonucleotide which would meet all three of these criteria less laborious.Furthermore, calculating the duplex dissociation temperature is critical to characterize oligonucleotides.
     
    The dissociation temperature (Td) can be calculated with the following formula:
     
    Td = 2°C x number of AT bp + 4°C x number of GC bp
     
    The “nearest neighbor model” allows for the use of nearest neighbor thermodynamic values with the following equation:
     
    Td = [ΔH / ΔS + R x ln(Ct/4)] – 273.15 °C –t
     
    Where ΔH and ΔS are the enthalpy and entropy for helix formation. R is the molar gas constant [1.987 (cal/°C x mol)], and Ct is the concentration of the probe. The constant t is a temperature correction for filter hybridization with a value equal to 7.6 °C.
     
    The melting temperature (Tm) of a duplex can be calculated with the following equation:
     
    Melting Temperature Calculatiion
     
    This model also takes into account the influence of salts present in the sample. The next equation shows how the Tm can be estimated in the presence of sodium ions.

    Melting Temperature Algorithm
     

    Reference: Rychlik and Rhoads in Nucleic Acid Research 1989, 17, 21, 8543-8551.
     

    Determination of the melting point

    The common way to determine the actual melting point of an oligonucleotide duplex is to use a cell or quartz cuvette in a thermostat as part of a UV spectrometer. The temperature is plotted versus the absorbance resulting in an S-shaped curve with two observed plateaus. The absorbance reading halfway between the plateaus correspond to the Tm.

    A real-time PCR instrument allows the determination of the Tm as well, for example, using SYBR green to stain the resulting PCR products.


    Testing different web based oligonucleotide calculators for consistency in results

    Chavali et al. in 2005 published a paper in the Journal Bioinformatics in which the researchers reported their findings for their study in which they compared different oligonucleotide calculators for their ability to predict the best melting temperature with the least deviation.
     
    The study was divided into three sections.
     
    1)            The identification of the best oligonucleotide properties calculator to predict the best Tm to allow for the calculation of the optimal annealing temperature for PCR amplifications.
     
    2)            The evaluation of the secondary structure predictions.
     
    3)      Testing the efficiency of primer designing software and identifying the best one.
     
    Experimental methods employed were thermal melting studies, Tm predictions, statistical analysis, and calculation of optimal annealing temperature, secondary structure studies and primer design studies. In this study the Tm of 108 oligonucleotides was predicted using 25 oligonucleotide properties calculators. Tm deviation values in the range of 7°C were observed.
     
    Reference: Chavali et al. in Bioinformatics. 2005 Oct 15;21(20): 3918-25. Epub 2005 Aug 16.
     
    These results indicate that the theoretical methods are good to estimate the Tm but may not be very accurate and that it is advisable to verify the predicted results experimentally for selected oligos when high throughput methods are used.
     
    Note 1: A precise optimum annealing temperature must be determined empirically.
     
    Most of the calculator software use similar algorithms and models for more details please review the paper. 
     
    Note 2: Prediction of Tm value of BNA/DNA chimeric oligonucleotides.
     
    The R&D group at BSI has recently developed our own Tm value prediction calculator based on a modified nearest-neighbor themodynamic model and experimental data. To obtain accurate Tm value prediction, oligonucleotides ranging from 8 to 20 nucleotides are recommended. Currently for our customers, we are providing the predicted Tm values. If the predicted Tm value is needed for your research, please contact info@biosyn.com.

     
    Glossary
     
    Tm     The melting temperature is the temperature in °C at which 50% of the oligonucleotide and its perfect complement are in a duplex.
     
    Td     The dissociation temperature is the temperature in °C at which 50% of an oligonucleotide and its perfect filter-bound complement are in duplex at the particular salt concentration and total strand concentration.

    ΔH    The Enthalpy change can be calculated by subtracting the bond energies of the product from the bond energies of the reactions.

    ΔS  The change in entropy is the tendency for randomness in a system. Natural systems have the tendency toward low enthalpy and high entropy.  

    0 0
  • 06/30/14--00:00: Gene Synthesis
  • More than 30 years after the first complete gene, a yeast tRNA, was synthesized Har Gobind Khorana and coworkers in 1972 and the synthesis of the first peptide- and protein-coding genes was performed in the laboratories of Herbert Boyer and Alexander Markham, DNA synthesis methods have now become the dominating techniques of modern molecular biology where they play a pivotal role in synthetic biology. Since 2007 synthetic biologists have demonstrated that not only can partial or whole chromosomes of bacteria be transplanted between bacterial cells, but also a whole bacterial chromosome could be successfully synthesized. More recently, in March 2014, research scientists in Jef Boeke's group in the Langone Medical Centre at New York University, reported in a paper in the Journal Science that his team was able to synthesize one of the S. cerevisiae 16 yeast chromosomes, the chromosome III, the researchers named synIII. How was this possible?  The experimental strategy used involved replacing the genes in the original chromosome with synthetic gene versions and finally integrating the finished human made chromosome into a yeast cell. However, the design and creation of 273,871 base pairs of DNA, which contained fewer base pairs than the 316,667 pairs in the original chromosome, was required. These examples illustrate the power of existing gene synthesis techniques enabling the assembly of large DNA and RNA sequences from chemical synthetic oligonucleotide building blocks. As can be verified by reviewing the literature in the field of "Synthetic Biology" current oligonucleotide synthesis and enzymatic gene assembly methods and techniques have now been optimized to the point where small operons, plasmids, and viruses can be constructed from scratch with relative ease. Hughes et al. in 2011 argued that these techniques are quickly becoming a cornerstone of modern molecular and synthetic biology methods.

    Biosynthesis Inc. now possesses the specialized technical skills, expertise and equipment to help their customer to build large synthetic DNA and RNA sequences.


    Reference

    Annaluru, Narayana; et al. (March 27, 2014). "Total Synthesis of a Functional Designer Eukaryotic Chromosome"<http://www.sciencemag.org/content/early/2014/03/26/science.1249252>. Science. doi<http://en.wikipedia.org/wiki/Digital_object_identifier>:10.1126/science.1249252<http://dx.doi.org/10.1126%2Fscience.1249252>. Retrieved 2014-03-28.

    Edge MD, Green AR, Heathcliffe GR et al. (August 1981). "Total synthesis of a human leukocyte interferon gene". Nature 292 (5825): 756-62. Bibcode<http://en.wikipedia.org/wiki/Bibcode>:1981Natur.292..756E<http://adsabs.harvard.edu/abs/1981Natur.292..756E>. doi<http://en.wikipedia.org/wiki/Digital_object_identifier>:10.1038/292756a0<http://dx.doi.org/10.1038%2F292756a0>. PMID<http://en.wikipedia.org/wiki/PubMed_Identifier> 6167861<http://www.ncbi.nlm.nih.gov/pubmed/6167861>.

    Khorana HG, Agarwal KL, Büchi H et al. (December 1972). "Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast". J. Mol. Biol. 72 (2): 209-217. doi<http://en.wikipedia.org/wiki/Digital_object_identifier>:10.1016/0022-2836(72)90146-5<http://dx.doi.org/10.1016%2F0022-2836%2872%2990146-5>. PMID<http://en.wikipedia.org/wiki/PubMed_Identifier> 4571075<http://www.ncbi.nlm.nih.gov/pubmed/4571075>.

    Hughes RA, Miklos AE, Ellington AD.; Gene synthesis: methods and applications. Methods Enzymol.<http://www.ncbi.nlm.nih.gov/pubmed/21601682> 2011;498:277-309. doi: 10.1016/B978-0-12-385120-8.00012-7.

    Itakura K, Hirose T, Crea R et al. (December 1977). "Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin"<http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=412251>. Science 198 (4321): 1056-1063. Bibcode<http://en.wikipedia.org/wiki/Bibcode>:1977Sci...198.1056I<http://adsabs.harvard.edu/abs/1977Sci...198.1056I>. doi<http://en.wikipedia.org/wiki/Digital_object_identifier>:10.1126/science.412251<http://dx.doi.org/10.1126%2Fscience.412251>. PMID<http://en.wikipedia.org/wiki/PubMed_Identifier> 412251<http://www.ncbi.nlm.nih.gov/pubmed/412251>.

    Shukman, David (27 March 2014). "Scientists hail synthetic chromosome advance"<http://www.bbc.com/news/science-environment-26768445>. BBC News. Retrieved 2014-03-28.



    0 0

    Cell Penetrating Peptide Delivers Proteins Into Cells

    By Klaus D. Linse
     
    Cell-penetrating peptides, or CPPs, are short peptides that can facilitate cellular uptake of a variety of molecular cargo. CPPs (sometimes also called protein transduction domains, or PTDs, are a group of short, highly basic peptides. These peptides can penetrate cell membranes either alone or along with cargo molecules. This “cargo” can range from nano-sized particles to small chemical molecules, peptides, proteins and large fragments of DNA as well as RNA molecules such as siRNA duplexes. Often the "cargo" is co-inserted into target cells together with the CPPs either via covalent attachment or through non-covalent interactions. The function of the CPP is to help deliver the cargo into cells.
     
    In a paper published online on June 2014 in the Journal Nature Methods, a research group at Texas A & M (Erazo-Oliveras et al. 2014) reported the development of a protein transduction approach using a cell-penetrating peptide (CPP) to deliver proteins as cargo across the cell membrane into living cells. The TAT sequence derived from the HIV-1 trans-activator gene product was used as the template for the design of a dimeric delivery molecule or vehicle. The peptide was modified with a lysine and fluorophore tetramethylrhodamine (TMR) to allow for fluores­cence imaging as well as a cysteine, both added at the N-terminal end of the TAT peptide to permit dimerization by disulfide bond formation. The researchers used this design since it is known that disulfide bonds are relatively stable inside endosomes but are cleaved following endosomal escape and upon entry into the reducing cytosol which releases the monomeric molecule. It was found that the CPP dfTAT can penetrate live cells by escaping from endosomes with high efficiency. The scientists demonstrated that this peptide allows the delivery of proteins, and potentially other cargo molecules, into the cells with the help of a mechanism called endosomal leakage. Furthermore, the cytosolic delivery of proteins as cargo into several cell lines was achieved with the help of the dfTAT peptide.
     
    The structures of the modified peptides as reported by Erazo-Oliveras et al. are shown below.
     
     ckTAT
    ckTAT
     
     
     fk(TMR)TAT
     
    fk(TMR)TAT
     
     
     
     dfTAT
    dfTAT
     
    Figure 1: Structures of ckTAT, fK(TMR)TAT and dfTAT: CKRKKRRQRRRG (Upper panel); CK(ε-NH-TMR)RKKRRQRRRG (Middle panel); [CK(ε-NH-TMR)RKKRRQRRRG]2 (Lower panel) are depicted. The arginine side chains are shown in blue, the TMR fluorophore is shown in red and the disulfide bond forming the dimer is shown in magenta.
     
    Scientists observed that the delivery did not require a binding interaction between the peptide and the proteins. In addition, this type of delivery did not noticeably affect cell viability, cell proliferation or gene expression. However, only the future will tell if this intracellular delivery method becomes a useful application for cell-based assays, cellular imaging and/or ex vivo manipulations (manipulations performed outside) of cells. On the other hand, if this approach could be used to selectively deliver a tumor suppressor protein into cancer cells, a new method to treat cancer with potentially less side effects could be available soon.

     
    How does endosome mediated cell delivery work?
     
    Endosomes are smooth-surfaced membrane containing vesicles or compartments found inside eukaryotic cells. Endosomes are the intermediate transporters of particles brought into a cell from outside and are part of the endocytosis pathway. Endocytosis is an energy-using process which allows cells to take materials from the outside into the cell by engulfing and fusing them with the cell or plasma membrane. Furthermore, molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes, the last compartment of the endocytic pathway, where they are degraded.  However, in an opposite process called exocytosis, these molecules can be recycled back to the plasma membrane.

    Endocytosis has been proposed as one of the primary mechanisms which allow cell-penetrating peptides (CPP) and their cargos to enter a cell. Unfortunately, one major limitation of this pathway is the entrapment of the CPP-cargo in intracellular vesicles. To reach the targets located in the cytoplasm the cargo needs to escape the vesicles in order to exert its biological function.

    Endocytosis can be divided into two major categories:

    1)         Phagocytosis - the ingestion of a smaller cell or cell fragments, a microorganism, or foreign particles,
                involves the uptake of large particles

    2)         Pinocytosis - the transport of fluids into a cell, involving solute uptake.

    Also, pinocytosis can be further divided into macropinocytosis, clathrin-dependent, caveolin-dependent and clathrin/caveolin-independent pathways. 
    Experimental data suggest that numerous factors appear to influence the route of cellular uptake of the CPPs, some of which may need further elucidation.

    Over the years, several strategies for the controlled cellular delivery of bioactive macromolecules with therapeutic potential have been developed. Among these, several non-viral carrier systems such as liposomes, polycationic carrier, nanomaterials and peptides have been investigated for their ability to penetrate or transduce cell membranes efficiently.

    Protein transduction is an emerging technology with potential applications in gene therapy. It can be described as the internalization of proteins from the outside into the cell. For many current gene therapy strategies, for example for the gene therapy of cancer, a sustained and regulated expression of the transgene is not necessarily required. Therefore, it appears possible that a short term delivery of the gene product, rather than the gene itself, maybe all that is needed. The observation that some proteins, when added to the outside of the cell, can be taken up by the cell has resulted in detailed studies of the fundamental mechanism that allows this to happen. As a result protein transduction domains were identified. Additionally, it was found that these domains when fused to other proteins allowing these proteins to enter the cell as well, and sometimes even the nucleus.

    The Drosophila antennapedia peptide, the herpes simplex virus VP22 protein and the HIV TAT protein transduction motif are the three most widely studied transduction motifs. Furthermore, there are indications that other proteins may have similar properties. Some of these are peptide motifs present in haemagglutinin from influenza, lactoferrin, fibroblast growth factor and the homeodomain (HD) of engrailed, Hoxa-5, Hoxb-4 and Hoxc-8 proteins.

    The cytosolic delivery of proteins into several cells with the help of the dfTAT peptide appears to be similar to the transfection of cells with lipofectamine. Lipofectamine, or Lipofectamine 2000, a common transfection reagent is produced and sold by Invitrogen. It helps to increase the transfection efficiency of RNA, including mRNA and siRNA, or plasmid DNA into in vitro cell cultures. This is called lipofection. Lipid subunits present in the reagent can form liposomes in aqueous environments that can entrap the transfection materials, for example DNA plasmids. The DNA-containing liposomes having a positive charge on their surfaces, can fuse with the negatively charged plasma membrane of living cells, allowing nucleic acid to cross into the cytoplasm now available to the cell for replication or expression.

    Several approaches for the cytosolic delivery of liposomal macromolecules have also been developed.
     
    These include:
     
    1)                  The co-encapsulation of fusogenic peptides into targeted drug-containing liposomes

    2)                  The coupling of HIV-1 derived cell penetrating peptide TAT to the surface of liposomes

    3)                  Photochemical internalization, based on photochemically inducible permeabilisation of
                         endocytic vesicles.

    In addition, several endosome-disrupting peptides that destabilize endosome membranes have been derived from certain pathogens in recent years, including viruses and bacterial toxins. These membrane-disrupting peptides are known to promote endosomal escape after endosomal acidification and already  some of them have been used for the design of fusion peptides.


    Table 1: Sequences of cell-penetrating peptide based tags for fusion peptides

    Tag
    Amino acid sequence
     (single letter code)
    Function
    Hemagglutinin
    HA2
    GLFGAIAGFIENGWEGMIDGWYG
     
     
    The N-terminal domain of the hemagglutinin HA2 subunit possesses 23 amino acids and a relatively hydrophobic region referred to as fusion peptide. The fusion peptide domain is buried in the HA trimer in its resting conformation and acidification in the endosome triggers an irreversible conformational change of HA2. This exposes the fusion peptide and allows it to insert itself into endosomal membranes. Subsequent formation of a fusion pore results in membrane fusion and leading to transfer of viral genome into the cytosol.
    Hemagglutinin
    INF7
    GLFEAIEGFIENGWEGMIDGWYG
    INF7 peptide, a glutamic acid-enriched HA2 analog, was identified as a more potent endosome membrane-destabilizing peptide. In this peptide, two glutamic acid moieties (underlined in peptide sequence) were introduced into the original HA2 fusion peptide to extend the α-helix structure, which increased pH sensitivity. HA2 was used to enhance CPP-mediated endosomal escape of cargoes.
    HIV-1 trans-activator gene product, TAT
    YGRKKRRQRRR
     
    This region corresponds to amino acids 47–57 of TAT and has a high net positive charge at physiological pH with nine out of 11 of its amino acids being either arginine or lysine. TAT has been shown to be a regulator of transcription in latent HIV and to be essential for HIV replication. It is an 86 amino acid protein made from two exons of 72 and 14 amino acids, respectively. It was first demonstrated independently by Green and Loewenstein and Frankel and Pabo that TAT added exogenously in culture was taken up rapidly by cells. The proteins transduction property was shown to reside in amino acids 37–72.
    NLS
    PKKKRKV
    The nuclear localization signal or sequence (NLS) is a peptide sequence that 'tags' a protein for import into the cell nucleus by nuclear transport.
    R9
    RL9
    RW9
    RRRRRRRRR-NH2
    RRLLRRLRR-NH2 RRWWRRWRR-NH2
    The RW9 and RL9 peptides derive from the 16-mer sequences RW16 andRL16 respectively, which were designed from penetratin, a Antennapedia homeoprotein.
    Herpes simplex virus VP22 protein
    VP22 protein is a small basic
    protein, approximately 38 kDa in size.
    The herpes simplex virus VP22 protein is encoded by the
    UL49 gene. In vivo, VP22 exists in two distinct forms as
    assayed by gel electrophoresis, phosphorylated and nonphosphorylated. VP22 is exported from cells in which
    it is synthesized, despite lacking a signal sequence, by
    ‘nonclassical’ Golgi-independent secretion. Upon export,
    VP22 can enter cells with high efficiency. Re-internalised VP22 is targeted to the nucleus despite having no recognised nuclear localisation signal, binds to chromatin and segregates to daughter cells.

    A list of more peptides that facilitate translocation into cells can be found at the following link:  
    http://www.biosyn.com/tew/Cell-Penetrating-or-Trojan-Peptides-CPP.aspx


    References

    Alfredo Erazo-Oliveras, Kristina Najjar, Laila Dayani, Ting-Yi Wang, Gregory A Johnson & Jean-Philippe Pellois; Protein delivery into live cells by incubation with an endosomolytic agent.  nature methods | ADVANCE ONLINE PUBLICATION Received 31 May 2013; accepted 9 May 2014; published online 15 June 2014; doi:10.1038/nmeth.2998

    KG Ford, BE Souberbielle, D Darling and F Farzaneh; Protein transduction: an alternative to genetic Intervention. Review. Gene Therapy (2001) 8, 1–4.  http://www.nature.com/gt/journal/v8/n1/full/3301383a.html
     
    Marjan M. Fretz, Enrico Mastrobattista, Gerben A. Koning Wim Jiskoot and Gert Storm;
    Strategies for cytosolic delivery of liposomal macromolecules. Published in the International Journal of Pharmaceutics. 298: 305-309 (2005).
     
    Ji-Sing Lioua, Betty Revon Liua, Adam L. Martin, Yue-Wern Huangd, Huey-Jenn Chiang, Han-Jung Lee; Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence. Peptides 37 (2012) 273–284.
     
    Astrid Walrant, Isabelle Correia, Chen-Yu Jiao, Olivier Lequin, Eric H. Bent, Nicole Goasdoué,
    Claire Lacombe, Gérard Chassaing, Sandrine Sagan, Isabel D. Alves; Different membrane behaviour and cellular uptake of three basic arginine-rich peptides. Biochimica et Biophysica Acta 1808 (2011) 382–393.

    0 0
  • 07/03/14--00:00: Peptide Libraries
  • Peptide libraries allow for the search of peptide based bioactive molecules by screening large numbers of peptides for their activity. Often, this is not possible with the help of traditional chemical approaches. Therefore combinatorial methods have been designed over the years with the using peptide chemistry. Synthetic peptide libraries can be prepared as either mixtures or sets of individual peptides. In the past both, tert-butyloxycarbonyl (tBoc) or 9-flurenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis methods (SPPS) have been used for the synthesis off large numbers of individual peptides. However, in recent years the automated Fmoc-based SPPS method appears to have become the dominant synthesis method. The availability of commercial automated SPPS instruments and methods makes the development of assays and screening strategies now much easier.
    Combinatorial chemistry is a branch of applied chemistry using rapid synthesis and screening methods and strategies to screen large mixtures of individual but related molecules for their biological activities. Since combinatorial chemistry in drug discovery represents a merging of chemistry and biology it generally involves the use of chemical synthetic methods coupled with biological screening assays. The resulting compound libraries can be mixtures or sets of individual molecules or chemical structures such as peptides of defined length. However, combinatorial chemistry is not only useful for the synthesis of peptide libraries but also for a whole host of other molecular libraries such as oligonucleotides, either made up of natural or modified nucleotide monomers. In pharmaceutical research combinatorial chemistry is used for the development of biologically active compounds such as new drugs and catalysts, some of which can be peptide based.

    Peptide libraries have now become key research tools for the screening of bioactive compounds by using large numbers, tens of thousands or even billions, of peptides that is not practically attainable with the help of traditional chemical approaches.
    The use of libraries containing large collections of peptides that can range from hundreds to millions or even to billions of different sequences allows for high-throughput screening of biological functions using monoclonal and polyclonal antibodies, receptors, enzymes or other target molecules. The chemical diversity of amino acids used coupled with the large number of sequences in a library results in the power of this technology. For drug screening and development, peptide libraries are useful for identifying of ligands that can serve as leads for pharmaceutical development or other purposes.

    Bio-Synthesis has implemented Peptide Library Tools to its rapid high-throughput parallel peptide synthesis platform. These libraries can be used to screen highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds and enzyme inhibitors.

    Applications:

    • Epitope mapping studies
    • Vaccine research
    • High-throughput protein interaction analysis
    • Customized peptide microarray production
    • Kinase assays
    • G-protein-coupled receptor (GPCR) ligand screening
    • Drug discovery
    • Protein-Protein interaction
    • Orphan receptor identification
    • Functional proteome
    • Nucleic acid binding
    • Enzyme substrate or inhibitor screening
    • Antigen and epitope screening
    • Autoantibody screening
    • Antibody cross reaction screening
    • Peptide/Protein Cross talk screening
    • Pre-absorption of antibody
    • Discovery of signalling molecules


    0 0

    Amino Acid Analysis of Tyramine

    By Klaus Linde


    Amino acid analysis allows for the analysis and quantitative detection of tyramine using a standard amino acid analyzer. Amino acid analysis of Tyramine can be performed either with pre-column or post-column modification or labeling since this naturally occurring monoamine contains a primary amino group. In addition, this technique lends itself to quantitatively analyze tyramine levels in food or feed products.

    Tyramine can trigger headaches and migraines in some individuals. People who take monoamine oxidase inhibitors or are especially sensitive to tyramine should eat a diet containing low amounts of this amine. High levels of tyramine have been found in aged foods such soy sauce, salami and sauerkraut. The solution to preventing migraines is to eat fresh foods and cut down on the use of prepared and processed foods.

    Tyramine, 2-(4-Hydroxyphenyl)ethylamine, 4-(2-Aminoethyl)phenol, or 4-Hydroxyphenethylamine, is a naturally occurring monoamine and trace biogenic and sympathomimetic amine derived from the amino acid tyrosine. Tyramine has the formula HOC6H4CH2CH2NH2 , and a molecular weight of 137.18. Molecular identification numbers of tyramine are the CAS Number 51-67-2, the Beilstein Registry Number 1099914, the EC Number 200-115-8, the MDL number MFCD00008193, and the PubChem Substance ID 24900582.


    Tyramine Structure

    Figure 1: Tyramine Structure.

    The biogenic amine tyramine can act as a catecholamine releasing agent. Catecholamines (CA) are monoamines, organic compounds that have a catechol group, which contain a benzene ring with two hydroxyl side groups and a side-chain amine. The term catechol refers to the 1,2-dihydroxybenzene group. Catecholamines are derived from the amino acid tyrosine and are water-soluble and approximately 50% of catecholamines are bound to plasma proteins when they circulate in the bloodstream. Tyramine by itself is unable to cross the blood-brain barrier and causes only non-psychoactive peripheral sympathomimetic effects. Some of its effects resemble effects of epinephrines. However, a hypertensive crisis can result from ingestion of tyramine-rich foods together with monoamine oxidase inhibitors (MAOIs).

     
    Tyrosine decarboxylation reaction
     

                                 Tyrosine                                                                         Tyramine

    Figure 2: Tyrosine decarboxylation reaction. According to the wormatlas, tyrosine is converted to tyramine via a decarboxylation reaction by the enzyme tyrosine decarboxylase. Tyramine is further converted to octapamine by the enzyme tyramine β–hydroxylase.

    Tyrosine is the precursor to catecholamines and tyramine is a breakdown product of tyrosine. In the gut and during fermentation, tyrosine is decarboxylated to tyramine. During normal metabolism in humans and mammals tyramine is deaminated in the liver to an inactive metabolite. David et al. in 1974 showed using MF1 mice that, as tissue concentrations of tyrosine are increased, decarboxylation becomes increasingly important and, at very high tissue levels, is the predominant route of metabolism. However, when the hepatic monoamine oxidase is inhibited, the clearance of tyramine is blocked and circulating tyramine levels can increase. Elevated levels of tyramine can compete with tyrosine for transport across the blood–brain barrier where it can then enter adrenergic nerve terminals.

    Biogenic amines, including tyramine, are molecules that have allergenic properties and are often found in many fermented products. The decarboxylation of tyrosine during fermentation or decay in foods can lead to its presence in many food matrices. The amines are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. However, the concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors. In addition, it is now known that peptides containing amino acids precursors of biogenic amines can be used by bacteria to produce these biogenic amines. For example, tyramine can be produced from peptides containing tyrosine and free tyrosine is not the only precursor for tyramine production. Tyramine, together with histamine, if present in wine can cause headaches and migranes. The lactobacillus plantarum IR BL0076 strain isolated from wines of the Rhône Valley during aging is known to produce tyramine. Some enological practices, the art of wine making, which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Furthermore, tyramine is found in many plants and animals. The molecule is metabolized by the enzyme monoamine oxidase. Tyramine is also present in ergot, mistletoe and putrefied animal matter. Foods that may contain tyramine include ripe cheeses, beers, red wines, meats that are potentially spoiled or pickled, aged, smoked, fermented, or marinated, pork, chocolate, alcoholic beverages, and many fermented foods, such as cheeses, sour cream, yogurt, shrimp paste, soy sauce, and many others. In general tyramine is produced in foods from the natural breakdown of the amino acid tyrosine or tyrosine containing peptides but is not added to foods. Tyramine levels can increase in aging foods, or foods that are fermented or stored for long periods of time. Some fermented foods, and especially cheese, are within the food products more often related with biogenic amines poisoning. During the last century, a close relation between migraine crisis and ingestion of tyramine-rich food, especially cheese, was observed and these effects of tyramine consumption were coined as cheese-reaction.

    Tyramine plasma levels, together with plasma tyrosine and methionine levels are significantly elevated in individuals with type II tyrosinemia. This is caused by a deficiency of the enzyme tyrosine aminotransferase. The disorder can affect the eyes, skin, and mental development.

    References

    Maryse Bonnin-Jusserand, Cosette Grandvalet, Aurélie Rieu, Stéphanie Weidmann and Hervé Alexandre; Tyrosine-containing peptides are precursors of tyramine produced by Lactobacillus plantarum strain IR BL0076 isolated from wine. BMC Microbiology 2012, 12:199.

    JEAN-CLAUDE DAVID, WALLACE DAIRMAN, AND SIDNEY UDENFRIEND; Decarboxylation to Tyramine: A Major Route of Tyrosine Metabolism in Mammals. Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 1771-1775, May 1974.

    Daniel M. Linares, Beatriz del Río,Victor Ladero, Noelia Martínez, María Fernández, María Cruz Martín and Miguel A. Álvarez; Factors influencing biogenic amines accumulation in dairy products. Frontiers in Microbiology. 2012, Volume 3, Article 180, 1-10.

    http://www.biomedcentral.com/1471-2180/12/199.

    http://www.wormatlas.org/neurotransmitterstable.htm


    0 0

    Tyramide Signal Amplification (TSA)

    By Klaus D. Linse

    Tyramide Signal Amplification is a novel technique using a biotinylated tyramine to detect specific proteins or nucleic acid sequences in-situ, where tyramide, a phenolic compound, has the ability to bind to the electron rich surface of targets.  Signal amplification is the use of specific detection methodologies to directly increase the signal in proportion to the amount of target in a reaction by simultaneously minimizing the possibility of contamination by target amplification products. In general, a reporter group or enzyme is used for this type of reaction. For example, during gene amplification the number of gene copies is increased in cells. One powerful technique used in molecular biology is the polymer chain reaction (PCR) that amplifies target DNA allowing the production of increased quantities of DNA. Some commonly used signal amplification technologies include branched DNA (bDNA) and hybrid capture (HC) assays. Signal amplification is a common method used nowadays to amplify the target signal in in-situ hybridization assays, such as CARD-FISH, with the help of haptens. A hapten can be any small molecule that, when combined with a larger carrier such as a protein, induces the production of antibodies that specifically bind to it either in the free form or connected to carrier molecule.

    The general principle of the CARD-FISH or TSA method is illustrated in Figure 1.

     CARD-FISHCARD-FISH

    Figure 1:  Principle of CARD-FISH or catalyzed reported deposition-fluorescence in-situ hybridization.  

    Legend:
    H = hapten = biotin, digoxigenin, dinitrophenyl, fluorochrome-labled probe
    HRP = horseradish peroxidase; hapten H = fluorochrome = F; B = biotin; stars = flurochrome or enzyme. 
     

    1.         Hybridization in-situ involves the use of a hapten such as biotin, dioxigenin, dinitrophenyl, and or a fluorochrom-labeled probe.

    2.         The use of a probe covalently conjugated to horseradish peroxidase (HRP) such as an anti-hapten antibody or oligonucleotide.

    3.         CARD signal amplification is achieved with hapten-labeled tyramine or a probe labeled with a single flourochrome that enables the use of radicalization of multiple tyramide molecules and hydrogen peroxide.

    4.         Deposition of tyramide radicals to tyrosine moieties of proteins in-situ in the vicinity of the site of synthesis.

    5.         Direct visualization of flouorochrome-labled tyramines.

    6.         Indirect visualization of biotin-labeled or hapten-labeled tyramides with strepavidin, avidin or anti-hapten antibody conjugates labled with fluorochromes. In addition, the use of enzyme precipitation reactions allows the visualization of enzyme activities.


    What is TSA or CARD?

    Tyramide Signal Amplification, or TSA, sometimes also called Catalyzed Reporter Deposition, or CARD, or tyramine amplification technique (TAT), is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) for the generation of a high-density labeled target protein or nucleic acid sequence in-situ. Signal amplification in TSA is enabled via binding of biotinylated tyramine to proteins near the site of peroxidase-labeled antibodies. This technique enables reliable detection and quantitation of proteins as well as nucleic acids. In comparison to conventional standard avidin-biotinylated enzyme complex (ABC) based assays, TSA has been reported to increase detection sensitivity up to 100-fold. In addition, TSA can be combined with several other technologies such as nucleic acid labeling, primary and secondary antibodies, avidin and lectin conjugates, cytoskeletal stains, organelle probes and cell tracers based detection techniques. TSA can be used to improve current immunohistochemistry, (IHC), immunofluorescence , (IF), or in-situ hybridization, (ISH), based protocols using existing imaging hardware, for example microscopes.

    This amplification method is based on the characteristic ability of tyramine to become chemically sticky after oxidative radicalization. In TSA, HRP reacts with hydrogen peroxide and the phenolic part of tyramine to produce quinone-like structures that carry a radical on the C2 group. But first, the targeted epitope is detected with HRP with the help of specific antibodies. The incubation of the labeled tissue with biotinylated tyramine and hydrogen peroxide (H2O2) results in a peroxide enzyme catalyzed reaction that adds radicals to tyramine. Peroxidases catalyze dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. Enols are alkenes containing a hydroxyl group connected to one of the carbon atoms at the double bond. Horseradish peroxidase (HRP) can also catalyze a third type of reaction that results in the production of hydroxyl radicals (·OH) from H2O2 in the presence of O2·-. The radicalized tyramine can now bind covalently to nearby tissue molecules, thereby amplifying the signal. The biotin on the bound tyramine serves as a tracer molecule that can be visualized using standard techniques that use avidin-biotin-enzyme complex formation reactions. The conjugation of tyramine molecules to a hapten or fluorochrome make the indirect and direct fluorescence detection of enzymatically deposited tyramides possible.

     

    To illustrate the HRP reaction the conjugation of fluorescein-tyramide to a protein as catalyzed by peroxides is shown in Figure 2.

    Conjugation of fluorescein-tyramine to a protein

    Figure 2:  Conjugation of fluorescein-tyramine to a protein. The fluorescein-tyramide is covalently linked to a protein tyrosine side chain via peroxidase-mediated formation of the dityrosine adduct.


    Principles of Tyramide Signal Amplification

    TSA labeling is a combination of several elementary chemical and enzymatic processes (See Figure 1).

    • The selected probe is bound to the target using immunoaffinity or hybridization. Secondary detection of the probe is achieved with a HRP-labeled antibody or streptavidin conjugate. Peroxidase conjugates of other targeting proteins such as lectins and receptor ligands may also be suitable for labeling targets as well as endogenous peroxidase activity. In addition, unconjugated HRP can be used as a neuronal tracer often in combination with TSA.

    • Multiple copies of a labeled tyramide derivative are activated with HRP. For this, fluorescent or biotinylated tyramide is used. In addition, other hapten-conjugated tyramides, tyramide-conjugated gold particles, as well as other similar molecules may be used.

    • The covalent coupling of the resulting highly reactive, short-lived tyramide radicals to residues at the phenol moiety of protein tyrosine residues in the vicinity of the HRP-target interaction site in proximity to the target results in signal localization.

     

    Applications of CARD or TSA

    • Fluorescein-labeled tyramine can been used to detect protein oxidation by reactive oxygen species in tissue.

    • Detection and quantification of low abundance analytes such as peptide, proteins, DNA or RNA molecules.

    • Improving the performance of weakly binding primary antibodies.

    • Improving of background by reducing the amount of antibodies or probes needed for the detection.

    • Reduction of scanning times which results in the faster production of images.

    • Mapping of DNA probes of less than 1 kb size.

     

    References

    Rudolf Amann and Bernhard M. Fuchs; Single-cell identification in microbial communities by improved fluorescence in-situ hybridization techniques. Nature Reviews 2008, Volume 6, 339-348.

    L.M. Schriml, H.M. Padilla-Nash, A. Coleman, P. Moen1, W.G. Nash2, J. Menninger, G. Jones, T. Ried and M. Dean; Tyramide Signal Amplification (TSA)-FISH Applied to Mapping PCR-Labeled Probes Less than 1 kb in Size. BioTechniques 27:608-613 (September 1999).

    Ernst J.M. Speel, Anton H.N. Hopman and Paul Komminoth; Amplification Methods to Increase the Sensitivity of In-Situ Hybridization: Play CARD(S). J Histochem Cytochem 1999, 47: 281.

    Reinhard von Wasielewski, Michael Mengel, Suzanne Gignac, Ludwig Wilkens, Martin Werner and Axel Georgii; Tyramine Amplification Technique in Routine ImmunohistochemistryThe Journal of Histochemistry & CytochemistryVolume 45(11): 1455–1459, 1997.


    0 0
  • 07/22/14--00:00: Protein Methylation
  • Protein Methylation

    By Klaus D. Linse

    Proteins are controlled or regulated by a large battery of post-translational modifications (PTMs). Post-translational modification of proteins refers to biochemical events that proteins undergo after translation. Examples of these modification types are methylation, phosphorylation, glycosylation, sulfation, sumoylation, ubiquitinylation, glycosylation, disulfide bonding and others. Post-translational modifications create sites for specific protein-interaction domains. Biochemical research performed during the last decades revealed that in cells protein domains interact in a controlled but dynamic manner and are an important part of regulatory cellular metabolic pathways. Furthermore, post-translational modifications of protein domains define the state of the proteome, a part of cellular organization.

    More recently, the inhibition or modulation of protein-protein interactions (PPIs) has become popular in the drug discovery industry. In contrast to traditional drug discovery approaches, the goal is to use the knowledge gained from the study of protein-protein interactions to find new drug candidates or inhibitors. One example for targeted proteins is the protein kinase family. Another example is the search for drugs that allow the treatment of so called “undruggable” diseases.

    Methylation alters protein domains or peptide motifs

    Proteins can be altered by a diverse set of post-translational modifications. These include the methylation of arginine residues, the methylation, acetylation, ubiquitylation or sumoylation of lysine residues, or prolyl-hydroxylation, and many others. In addition, the presence of multiple post-translational modifications on one protein can have combinatorial effects which can fine tune the regulation of intra-molecular interactions of domain containing proteins.

    Today, protein methylation is of wide interest to the scientific field. This hasn’t been always so. Researchers began to study protein methylation in the 1960s, and by the early 1980s, it was known that lysine, arginine, histidine and dicarboxylic amino acids were post-translationally modified. Highly specific enzymes called methyltransferases are known to be responsible for the selective transfer of a methyl group to a targeted molecule. With the availability of modern molecular biology techniques starting in the mid 1990s, is has now become clear that protein methylation is involved in many important functions, including gene regulation and signal transduction.

    What is protein methylation?

    In chemistry, biochemistry, and biology methylation refers to the addition of methyl groups (-CH3; the addition of 12 mass units or delta mass = 15 dalton) to organic compounds. This reaction type is a specific case of alkylation. The term alkylation refers to the transfer of an alkyl group, such as the isopropyl group, –CH(CH3)2, or the methyl group, -CH3, from one molecule to another. Alkyl groups may be transferred as alkyl groups with a positively charged carbon atom, a free radical, a carbanion or a carbine. In chemistry, some typical methylation reagents are dimethylsulfate, methanol, methyl halides and diazomethane.

    However, it is well established now that many different molecules present in a cell can be methylated. For example, scientists now know that methylation of DNA is epigentically inherited. The methylation of DNA turned out to be an important regulator of gene transcription. The addition of methyl groups to DNA typically occurs at CpG islands or regions. In addition, methylation can also occur in RNA molecules, for example at conserved sequence regions in 18S rRNA. Methylation of cytosine residues in DNA to form 5-methylcytosine has widespread effects on gene expression due to recruiting specific DNA-binding proteins.

    In cellular proteins many protein or peptide motifs that contain lysine residues can be methylated or acetylated. These modified motifs can lead to the recognition by other protein domains such as chromo-domains or bromo-domains. Such domains are found in proteins regulating chromatin structure and gene expression. For example, the flexible N-terminal and C-terminal ends of histones are known to contain lysine modifications important for the coupling of histones to changes in chromatin organization and the epigenetic control of gene expression. Typically, a single chromo- or bromo-domain recognizes a suitable modified lysine residue within a short peptide motif sequence.

    The monomethylation of a lysine side chain is illustrated in Figure 1 and the structure of S-Adenosyl methionine (SAM), the primary methyl donor molecule in cellular metabolism for numerous biochemical reactions is shown in Figure 2.
     

    Lysine monomethylation

    Figure 1: Monomethylation of a lysine on its ε-amino group. Adenosyl methionine (SAM), a molecule composed of adenosine and methionine, is the primary methyl group donor in metabolism. Donation of the methyl group, in this reaction to the ε-amino group of a lysine, transforms SAM into S-adenyl homocysteine (SAH).

     

    SAM          SAM Model

    Figure 2: S-Adenosyl methionine (SAM), the primary methyl donor molecule in cellular metabolism for numerous biochemical reactions. During the methionine cycle, methionine is converted to SAM. After transfering its methyl group to a target molecule, SAM is converted to S-adenosyl homocysteine (SAH), which is then further converted to homocysteine. Homocysteine is either converted back to methionine, or enters the trans-sulfuration pathway to form other sulfur-containing amino acids.

    The study of lysine methylation of histones has been quite fruitful in recent years after histone lysine methyltransferases were discovered. Histone lysine methylation, either activate or repress gene expression depending on the status of the methylated lysine and its position. However, some of the recently discovered lysine methyl transferases target not only histones but other proteins as well. For example, Set9, a SET domain-containing lysine methyltransferase, was initially found to target histone H3 lysine 4 for mono-methylation but was subsequently shown to target a variety of non-histone proteins as well. Some of its targets are transcription factors.

    The SET domain is a 130 to 140 amino acid, evolutionary well conserved sequence motif initially characterized in the Drosophila proteins Su(var)3-9, Enhancer-of-zeste and Trithorax, from which the acronym SET is derived. In addition, the SET domain is found in proteins of diverse functions ranging from yeast to mammals, but also in some bacteria and viruses. Several structures of SET domain proteins have been reported over the past year.  SET domains are folded in a novel way, and adjacent domains are used for both structural stabilization and the completion of their active sites. In addition, the cofactor S-adenosyl-L-methionine and peptide substrates bind on opposite faces of the SET domain. Furthermore, the side chain of the target lysine approaches the transferred methyl group through a narrow channel that passes through the middle of the domain.

    Reference

    Paik WK, Paik DC, Kim S.; Historical review: the field of protein methylation. Trends Biochem Sci. 2007 Mar;32(3):146-52. Epub 2007 Feb 8.

    Bruce T. Seet, Ivan Dikic, Ming-Ming Zhou and Tony Pawson; Reading protein modifications with interaction domains. Nat Rev Mol Cell Biol. 2006 Jul;7(7):473-83.

    http://smart.embl.de/smart/do_annotation.pl?DOMAIN=SM00317

    Xiao B, Wilson JR, Gamblin SJ.; SET domains and histone methylation. Curr Opin Struct Biol. 2003 Dec;13(6):699-705.

    Yang XD, Lamb A, Chen LF; Methylation, a new epigenetic mark for protein stability. Epigenetics. 2009 Oct 1;4(7):429-33. Epub 2009 Oct 10.


    0 0

    Mechanisms of Protein Lysine and Arginine Modifications
    By Klaus D. Linse

    Arginine and lysine are abundant amino acids found in basic proteins such as histones and protamines. Histones belong to the family of basic proteins associated with DNA in the nucleus and protamines are small, nuclear proteins that replace histones late in the haploid phase of spermatogenesis believed to be essential for sperm head condensation and DNA stabilization. Arginine is a dibasic amino acid wheras lysine contains a ε-amino group. Arginine is considered to be a conditionally essential amino acid and lysine is an essential amino acid needed in the human diet. In addition, L-arginine is frequently added to energy beverages and supplements designed to support sexual performance. Protein arginine and lysine residues, particularly in histones, are subject to post-translational modifications. These modifications include acetylation, citrullination, methylation, sumoylation, and ubiquitination. The post-translational addition of methyl groups to the amino-terminal tails of histone proteins have now been known for more than three decades. One of the surprising result of sequencing the whole human genome was the finding how relatively few genes are actually encoded by the human DNA or genome. Biochemical data now suggest that the complexity of cellular processes stem from post-transcriptional mechanisms including RNA splicing and protein post-translational modifications. For example, critical biological cellular processes such as DNA replication, repair, and transcription are regulated by the combinatorial effect of these modifications. Furthermore, enzymes that modify histone arginine and lysine residues appear to be important for normal cellular functions. The study of histones and their modifications indicates that wrongly modified lysine and arginine side chains in histones maybe the cause for a variety of diseases including arthritis, cancer, heart disease, diabetes, and neuro-degenerative disorders such as Parkinson’s disease and Alzheimer’s disease. Furthermore, it has been observed that arginine side chains in proteins can be converted to citrulline by peptidylarginine deiminase (PAD) enzymes. The structures of important epigenetic post-translational modifications observed for arginine and lysine side chains in proteins are shown in figure 1.

    Post-translational modifications for arginine and lysine

    Lysine acetylation

    Figure 1: Important epigenetic post-translational modifications on arginine and lysine protein side chains.


    To fully understand the importance and function of post-translational modifications it is important to understand detailed kinetic and chemical mechanisms of all proteins and enzymes involved. To achieve this, the use of protein domains together with peptide motifs, either in their natural state or modified with functional groups, studied with biophysical methods such as, nuclear magnetic resonance (NMR), mass spectrometry (MS), and/or kinetic isotope exchange experiments, and others, may allow to further elucidate many details of enzymatic mechanisms.

    The basic unit of chromosomes in eukaryotic organisms is the nucleosome composed of double-stranded DNA wrapped around a protein octamer containing two copies of each of the histone proteins H2A, H2B, H3, and H4. These core histone proteins are highly conserved and play a critical role in modulating chromatin structure and DNA accessibility important for replication, repair, and transcription. Intense research of histone proteins has resulted in the mapping and characterization of histone-post-translational modification guided chromatin remodeling. These modifications are now known as epigenetic modifications and define the “histone code”. Histone proteins are known to contain multiple post-translational modifications including methylation, citrullination (deimination), acetylation, phosphorylation, ubiquitination, and sumoylation. The modifications occur within the histone core region as well as on the N-terminal tails protruding from the core region. The combinatorial effect of the combined histone modifications in time and space is important for DNA regulatory processes including replication, repair, and transcription. Progress made in this field of research has resulted in the discovery of key enzymes that catalyze histone lysine acetylation and deacetylation as well as methylation and demethylation. The first specific histone lysine methyltransferases were discovered in 2000 and were found to be S-adenosyl methionine dependent enzymes. Several of the known methyltransferases are quite specific for their targeted lysine within a peptide motif and preferentially add one, two or three methyl groups. Recent research results indicate that methylation of certain core histones is catalyzed by a family of conserved proteins known as the histone methyltransferases (HMTs) and biochemical evidence suggests that the site-specific methylation is associated with various biological processes such as transcriptional regulation or epigenetic silencing via heterochromatin assembly. In addition, after the discovery of demethylation enzymes it has now become clear that lysine methyl groups can be removed or cleaved. Post-translational methylation is therefore now considered as an epigenetic protein marker that can be enzymatically added or written, read and erased. These types of enzymes are often referred to as protein mark writers, readers and erasers.

    The proposed general chemical mechanism of lysine modifications catalyzed by S-adenosyl-L-methionine (AdoMet)-dependent histone lysine methyltransferases is depicted in figure 2.

    AdoMet dependent protein methylation 

    Figure 2: Proposed general chemical mechanism of lysine modifications catalyzed by S-adenosyl-L-methionine (AdoMet)-dependent histone lysine methyltransferases. Apparently SET domain, Dot/KMT4, and protein arginine methyltransferases use a similar mechanism for the transfer of methyl groups from the donor AdoMet.


    S-adenosyl-L-methionine {SAM or AdoMet; S-(5’-deoxy-5’-adenosyl)-L-methionine)} is a condensation product of adenosine and L-methionine involving the replacement of the –OPO3H2 group of adenylic acid by –S+(CH3)-CH2CH2CH(NH3+)CO2 of methionine. The replacing group is a sulfonium compound bearing a methyl group that is transferred in transmethylation reactions, sometimes also called an active methionine.

    The SET domain was first recognized as a conserved sequence in three Drosophila melanogaster proteins. These proteins were a modifier of position-effect variegation, called Suppressor of variegation 3-9 or Su(var)3-9, the Polycomb-group chromatin regulator Enhancer of zeste (E(z)), and the trithorax-group chromatin regulator trithorax (Trx). The domain is approximately 130 amino acids long and was characterized in 1998. Many SET-domain proteins have now been found in all eukaryotic organisms studied.

    The histone H3 lysine 79 methyltransferase DOT1L/KMT4 has been found to promote oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia but its normal function in mammalian gene regulation is poorly understood.

    Protein arginine methyltransferases (PRMTs) are encoded in mammalian genomes and catalyze three types of arginine methylation; monomethylation and two types of dimethylation. Methylation of arginine residues in proteins is an abundant modification assumed to be important for transduction, gene transcription, DNA repair and mRNA splicing, among many others. Transduction refers to any process by which a cell can convert a signal from itself to another cell, or the process by which DNA is transferred from one bacterium to another by a virus. Three types of methy­larginine species have been found to exist: ω‑NG‑monomethylarginine (MMA), ω‑NG,NG‑asymmetric dimethylarginine (ADMA) and ω‑NG,N’G‑symmetric dimethylarginine (SDMA). Furthermore, recent studies have linked these modifications to carcinogenesis and metastasis and studies using newer sequencing technologies have not generally found alterations to the PRTMs. PRTMs are ubiquitously expressed and are involved in important cellular processes that affect cell growth, proliferation and differentiation. The deregulation of these enzymes is thought to cause the pathogenesis of several diseases, possibly including cancer. 

    The demethylation mechanism for the monoamine oxidase demethylase family LSD1/KDM1 is illustrated in figure 3.

     mechanism of demethylase enzyme LSD1/KDM1

    Figure 3: Proposed general chemical mechanism of demethylase enzyme LSD1/KDM1. FAD dependent demethylation, for example of Lys-4 of histone H3, proceeds through the hydrolysis of an iminium ion following a two oxidation of the amine by the flavin moiety. R = ribosyl adenine dinucleotide.

     

    The enzyme lysine (K)-specific demethylase 1A or LSD1 is encoded by the KDM1A gene and is a member of the monoamine oxidase family. This enzyme can demethylate mono- and di-methylated lysines. LSD1 can demethylate histone 3, lysines 4 and 9 (H3K4 and H3K9). However, the precise mechanism is still controversial. Structural studies of LSD1 in complex with histone analog suicide inhibitors allowed Culhane and Cole in 2007 to elucidated part of the catalytic mechanism. Histone 3 peptides were used to create modified peptide analogs called suicide inhibitors that contained propargylamine, aziridine, and cyclopropylamine groups on the epsilon position of lysine 4.

    Yang et al in 2007 reported the crystal structure of human LSD1 with a propargylamine-derivatized H3 peptide covalently tethered to FAD, depicted in figure 4. The analysis of the structure showed that H3 adopts three consecutive gamma-turns, enabling an ideal side chain spacing that places its N terminus into an anionic pocket and positions methyl-Lys4 near FAD for catalysis. Furthermore the scientists could show that the LSD1 active site cannot productively accommodate more than three residues on the N-terminal side of the methyllysine. This fact explained the enzymes H3-K4 specificity. This structure of LSD1-bound H3 may act as a model allowing the design of potent LSD1 inhibitors with therapeutic potential.

     Structural basis of histone demethylation by LSD1

    Figure 4: Structural basis of histone demethylation by LSD1 revealed by suicide inactivation. Yang et al in 2007 solved the crystal structure of human LSD1 with a propargylamine-derivatized H3 peptide covalently tethered to FAD (A). H3 adopts a protein fold of three consecutive gamma-turns. This protein domain enables an ideal side chain spacing that places its N terminus into an anionic pocket and positions methyl-Lys 4 near FAD for catalysis. The location of FAD and the modified H3 peptide is illustrated in B. The LSD1 protein structure was deselected to allow a closer look at the FAD and H3 peptide location. The structural data for 2UXN was downloaded from the NCBI structure database and rendered using Cn3D 4.3 to create the models.


    Iron (II) dependent a-ketoglutarate oxigenases can hydroxylate alkylgroups directly through a hydroxyl radical. This reaction is thought to provide a plausible path to demethylation of quaternary ammonium salts as indicated in figure 5.

    Proposed catalytic mechanism for the iron (II) dependent demethylation of trimethyl-lysine substrates
     

    Figure 5:Proposed catalytic mechanism for the iron (II) dependent demethylation of trimethyl-lysine substrates. The reaction proceeds through an iron (II), α-ketoglutarate, O2 derived hydroxyl radical oxidation of the methyl C-H bond.


    The enzymes called jumonji histone demethylases or JHDMs are conserved in organisms ranging from yeast to humans and are capable of demethylating trimethyl-lysine residues. JHDMs belong to the Fe (II)/oxoglutarate-dependent dioxygenase or hydroxylase superfamily. The proposed mechanism by which these enzymes catalyze the demethylation of trimethylated lysine residues with the help of a quaternary complex containing 2-oxoglutarate, Fe (II), and the conserved residues His-188, Glu-190, and His-276 is illustrated in figure 6. The JMJD2A/KDM4A numbering according to Allis et al. 2007 is used.


    mechanism for JHDM enzymes for the demethylation of tri-methylated lysine residues

    Figure 6:Proposed catalytic mechanism for JHDM enzymes for the demethylation of tri-methylated lysine residues.   In the active site of the enzyme, the conserved residues His-188, Glu-190, and His-276 chelate the Fe (II). The first chemical step is an electron transfer from Fe (II) to molecular oxygen generating a superoxide radical and Fe (III). The hypothetical model assumes that the hydrophobic active site assists to generate high oxidation states. Nucleophilic attack of the activated oxygen at the ketone carbon of 2-oxoglutarate results in an Fe (IV) peroxyhemiketal bicyclic intermediate. Subsequent decarboxylation results in a succinate, carbon dioxide (CO2), and an iron (IV)-oxo intermediate. The intermediate oxidizes the methyl carbon of the methylated lysine producing a hemiaminal intermediate and regereating iron (II). The resulting hemiaminal is thought to spontaneously decompose to demethylated lysine and formaldehyde. However, unlike the LSD1/KDM1 mechanism, this mechanism does not require a lone electron pair on the ε-nitrogen of the methylated lysine substrate.

    In recent years the importance of chromatin modifications in many aspects of cell biology has become apparent and several enzyme families that modify histones have now been identified. The majority of these proteins are conserved throughout evolution. To minimize the confusion in naming these enzymes Allies et al. in 2007 proposed a new nomenclature for all the characterized members of the families of lysine demethylases, acetyltransferases, and lysine methyltransferases.

    The enzymes have been given more generic names that reflect

    (1) the type of enzymatic activity they perform and

    (2) the type of residue they modify. 


    Table 1 contains a list of lysine demethylases now called KDMs (K-demethylases).
     
    Table 1:  K-Demethylases (KDMs; Formerly Lysine Demethylases)(Allis et al. 2007).

    Name
    Human
    D. melanogaster
    S. cerevisiae
    S. pombe
    Substrate
    Specificity
    Function
     
    KDM1
    LSD1/BHC110
    Su(var)3-3
     
    SpLsd1/
    Swm1/
    Saf110
     
    H3K4me1/2,
    H3K9me1/2
     
    Transcription activation
    and repression, heterochromatin
    formation
    KDM2
     
     
    Jhd1
     
    H3K36me1/2
    Transcription elongation
    KDM2A
    JHDM1a/FBXL11
     
     
     
    H3K36me1/2
     
    KDM2B
    JHDM1b/FBXL10
     
     
     
    H3K36me1/2
     
    KDM3A
    JHDM2a
     
     
     
    H3K9me1/2
    Androgen receptor gene activation,
    spermatogenesis
    KDM3B
    JHDM2b
     
     
     
    H3K9me
     
    KDM4
     
     
    Rph1
     
    H3K9/
    K36me2/3
    Transcription elongation
    KDM4A
    JMJD2A/JHDM3A
     
     
     
    H3K9/
    K36me2/3
    Transcription repression,
    genome integrity
    KDM4B
    JMJD2B
     
     
     
    H3K9/
    H3K36me2/3
    Heterochromatin formation
    KDM4C
    JMJD2C/GASC1
     
     
     
    H3K9/
    K36me2/3
    Putative oncogene
    KDM4D
    JMJD2D
     
     
     
    H3K9me2/3
     
    KDM5
     
    Lid
    Jhd2
    Jmj2
    H3K4me2/3
     
    KDM5A
    JARID1A/RBP2
     
     
     
    H3K4me2/3
    Retinoblastoma-interacting
    protein
    KDM5B
    JARID1B/PLU-1
     
     
     
    H3K4me1/2/3
    Transcription repression
    KDM5C
    JARID1C/SMCX
     
     
     
    H3K4me2/3
    X-linked mental retardation
    KDM5D
    JARID1D/SMCY
     
     
     
    H3K4me2/3
    Male-specific antigen
    KDM6A
    UTX
     
     
     
    H3K27me2/3
    Transcription activation
    KDM6B
    JMJD3
     
     
     
    H3K27me2/3
    Transcription activation
     
     
    Histone acetyl-transferases (HATs or KATs) catalyze the transfer of the acetyl moiety from acetyl-coenzyme A (acetyl-CoA or AcCoA) to the ε-amino group of histone lysine residues. The result of this reaction is an acetylated lysine side chain and a free CoA. AcCoA is the sole acetylgroup donor for protein acetylation and its concentration in cells affects the level of protein acetylation. CoA is a coenzyme containing pantothenic acid, adenosine 3’-phosphate 5’-pyrophosphate, and cysteamine and is a key intermediate in the mitochondrial metabolism of pyruvate. 

    HATs are grouped into three main groups or families

    (1) the glucosamine-6-phosphate N-acetyltransferase or Gcn5-related N-acetyltransferase  (GNAT) family (founding member in yeast Gcn5/ScKAT2), and 

    (2) the monocytic leukemia 3, also known as MYST3 (MYST; founding members MOZ, Ybf2/Sas2, and Tip60) family, and 

    (3) the transcriptional coactivators p300 and the cyclic adenosine mono-phosphate response element-binding protein (CREB) histone acetyltransferase (p300/CBP) family.

    The structure of acetyl-coenzyme A is illustrate in figure 7 and the proposed mechanism for class I/II/IV histone acetyltransferases catalyzing the transfer of the methyl group from the acetylgroup donor AcCoA to the lysine residue is depicted in figure 8.

    Acetyl-coenzyme A (AcCoA)

    Figure 7: Acetyl-coenzyme A (AcCoA), an important intermediate in the metabolism of pyruvate, fatty acids and many amino acids. Pyruvate generated in the cytosol during glycolysis is transported across the mitochondrial memebranes to the matrix wher it reacts with HSCoA to form carbon dioxide and acetyl CoA.  The reaction is catalyzed by the enzyme pyruvate dehydrogenase which is a soluble component of the matrix. The mitochondria contain an outer membrane, an inner membrane with extensive cristae, the inter-membrane space, and the matrix with small granules, as was revealed by electron microscopy. The matrix contains the mitochondrial DNA and ribosomes. 

    Proposed chemical mechanism of histone acetyl-transferasesFigure 8: Proposed chemical mechanism of histone acetyl-transferases.


    Histone deacetylases remove the acetyl group form lysine side chains in histone proteins. Class I/II/IV histone deacetylases (HDACs) have homologous active-sites and are thought to proceed through a catalytic mechanism in which the two active-site His-Asp dyads work as a general acid-base catalytic pair. The proposed chemical mechanism is illustrated in figure 9.    

    Proposed chemical mechanism of class I/II/IV histone deacetylases (HDACs)

    Figure 9: Proposed chemical mechanism of class I/II/IV histone deacetylases (HDACs). In this mechanism an active-site metal ion and Tyr-306 (HDAC8 numbering) polarize the carbonyl by coordinating to the acetyl oxygen. The metal appears to be zinc (II) but could be iron (II) as well. The metal ion is coordinated by Asp-178, His-180, and Asp-267. Structural studies suggested that the metal ion also coordinates and activates the water molecule for attack of the acetyl carbonyl carbon. Apparently, both His-142 and His-143 activates the water molecule for nucleophilic attack of the acetyl carbonyl carbon. Subsequently, the tetrahedral intermediate collapses to form acetate and the lysine products.


    Table 1 contains a list of lysine demethylases now called KDMs (K-demethylases).

     
    Table 1:  K-Demethylases (KDMs; Formerly Lysine Demethylases)(Allis et al. 2007).

    Name
    Human
    D. melanogaster
    S. cerevisiae
    S. pombe
    Substrate
    Specificity
    Function
     
    KDM1
    LSD1/BHC110
    Su(var)3-3
     
    SpLsd1/
    Swm1/
    Saf110
     
    H3K4me1/2,
    H3K9me1/2
     
    Transcription activation
    and repression, heterochromatin
    formation
    KDM2
     
     
    Jhd1
     
    H3K36me1/2
    Transcription elongation
    KDM2A
    JHDM1a/FBXL11
     
     
     
    H3K36me1/2
     
    KDM2B
    JHDM1b/FBXL10
     
     
     
    H3K36me1/2
     
    KDM3A
    JHDM2a
     
     
     
    H3K9me1/2
    Androgen receptor gene activation,
    spermatogenesis
    KDM3B
    JHDM2b
     
     
     
    H3K9me
     
    KDM4
     
     
    Rph1
     
    H3K9/
    K36me2/3
    Transcription elongation
    KDM4A
    JMJD2A/JHDM3A
     
     
     
    H3K9/
    K36me2/3
    Transcription repression,
    genome integrity
    KDM4B
    JMJD2B
     
     
     
    H3K9/
    H3K36me2/3
    Heterochromatin formation
    KDM4C
    JMJD2C/GASC1
     
     
     
    H3K9/
    K36me2/3
    Putative oncogene
    KDM4D
    JMJD2D
     
     
     
    H3K9me2/3
     
    KDM5
     
    Lid
    Jhd2
    Jmj2
    H3K4me2/3
     
    KDM5A
    JARID1A/RBP2
     
     
     
    H3K4me2/3
    Retinoblastoma-interacting
    protein
    KDM5B
    JARID1B/PLU-1
     
     
     
    H3K4me1/2/3
    Transcription repression
    KDM5C
    JARID1C/SMCX
     
     
     
    H3K4me2/3
    X-linked mental retardation
    KDM5D
    JARID1D/SMCY
     
     
     
    H3K4me2/3
    Male-specific antigen
    KDM6A
    UTX
     
     
     
    H3K27me2/3
    Transcription activation
    KDM6B
    JMJD3
     
     
     
    H3K27me2/3
    Transcription activation
     

    Histone acetyl-transferases (HATs or KATs) catalyze the transfer of the acetyl moiety from acetyl-coenzyme A (acetyl-CoA or AcCoA) to the ε-amino group of histone lysine residues. The result of this reaction is an acetylated lysine side chain and a free CoA.AcCoA is the sole acetylgroup donor for protein acetylation and its concentration in cells affects the level of protein acetylation. CoA is a coenzyme containing pantothenic acid, adenosine 3’-phosphate 5’-pyrophosphate, and cysteamine and is a key intermediate in the mitochondrial metabolism of pyruvate.

    HATs are grouped into three main groups or families

    (1) the glucosamine-6-phosphate N-acetyltransferase or Gcn5-related N-acetyltransferase  (GNAT) family (founding member in yeast Gcn5/ScKAT2), and

    (2) themonocytic leukemia 3, also known as MYST3 (MYST; founding members MOZ, Ybf2/Sas2, and Tip60) family, and

    (3) the transcriptional coactivators p300 and the cyclic adenosine mono-phosphate response element-binding protein (CREB) histone acetyltransferase (p300/CBP) family.

     

    Table 2 contains a list of lysine acetyl-transferases now called KATs (K-acetyl-transferases).
     
    Table 2 :  K-Acetyl transferases(KATs; Formerly Lysine Acetyl-transferases) (Allis et al. 2007).

     
    Name
    Human
    D. melanogaster
    S. cerevisiae
    S. pombe
    Substrate
    Specificity
    Function
     
     KAT1
    HAT1
    CG2051
    Hat1
    Hat1/
    Hag603
    H4 (5, 12)
    Histone deposition, DNA
    repair
    KAT2
     
    dGCN5/PCAF
    Gcn5
    Gcn5
    H3 (9, 14, 18, 23, 36)/
    H2B; yHtzl (14)
    Transcription activation,
    DNA repair
    KAT2A
    hGCN5
     
     
     
    H3 (9, 14, 18)/H2B
    Transcription activation
    KAT2B
    PCAF
     
     
     
    H3 (9, 14, 18)/H2B
    Transcription activation
    KAT3
     
    dCBP/NEJ
     
     
    H4 (5, 8); H3 (14, 18)
    Transcription activation,
    DNA repair
    KAT3A
    CBP
     
     
     
    H2A (5); H2B (12, 15)
    Transcription activation
     
    KAT3B
    P300
     
     
     
    H2A (5); H2B (12, 15)
    Transcription activation
     
    KAT4
    TAF1
    dTAF1
    Taf1
    Taf1
    H3 > H4
    Transcription activation
     
    KAT5
    TIP60/PLIP
    dTIP60
    Esa1
    Mst1
    H4 (5, 8, 12, 16); H2A
    (yeast 4, 7; chicken 5, 9, 13, 15); dH2Av/yHtzl
    (14)
    Transcription activation,
    DNA repair
    KAT6
     
    (CG1894)
    Sas3
    (Mst2)
    H3 (14, 23)
    Transcription activation
    and elongation, DNA replication
    KAT6A
    MOZ/MYST3
    ENOK
     
     
    H3 (14)
    Transcription activation
    KAT6B
    MORF/MYST4
     
     
     
    H3 (14)
    Transcription activation
    KAT7
    HBO1/MYST2
    CHM
     
    (Mst2)
    H4 (5, 8, 12) > H3
    Transcription, DNA replication
    KAT8
    HMOF/MYST1
    dMOF (CG1894)
    Sas2
    (Mst2)
    H4 (16)
    Chromatin boundaries,
    dosage compensation,
    DNA repair
    KAT9
    ELP3
    dELP3/
    CG15433
    Elp3
    Elp3
    H3
     
    KAT10
     
     
    Hap2
     
    H3 (14); H4
     
    KAT11
     
     
    Rtt109
     
    H3 (56)
    Genome stability,
    Transcription, elongation
    KAT12
    TFIIIC90
     
     
     
    H3 (9, 14, 18)
    Pol III transcription
    KAT13A
    SRC1
     
     
     
    H3/H4
    Transcription activation
    KAT13B
    ACTR
     
     
     
    H3/H4
    Transcription activation
    KAT13C
    P160
     
     
     
    H3/H4
    Transcription activation
    KAT13D
    CLOCK
     
     
     
    H3/H4
    Transcription activation
     
     
    Table 3 contains a list of lysine methyl-transferases now called KMTs (K-methyl-transferases).
     
    Table 3 :  K-methyl transferases (KMTs; Formerly Lysine Methyl-transferases) (Allis et al. 2007).

     

    Name

    Human

    D. melanogaster

    S. cerevisiae

    S. pombe

    Substrate

    Specificity

    Function

     

    KMT1

     

    Su(Var)3-9

     

    Clr4

    H3K9

    Heterochromatin formation/silencing

    KMT1A

    SUV39H1

     

     

     

    H3K9

    Heterochromatin formation/silencing

    KMT1B

    SUV39H2

     

     

     

    H3K9

    Heterochromatin formation/silencing

    KMT1C

    G9a

     

     

     

    H3K9

    Heterochromatin formation/silencing

    KMT1D

    EuHMTase/GLP

     

     

     

    H3K9

    Heterochromatin formation/silencing

    KMT1E

    ESET/SETDB1

     

     

     

    H3K9

    Transcription repression

    KMT1F

    CLL8

     

     

     

     

     

    KMT2

     

     

    Set1

    Set1

    H3K4

    Transcription activation

    KMT2A

    MLL1

    Trx

     

     

    H3K4

    Transcription activation

    KMT2B

    MLL2

    Trx

     

     

    H3K4

    Transcription activation

    KMT2C

    MLL3

    Trr

     

     

    H3K4

    Transcription activation

    KMT2D

    MLL4

    Trr

     

     

    H3K4

    Transcription activation

    KMT2E

    MLL5

     

     

     

    H3K4

    Transcription activation

    KMT2F

    hSET1A

     

     

     

    H3K4

    Transcription activation

    KMT2G

    hSET1B

     

     

     

    H3K4

    Transcription activation

    KMT2H

    ASH1

    Ash1

     

     

    H3K4

    Transcription activation

    KMT3

     

     

    Set2

    Set2

    H3K36

    Transcription activation

    KMT3A

    SET2

     

     

     

    H3K36

    Transcription activation

    KMT3B

    NSD1

     

     

     

    H3K36

     

    KMT3C

    SYMD2

     

     

     

    H3K36 (p53)

    Transcription activation

    KMT4

    DOT1L

     

    Dot1

     

    H3K79

    Transcription activation

    KMT5

     

     

     

    Set9

    H4K20

    DNA-damage response

    KMT5A

    Pr-SET7/8

    PR-set7

     

     

    H4K20

    Transcription repression

    KMT5B

    SUV4-20H1

    Suv4-20

     

     

    H4K20

    DNA-damage response

    KMT5C

    SUV4-20H2

     

     

     

     

     

    KMT6

    EZH2

    E(Z)

     

     

    H3K27

    Polycomb silencing

    KMT7

    SET7/9

     

     

     

    H3K4 (p53 and TAF10)

     

    KMT8

    RIZ1

     

     

     

    H3K9

    Transcription repression

     

     


    Reference

    Allis CD, Berger SL, Cote J, Dent S, Jenuwien T, Kouzarides T, Pillus L, Reinberg D, Shi Y, Shiekhattar R, Shilatifard A, Workman J, Zhang Y.; Newnomenclature for chromatin-modifyingenzymes.Cell. 2007 Nov 16;131(4):633-6.

    Jeffrey C. Culhane and Philip A. Cole; LSD1 and The Chemistry of Histone Demethylation. Curr Opin Chem Biol. Oct 2007; 11(5): 561–568. Published online Sep 11, 2007. doi:  10.1016/j.cbpa.2007.07.014, PMCID: PMC2112775. NIHMSID: NIHMS33479.

    Darnel, J., Lodish, H., Baltimore D.; Molecular Cell Biology. 2nd edition. Scientific American Books. W.H. Freeman and Company, New York, 1990.

    Shane C Dillon,Xing Zhang, Raymond C Trievel,and Xiaodong Cheng; The SET-domain protein superfamily: protein lysine methyltransferases Genome Biol. 2005; 6(8): 227.

    Fred Dyda, David C Klein, and Alison B Hickman; GCN5-Related N-Acetyltransferases: A Structural Overview. http://www.ncbi.nlm.nih.gov/books/NBK2231/

    Rice JC, Allis CD; Histone methylation versus histone acetylation: new insights into epigenetic regulation. Curr Opin Cell Biol. 2001 June; 13(3):263-73.



    Sabrina Schroeder, Tobias Pendl, Andreas Zimmermann, Tobias Eisenberg, Didac Carmona-Gutierrez, Christoph Ruckenstuhl, Guillermo Mariño, Federico Pietrocola, Alexandra Harger, Christoph Magnes, Frank Sinner, Thomas R Pieber, Jörn Dengjel, Stephan J Sigrist, Guido Kroemer, Frank Madeo;
    Acetyl-coenzyme A: A metabolic master regulator of autophagy and longevity. Autophagy 10:7, 1335–1337; July 2014; © 2014 Landes Bioscience.



    Brian C. Smith and John M. Denu;Chemical mechanisms of histone lysine and arginine modifications.
    Biochim Biophys Acta. Jan 2009; 1789(1): 45–57.



    David J. Steger, Martina I. Lefterova,Lei Ying,Aaron J. Stonestrom,Michael Schupp,David Zhuo,Adam L. Vakoc,Ja-Eun Kim,Junjie Chen,Mitchell A. Lazar,Gerd A. Blobel, and Christopher R. Vakoc; DOT1L/KMT4 Recruitment and H3K79 Methylation Are Ubiquitously Coupled with Gene Transcription in Mammalian Cells. Mol Cell Biol. Apr 2008; 28(8): 2825–2839.



    Lawrence M. Szewczuk, Jeffrey C. Culhane, Maojun Yang,
    Ananya Majumdar,Hongtao Yu, and Philip A. Cole; Mechanistic Analysis of a Suicide Inactivator of Histone Demethylase LSD1. Biochemistry. Jun 12, 2007; 46(23): 6892–6902.

    Yang M, Culhane JC, Szewczuk LM, Gocke CB, Brautigam CA, Tomchick DR, Machius M, Cole PA, Yu H; Structural Basis of Histone Demethylation by Lsd1 Revealed by Suicide Inactivation. Nat.Struct.Mol.Biol. (2007) 14 p.535.

    Yanzhong Yang& Mark T. Bedford; Protein arginine methyltransferases and cancer. Nature Reviews Cancer13, 37-50(January 2013).


     


    0 0
  • 07/24/14--00:00: Quantification of dsRNA
    1. Gel electrophoresis: Run total of 0.5 µl of the unpurified and 1 µl of the purified dsRNA in 10 µl total volume on a 1% agarose gel. Run 2 µl low mass ladder on a side. Confirm yield and size of single band product.
    2. Absorbance measurement at 260 and 280 nm: Concentration can be measured using absorbance at 260 nm because it is well established that a solution of RNA with an optical density of 1.0 (1.0 Absorbance Unit) has a concentration of 50 µg/ml in a 10 mm pathlength cell*. A wavelength of 320 nm is used to compensate for the effects of background absorbance due to, for example, turbidity or high absorbance buffer solution.
    Concentration = (A260/A320) x 50 x dilution factor, µg/ml

    Absorbance ratio can be used to establish the presence of impurities in a sample preparation, relative to a pure sample. The two wavelength of interest to the Molecular Biologist are the absorbance maxima of the nucleic acid, 260 nm, and the protein impurity, 280 nm.

    Absorbance ratio = (A260-A320)/ (A280-A320)

    The absorbance ratio 1.7 is known for pure nucleotide, enabling rapid assessment of quality. An absorbance ratio of the two wavelengths below the expected 1.7 for the pure substance indicates the presence of impurity in the sample.

    To determine concentration and purity of dsRNA, follow simple procedure:
    1. Fill microvolume cell with water. Set absorbance at 320 nm to zero. This is your background reading.
    2. Add 2 µl dsRNA to 78 µl water in microvolume cell. Mix by pipetting.
    3. Measure absorbance at 260, 280 and 320 nm.
    4. Use formulas for the concentration and for the absorbance ratio to determine concentration and purity of dsRNA.

    *Reference: Molecular Cloning, Maniatis et al.



    0 0
  • 07/25/14--00:00: BNA based FISH Probes
  • BNA based FISH probes

     
    Recent advancements in the design and development of molecular probes and image analysis has made fluorescence in situ hybridization (FISH) a powerful tool. Although being a useful technique FISH is a fairly time-consuming procedure with limitations in sensitivity. Probes that exhibit higher DNA affinities promise to potentially improve the sensitivity of the technique. Artificial nucleotides such as bridged nucleic acids (LNAs) have been described for the development of chimeric LNA/DNA oligonucleotides as probes for fluorescence in situ hybridization on metaphase chromosomes and interphase nuclei.
     
    Bridged nucleic acids (BNA3) are artificial bicyclic oligonucleotides that contain a five-membered or six-membered bridged structure with a “fixed” C3’-endo sugar puckering (Saenger 1984). The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoamidite chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability. Oligonucleotide modifications are characterized by the presence of one or more bicyclic ribose analogs. The structural similarity to native nucleic acids and the presence of a nitrogen atom within the bicyclic ring leads to very good solubility in water and allows for easy handling of synthetic primers and probes. In contrast to peptide nucleic acids (PNAs) and minor groove binders (MGBs), but similar to LNAs, BNA monomers can be used for both primers and probes in real time quantitative polymerase chain reaction (RT-Q-PCR) assays. Compared to locked nucleic acids (LNAs) the substitution of DNA monomers with BNA monomers in oligonucleotides adds exceptional biological stability, resistance to nucleases and a significantly increased affinity to their complementary DNA targets.
     
    Silahtaroglu et al. in 2003 reported results for a study where LNA substituted oligonucleotides of either the 23-bp human satellite-2 repeat sequence (ATT CCA TTC GAT TCC ATT CGA TC) or the 24-bp sequence composed of four blocks of the 6-bptelomere repeat (TTAGGG) have been used. The different LNA designs of the chimeric LNA/DNA probes for the human satellite–2 repeat used here are listed in table I. The desigin of a BNA based FISH probe is shown in table II.
     

    Table I. The LNA/DNA mixmer FISH probes for human satellite-2 repeat sequence used in the study*.

    Name

    LNA/DNA mixmers

    LNA monomers

    DNA oligo

    attccattcgattccattcgatc

    0

    Dispersed LNA

    aTtccatTcgaTtccAttcgaTc

    5

    LNA-3

    aTtcCatTcgAtTccAttCgaTc

    8

    LNA Blocks

    aTTCcattcgATTccattcGATc

    9

    LNA-2

    ATtCcAtTcaGaTtCcAtTcGaTc

    11

     

    *LNA substitutions are depicted in capital letters

    Table II. The BNA/DNA mixmer FISH probes for human satellite-2 repeat sequence#.

    BNA  probe

    ATtCcAtTcaGaTtCcAtTcGaTc

    11

    #BNA substitutions are depicted in capital letters.


    For the telomere specific LNA probe, only the LNA-2 design, with an LNA substitution at every second nucleotide position, was synthesized together with a DNA control. Oligonucleotide FISH probes with different LNA substitution patterns, labels and hybridization conditions were used in a comparative study and subsequently the optimal conditions were determined for an efficient LNA-FISH protocol. All LNA-containing oligonucleotides for human satellite-2 and the telomere repeats gave prominent signals when used as FISH probes. For the human satellite-2 sequence, the LNA-2 design gave the best hybridisation results in the experiments performed. The LNA-3 probe, with every third oligonucleotide substituted with LNA, also gave hybridisation signals, albeit weaker than those obtained with the LNA-2 probes.

    Repetitive elements comprise ~45% of the human genome and consist of interspersed repeats derived from non-autonomous or autonomous transposable elements and tandem repeats of simple sequences (satellite DNA) or complex sequences. The most abundant short interspersed nucleotide element (SINE) in human DNA is the Alu repeat, an ~282 bp non-LTR (Long Terminal Repeat) DNA sequence, which comprises 10% of the human genome and is present in ~1 million copies per haploid genome. Other abundant non-LTR sequences are long interspersed nucleotide elements (LINEs) of up to 6 kb that comprise ~20% of the human genome. LINE-1 elements are present at over 500 000 copies in the human genome. Only 3000–4000 are full length and 30–100 are active retrotransposons.

    LINE-1 elements are usually methylated in somatic tissues, and LINE-1 hypomethylation is a common characteristic of human cancers. Alu sequences are also normally methylated in somatic tissues and are thought to become hypomethylated in human cancer cells. Not all Alus are hypomethylated in human cancers. Alu sequences located upstream of the CDKN2A promoter were found to be hypermethylated in cancer cell lines, and an Alu sequence located in intron 6 of TP53 showed extensive methylation in normal and cancer cells. LINEs and SINEs are interspersed throughout the genome, whereas satellite DNA is largely confined to the centromeres or centromere-adjacent (juxtacentromeric) heterochromatin and to the large region of heterochromatin on the long arm of the Y chromosome. Satellite a (Sata) repeats are composed of 170 bp DNA sequences and represent the main DNA component of every human centromere. Satellite 2 (Sat2) DNA sequences are found predominantly in juxtacentromeric heterochromatin of certain human chromosomes and are most abundant in the long juxtacentromeric heterochromatin region of chromosome (Chr) 1. Sat2 sequences are composed of variants of two tandem repeats of ATTCCATTCG followed by one or two copies of ATG. Both Chr1 Sata and Chr1 Sat2 sequences, as well as Sata repeats present throughout all the centromeres, are highly methylated in normal postnatal tissues, hypomethylated in sperm and often hypomethylated in various cancers (26–29). In addition, Sat2 sequences on Chr1 and Chr16 are also hypomethylated in the ICF (immunodeficiency, centromeric region instability and facial abnormalities) syndrome, which usually involves mutations in DNMT3B.

    Probe design and preparation


    The 23-bp human satellite-2 repeat sequence, attccattcgattccattcgatc, or a 24-bp telomere sequence (ttagggttagggttagggttaggg) representing 4 blocks of 6-bp telomere repeat (ttaggg) are used for the BNA/DNA mixmers with different BNA substitution patterns (Table I). All mixmers oligonucleotides are both synthesized either with a Cy3 or a biotin group at the 5’ end. An unsubstituted DNA oligonucleotide probe is used as a control in each experiment. All the oligonucleotide probes are kept frozen until used in aliquots of distilled water.

    Fluorescence in situ hybridization


    FISH is carried out as described previously (Silahtaroglu et al., 1998) with the following modifications. The amount of probe is 6.4, 10, 13.4 and 20 pmoles. Denaturation of the target DNA and the probe are performed at 75oC for 5 minutes either separately using 70% formamide or simultaneously under the coverslip in the presence of hybridization mixture containing 50% formamide. In addition, the effect of denaturation is also tested. Two alternative hybridization mixtures are used: 50% formamide/2xSSC (pH 7.0) /10% dextran sulphate or 2xSSC (pH 7.0) /10% dextran sulphate. Hybridization times include 30 min, 1 hr, 2 hrs, 3 hrs and overnight. Hybridization temperatures include: 37°C, 55°C, 60°C and 72°C. Post-washing is either as for standard FISH, or with 50% formamide/2xSSC at 60°C, or without formamide. Hybridization signals with biotin labeled LNA/DNA mixmers are visualized indirectly using two layers of fluorescein labeled avidin (Vector Laboratories, USA) linked by a biotinylated anti-avidin molecule, which amplifies the signal 8-64 times. The hybridization of Cy3-labeled molecules is visualized directly after a short washing procedure. Slides are mounted in Vectashield (Vector Laboratories, USA) containing 4´-6´-diamidino-2-phenylindole (DAPI). The whole procedure is carried out in the dark. The signals are visualized using a Leica DMRB epifluorescence microscope equipped with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ, USA), and IPLAB Spectrum Quips FISH software (Applied Imaging international Ltd., Newcastle, UK) within two days after hybridization. 20 metaphases are analyzed after each hybridization experiment.

    Repetitive element in the human genome




    Description, primers and probes of repetitive elements  


    Primers for repeptitive elements
    (Source: Weisenberger et al. 2005)
     

    MethyLight


    MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR method to detect and quantify DNA methylation in genomic DNA with high sensitivity. The technique relies on methylation-specific priming combined with methylation-specific fluorescent probing. The combination of the two methylation-specific detection principles results in a highly methylation-specific detection technology. It allows to detect very low frequencies of hypermethylated alleles. The high sensitivity and specificity of MethyLight make it uniquely well suited for detection of low-frequency DNA methylation biomarkers. DNA and RNA based biomarker such as cell-free nucleic acids, mRNA and microRNA releases into the blood stream of cancer patients are increasingly used as target molecules to detect different tumor types or diseases. The quantitative accuracy of real-time PCR and the flexibility to design bisulfite-dependent, methylation-independent control reactions allows for a quantitative assessment of low-frequency methylation events.


    References

    Jeanpierre, M. (1994). Human satellites 2 and 3. Annals of Genetics 37, 163-171.

    Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu JR. (1988) A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci U S A. 85(18):6622-6626.

    Sanger Wolfram; Principles of Nucleic Acid Structure. Springer-Verlag, New York, Berlin, Heidelberg, London, Paris, 1984.

    Silahtaroglu, A.N., Hacihanefioglu, S., Guven, G.S., Cenani, A., Wirth, J., Tommerup, N., Tumer, Z.(1998) Not para-, not peri-, but centric inversion of chromosome 12. Journal of Medical Genetics 35(8), 682-684.

    Silahtaroglu, A.N., Tommerup, N., Vissing, H. (2003) FISHing with Locked Nucleic Acids (LNA): Evaluation of different LNA/DNA Mixmers. Molecular and Cellular Probes 17(4), 165-169.

    Daniel J. Weisenberger, Mihaela Campan, Tiffany I. Long, Myungjin Kim, Christian Woods, 

    Emerich Fiala, Melanie Ehrlich and Peter W. Laird; Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Research, 2005, Vol. 33, No. 21 6823–6836. doi:10.1093/nar/gki987.



older | 1 | .... | 3 | 4 | (Page 5) | 6 | 7 | .... | 16 | newer