8-Bromo-2'-deoxyadenosine, 8-Br-dA, Mw  330.14 g/mol , CAS 14985-44-5, (2R,3S,5R)-5-(6-Amino-8-bromo-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol.

The organobromine compound 8-bromo-2’-deoxyadenosine is a modified 2’-deoxyadenosine having a bromo substituent at position 8 of the adenosine ring system and a member of adenosines.

Similar to 8-Br-dG, 8-Br-dA is a useful molecule for crystallography based structural studies of oligonucleotides. Brominated as well as iodinated nucleosides are photolabile. This feature makes them ideal candidates for cross-linking studies as well.

The photo-active 8-Br-dA can be incorporated into oligonucleotides as a phosphoramidite during automated oligonucleotide synthesis. Photolytic cleavage of the C–Br bond leads to formation of the C8 radical. In aqueous conditions, the major reaction path generates a C5′ radical. The C5′ radical undergoes a cyclization reaction on the adenine, resulting in an aminyl radical with a rate constant of 1.8 × 105 s⁻¹. Steady-state photolysis studies in acetonitrile showed the conversion of 8-bromo-2′-deoxyadenosine to 5′,8-cyclo-2′-deoxyadenosine with a 65% yield and a diastereoisomeric ratio (5′R) ∶ (5′S) = 1.7.

8-bromoadenine present in DNA or RNA oligonucleotides can react with thiols under physiological conditions forming adducts. This reaction can be utilized to generate specific thiol modified oligonucleotides.

When tissues are inflamed, cellular DNA is damaged by hypobromous acid (Br-OH). Br-OH is generated by myeloperoxidase and eosinophil peroxidase. 

Reference

JiaLiu, Gregory LVerdin∗;  Synthesis of photoactive DNA: incorporation of 8-bromo-2′-deoxyadenosine into synthetic oligodeoxynucleotides. Tetrahedron Letters. Volume 33, Issue 30, 21 July 1992, Pages 4265-4268. [link] 

Liliana B. Jimenez,  Susana Encinas,  Miguel A. Miranda,  Maria Luisa Navacchia and Chryssostomos Chatgilialoglu; The photochemistry of 8-bromo-2′-deoxyadenosine. A direct entry to cyclopurine lesions. Photochemical & Photobiological Sciences. Issue 11-12, 2004  Photochem. Photobiol. Sci., 2004,3, 1042-1046. [link]

Mayeno, AN; Curran, AJ; Roberts, RL; Foote, CS (1989). "Eosinophils preferentially use bromide to generate halogenating agents". The Journal of Biological Chemistry. 264 (10): 5660–8. PMID 2538427. Retrieved 2008-01-12. [jbc] 

Sassa A, Ohta T, Nohmi T, Honma M, Yasui M.; Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases.  J Mol Biol. 2011 Mar 11;406(5):679-86. doi: 10.1016/j.jmb.2011.01.005. Epub 2011 Jan 15. [Pubmed]

The new coronavirus 2019-nCoV recently identified in patients with acute respiratory disease is genetically similar to SARS coronavirus and bat SARS-like coronavirus. Accurate molecular tests are needed for early detection and identification of infected patients. For this purpose, Chu et al. recently designed two 1-step quantitative real-time reverse-transcription PCR assays for the detection of regions ORF1B and N of the viral genome.

Real-time RT-PCR assays targeting the ORF1b and N gene regions of 2019-nCoV were designed based on the first publicly available sequence in Genbank (Accession number:  MN908947). Regions (ORF1b and N) that are highly conserved amongst sarbecoviruses were selected for primer and probe designs.

Primers and Probes

Typical RT-PCR Condition

Extraction

For RNA and DNA extractions use a viral RNA purification kit (e,g. QIAamp Viral RNA Mini Kit, Qiagen) and a DNA plasmid purification kit (e.g. QIAprep Spin Miniprep Kit, Qiagen). Follow the instructed of the manual from the manufacturer. Typical volumes are: RNA extractions, use ~140 µL of sample and eluted in 60 µL elution buffer containing poly(A) carrier RNA.

PCR

For a total volume of 20 μl reaction use 4 μl of 4X master reaction mixture (e.g. TagMan Fast Virus 1-Step Master Mix, ThermoFisher), 0.5 nmol/ml of forward primer, 0.5 nmol/ml of reverse primer, 0.24 nmol/ml of the probe, and 4 μl of RNA sample.

Use a RT-PCR system for PCR reactions as follows: Reverse transcription at 50 °C for 5 minutes, inactivation at 95 °C for 20 seconds, 30 to 40 cycles of PCR amplification (Denaturing at 95 °C for 5 seconds; Annealing/extension at 60 °C for 30 seconds). The duration of a RT-PCR usually is in the range of 1 hour 15 minutes to 2 hours.

Reference

Daniel K W Chu, Yang Pan, Samuel M S Cheng, Kenrie P Y Hui, Pavithra Krishnan, Yingzhi Liu, Daisy Y M Ng, Carrie K C Wan, Peng Yang, Quanyi Wang, Malik Peiris, Leo L M Poon, Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia, Clinical Chemistry, , hvaa029, https://doi.org/10.1093/clinchem/hvaa029

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RNA molecules, including siRNA, microRNA, and mRNA, enable immunomodulation and cancer immunotherapy. Both the innate and the adaptive immune system may respond to challenges from these molucles by silencing or upregulating immune-relevant genes.

Specifically designed nanomaterials enhance the delivery of RNA molecules to tumors and immune cells. RNA conjugates allowing the delivery to specific cellular targets, are crucial for the development of molecular diagnostic tests and targeted therapeutics.

RNA therapeutics are appealing for the treatment and prevention of a disease, such as cancer, a genetic disorder, diabetes, inflammation, or neurodegenerative diseases. However, RNA molecules by themselves are quite labile and are difficult to deliver through the various physiological barriers in the human body. For example, a human’s intrinsic defense system includes various exonucleases and RNases responsible for the degradation of RNA, organs, or tissues, as well as the innate immunes system for RNA clearance, making the delivery of RNA molecules challenging. Delivered RNAs may function by silencing of immune checkpoint genes, activating the innate or adaptive immune system by regulating cytokines expressions and acting as tumor antigen vaccines.

New RNA based approaches promise the development of nanoparticle-based platforms. These include liposomes, polymeric nanoparticles (NPs), and inorganic NPs. The hope is that these platforms will allow efficient delivery of RNAs to specific targets in cells or tissues.

Biosynthesis Inc. offers automated custom synthesis for un-modified and modified short, median length, and long DNA and RNA oligos.

Reference

Lin YX, Wang Y, Blake S, Yu M, Mei L, Wang H, Shi J. RNA Nanotechnology-Mediated Cancer Immunotherapy. Theranostics 2020; 10(1):281-299. doi:10.7150/thno.35568. Available from http://www.thno.org/v10p0281.htm


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siRNA

siRNA molecules are approximately twenty-two (22) nucleotides in length and are double-stranded. Initially, siRNA precursors are recognized by Dicer RNase and incorporated into the RNA-induced silencing complex (RISC). The siRNA-RISC complex binds the targeting site of mRNA, resulting in a sequence-specific cleavage by endonuclease Argonaute-2 (AGO2). The result is a decreased expression of a targeted protein.

(Elbashir SM, Lendeckel W, Tuschl T.; RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 2001;15:188-200).

MicroRNA

MicroRNAs are cellular RNA molecules that prevent the production of proteins by degrading the messenger (mRNA) of the proteins. MicroRNAs are short regulatory noncoding RNAs that block gene expression by binding to target sites in the 3'-untranslated regions (UTR) of protein-coding transcripts. The primary microRNAs (pri-microRNA) with a characteristic hairpin structure is recognized and processed by enzymes in Drosha and DGCR8 into ∼70 nucleotide long precursor microRNAs (pre-microRNAs). Resulting pre-microRNAs are further cleaved by Dicer RNase, forming mature dsRNAs (microRNAs). Finally, mature microRNAs are incorporated into RISC to induce cleavage of targeted mRNAs, such as siRNAs, or translational repression, resulting in a decrease of targeted proteins. Frequently, target sequences of microRNAs are found in the 3' UTR of mRNA, often within noncoding or intronic regions. Each microRNA can target hundreds of different mRNAs, thereby inducing regulation of the transcriptome.

In summary, microRNAs target multiple mRNAs, whereas siRNAs only possess a specific binding activity targeting only one mRNA at a time.

(Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215-33.)


Messenger RNA

Messenger RNA (mRNA) delivery upregulates the expression of targeted proteins. mRNA delivered into cells has a low risk for insertional mutagenesis. Furthermore, the delivery of mRNAs into cells is also more consistent. Also, the kinetics of protein expression are more predictable, and in-vitro synthesis is more convenient. The transfection efficiency with mRNA is higher than that of DNA, especially in immune cells. Each mRNA has an open reading frame (ORF) that includes two untranslated regions (UTRs) located at the 5' and 3' ends of mRNA. The 5' methyl cap and the 3' poly(adenosine) tail are crucial for efficient translation. The translational system (the ribosome) recognizes mRNAs during protein synthesis.

(Tan L, Sun X.; Recent advances in mRNA vaccine delivery. Nano Res. 2018;11:5338-54; Van Tendeloo VFI, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C. et al.; Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood. 2001;98:49-56. ; Matsui A, Uchida S, Ishii T, Itaka K, Kataoka K.; Messenger RNA-based therapeutics for the treatment of apoptosis-associated diseases. Sci Rep. 2015;5:15810. ; Xiong QQ, Lee GY, Ding JX, Li WL, Shi JJ.; Biomedical applications of mRNA nanomedicine. Nano Res. 2018;11:5281-309. )

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The extremely short half-lives, poor chemical stability, and natural degradation of RNAs by nucleases resulted in the development of carrier molecules at the nanoscale for RNA delivery into cells. Nanoparticle-based delivery systems protect RNA molecules from enzymatic degradation and immune system attacks and enable the accumulation of RNA in tumors. In general, nanoparticles range from 10 to 200 nanometers (nm), which enhances their tumor permeability and retention effect.

Currently, nanocarriers employed for RNA delivery are

(i)    lipid-based nano-systems,

(ii)   polymeric nanomaterials,

(iii)  inorganic nanoparticles, or

(iv)  Bio-inspired nanovehicles.

Table 1 :  

Nanoparticle-based platforms for RNA delivery (Lin et al. 2020).

.

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RNA interference (RNAi) is a conserved biological process for specific silencing of gene expression. Small interfering RNAs (siRNAs) are important tools for the control of gene expression, such as post-transcriptional gene silencing in mammals, including humans. However, to function as desired, they need to be taken up by cells or tissues and delivered to selected targets. For in vivo applications of siRNAs, delivery across plasma membranes remains the most significant obstacle.

Synthetic oligonucleotides can be modified via conjugation to lipophilic functional groups to allow efficient uptake of siRNAs by cells and tissues. Lipophilic groups are often attached to the 3′-end of the sense strand via oligomethylene linkers of various lengths. Alternatively, various commercially available lipophilic phosphoramidites also allow conjugation to oligonucleotides.

Covalent siRNA cholesterol conjugates are known to increase import into cellular where they induce RNAi resulting in the silencing of endogenous genes in vivo. Wolfram et al. in 2007 reported the preparation of a variety of lipophilic carrier molecules useful for optimized cell delivery. Long-chain fatty acid conjugates are helpful for cell delivery. However, for all these lipophilic molecules, efficient and selective cellular uptake depends on their interaction with lipoprotein particles, lipoprotein receptors, and transmembrane proteins. As Wolfrum et al. reported, the efficiency of siRNA delivery into cells depends on the combined length of the linker and the lipophilic group.

Figure 1:  Lipophilic siRNA conjugate structure. siRNAs when conjugated to different lipids have different in vivo activities. The desired lipophile (L) is conjugated to the 3’-end of the sense strand of the desired siRNA, here via a trans-4-hydroxyprolinol linker (Modified after Wolfrum et al. 2007).

Figure 2:  Structures of lipophilic siRNAs with various lipids conjugated to the core structure of the siRNA conjugate.

Reference

Petrova NS, Chernikov IV, Meschaninova MI, Dovydenko IS, Venyaminova AG, Zenkova MA, Vlassov VV, Chernolovskaya EL. Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group. Nucleic Acids Res. 2012 Mar;40(5):2330-44. doi: 10.1093/nar/gkr1002. [PMC]

Wolfrum, C., Shi, S., Jayaprakash, K. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 25, 1149–1157 (2007). [link]

A modified guanine-rich phosphodiester oligonucleotide prevents HIV-1 infection.

The 6mer sequence d(TGGGAG) is commonly called “Hotoda’s sequence”.

In the 1990s Japanese researchers studied oligodeoxynucleotides (ODNs) in which the 5’-end was covalently linked to a 4,4’-dimethoxytriphenylmethyl (DMT) residue for their ability to inhibit HIV-1 infection. The guanine-rich phosphodiester oligonucleotide with a dimethoxytrityl (DmTr) residue on its 5'-terminal, DmTr-TGGGAGGTGGGTCTG (SA-1042), was found as an inhibitor of HIV-1 infection in vitro.

SA-1042 interfered with the attachment of gp120 to the CD4 receptor and prevented the subsequent entry of the virus into cells. Furukawa et al. showed that guanine nucleosides at the 5'-terminal and modification of the 5'-terminal with DmTr are essential for anti-HIV-1 activity. The substitution of the guanine nucleoside close to the 5'-terminal with other nucleotides prevented antiviral activity. At least three consecutive guanine nucleotides adjacent to the 5'-terminal are required for the activity and modification of the 5'-terminal is essential for the activity. As a result of the study the hexanucleotide, DmTr-TGGGAG, was identified as a potent inhibitor of HIV-1 infection. The hexamer is capable of inhibiting the binding of the gp120 protein to its receptor the CD4 molecule. Furthermore, the hexamer inhibits the accessibility of anti-V3 monoclonal antibody to its ligand V3 peptide.

Sequences rich in guanosine nucleotides are known to form nonlinear structures known as guanine quadruplexes (G4s) stable under physiological conditions. A variety of G4 topologies are also known with variations in strand stoichiometry and polarity as well as different lengths of loop structures and locations the guanines within the sequence. Like aptamers, G4 structures are single-stranded DNA oligonucleotides that specifically bind to their target as a result of their unique 3-dimensional structure. Furthermore, G4 sequences appear to have a role in gene regulation, are important for telomerase maintenance and are also found in genomes, including mammalian genomes as well as the human genome. Several G-quadruplex-forming oligonucleotides with in vitro anti-HIV activity have already been discovered while using either the Systematic Evolution of Ligands by Exponential enrichment (SELEX) process or rational design approaches such as by using Synthetic Unrandomization of Randomized Fragments (SURF). Often evolved or natural occurring aptamers can be improved by chemical modification. Improvements to aptamers can be made by conjugation of large hydrophobic groups to terminal ends, the replacement of phosphodiester linkages with phosphorothioate bonds, as well as the addition of modified nucleic acids such as bridged nucleic acids (BNAs) at selected nucleotide position.

Several G-quadruplex forming aptamers are now known as HIV inhibitors. These quadruplexes function by inhibiting (i) virus binding and entry into the target cell, (ii) HIV reverse transcription, or (iii) virus integration, by interaction with HIV proteins such as envelope proteins, reverse transcriptase and integrase.

The Japanese researchers found that both the G4 structure and the cluster of large aromatic groups at the 5′-ends are essential for the anti-HIV activity. Oligonucleotide R-95288 with a 3,4-dibenzyloxybenzyl (DBB) residue at the 5’-end and a 2-hydroxyethylphosphate residue on the 3′-end of the d(5′TGGGAG3′) sequence was the most potent ODN identified.

With the goal to improve the G4 folding kinetics further Oliviero et al. reported the synthesis of stabilized, tetra-end-linked oligonucleotides (TEL-ODN) that form mononuclear G4s based on Hotoda's sequence. Figure 1 shows the molecular model of a TEL-ODN.

Figure 1:G-quadruplex structure of a monomolecular TEL-ODN. The TEL-ODN was modifed at the 5'-ends with a TBDPS group and the 3'-ends were connected using a tetra-end-linker. The adenosines were replaced with cytosines which resulted in a G4 molecule with a potent anti-HIV activity. The observed and reported EC₅₀ was 39 nM. 

The discovery of the high activity of the unmodified Hotoda's sequence revealed a potent G-quadruplex anti-HIV agent. However, the supramolecular structure is complex and more research maybe needed for it to become a sucessful HIV drug.

Reference

Burge S., Parkinson G.N., Hazel P., Todd A.K., Neidle S. Quadruplex DNA: Sequence, topology and structure. Nucleic Acids Res. 2006;34:5402–5415. doi: 10.1093/nar/gkl655. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

Furukawa H., Momota K., Agatsuma T., Yamamoto I., Kimura S., Shimada K. Identification of a Phosphodiester Hexanucleotide That Inhibits HIV-1 Infection In Vitro on Covalent Linkage of Its 5′-End with a Dimethoxytrityl Residue. Antisense Nucleic Acid Drug Dev. 1997;7:167–175. doi: 10.1089/oli.1.1997.7.167. [PubMed] [CrossRef] [Google Scholar]

Hotoda H., Koizumi M., Koga R., Momota K., Ohmine T., Furukawa H., Nishigaki T., Kinoshita T., Kaneko M. Biologically Active Oligodeoxyribonucleotides-IV 1: Anti-HIV-1 of TGGGAG having hydrophobic substituent at its 5′-End via phosphodiester linkage. Nucleosides Nucleotides. 1996;15:531–538. doi: 10.1080/07328319608002403. [CrossRef] [Google Scholar]

Hotoda H., Koizumi M., Koga R., Kaneko M., Momota K., Ohmine T., Furukawa H., Agatsuma T., Nishigaki T., Sone J., et al. Biologically active oligodeoxyribonucleotides. 5. 5′-End-substituted d(TGGGAG) possesses anti-human immunodeficiency virus type 1 activity by forming a G-quadruplex structure. J. Med. Chem. 1998;41:3655–3663. doi: 10.1021/jm970658w. [PubMed] [CrossRef] [Google Scholar]

Koizumi M., Koga R., Hotoda H., Momota K., Ohmine T., Furukawa H., Agatsuma T., Nishigaki T., Abe K., Kosaka T., et al. Biologically active oligodeoxyribonucleotides-IX. Synthesis and anti-HIV-1 activity of hexadeoxyribonucleotides, TGGGAG, bearing 3′- and 5′-end-modification. Bioorg. Med. Chem. 1997;5:2235–2243. doi: 10.1016/S0968-0896(97)00161-2. [PubMed] [CrossRef] [Google Scholar]

Na Li, Yuxuan Wang, Arti Pothukuchy, Angel Syrett, Naeem Husain, Siddharth Gopalakrisha, Pradeepa Kosaraju, Andrew D. Ellington, Aptamers that recognize drug-resistant HIV-1 reverse transcriptase, Nucleic Acids Research, Volume 36, Issue 21, 1 December 2008, Pages 6739–6751, [NAR]

Musumeci D, Riccardi C, Montesarchio D. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents. Molecules. 2015 Sep 22;20(9):17511-32. doi: 10.3390/molecules200917511. [PubMed].

Oliviero G., Amato J., Borbone N., D’Errico S., Galeone A., Mayol L., Haider S., Olubiyi O., Hoorelbeke B., Balzarini J., et al. Tetra-end-linked oligonucleotides forming DNA G-quadruplexes: A new class of aptamers showing anti-HIV activity. Chem. Commun. (Camb). 2010;46:8971–8973. doi: 10.1039/c0cc02866e. [PubMed] [CrossRef] [Google Scholar]

Romanucci V, Zarrelli A, Di Fabio G. Hotoda's Sequence and Anti-HIV Activity: Where Are We Now? Molecules. 2019 Apr 10;24(7):1417. doi: 10.3390/molecules24071417. [PubMed] 

Proximity ligation assay utilizing fluorescently labeled oligonucleotide probes allows the detection of protein-protein interactions implicated in Alzheimer’s disease (ApoE-C1q) and breast cancer (ErbB3-EGFR)

Within cells, a number of biological processes depend on protein-protein interaction.  The interaction could be either stable or transient depending on the nature of function it assumes.  For instance, a stable interaction of RNR1 and RNR2 subunits is required to form an active ribonucleotide reductase enzyme, which catalyzes the conversion of ribonucleotides to deoxyribonucleotides for DNA synthesis.  Among the strongest binding types is the interaction between RNase A and ribonuclease inhibitor, which exhibits dissociation constant (K_D) in the femtomolar (10^-15) range.  In contrast, the receptors involved in signal transduction interact transiently as the binding is reversible (Perkins et al., 2010).  An exemplary case is the ErbB family receptor, which undergoes conformational change (upon binding to a ligand) to dimerize with distinct ErbB receptor (Macdonald-Obermann et al.., 2013).

 It follows that the ability to detect protein-protein interaction could be highly useful to both basic science research as well as translational medicine.  A commonly used technique for in vitro application is based on affinity purification followed by mass spectrometry analysis of associating proteins for identification.  Alternatively, co-immunoprecipitation assay could be performed on cell extract using an antibody directed against the target protein to isolate its interacting proteins.  For in vivo detection, a ‘two-hybrid’ screening method can be employed, wherein the interaction between two separate proteins expressed by distinct plasmids leads to the transactivation of a reporter gene in transfected cells.  The latter method was used to uncover the interaction between the C-terminus and N-terminus of Rb (retinoblastoma) protein (Hensey et al., 1994_).

 Proximity ligation assay (PLA) represents an immunological method for detecting protein-protein interactions with a single molecule resolution (Soderberg et al., 2006).  In addition to the high degree of sensitivity and specificity, PLA could be used to confirm the protein-protein interaction detected by co-immunoprecipitation or co-localization assay in a short period of time.  To detect, two distinct primary antibodies are used to bind to each interacting protein separately.  Subsequently, it is bound to secondary antibody conjugated to unique oligonucleotides.  If the two interacting proteins are proximally located (not necessarily juxtaposed), then the two oligonucleotides can form a circular DNA strand in the presence of two additional circle-forming oligonucleotides.  After ligating to form a closed circle, polymerase is added to extend via rolling-circle DNA synthesis.  By hybridizing fluorescently labeled oligonucleotides to the “amplified” DNA, the protein-protein interaction within cells can be detected by fluorescence microscopy (Zatloukal et al., 2014).

 Alzheimer’s disease is a neurodegenerative disease affecting >5 million in the U.S and >29 million individuals globally.  It accounts for 60-70% of dementia and negatively impacts memory, speech, mood, behavior, body function, etc. with the life expectancy of 3-9 years following diagnosis.  Though most cases are sporadic, genetic analysis of hereditary cases has identified several genes that are associated with the early onset of Alzheimer’s disease.  Amyloid beta peptide (ABP) is responsible for the plaque formation in the brain, which is derived through the cleavage of amyloid precursor protein encoded by APP gene (Findeis, 2007).  Apolipoprotein E (ApoE) represents another gene linked to Alzheimer’s disease as ApoE binds to ABP and may affect ABP aggregation or clearance from the brain (Holtzman et al., 2012). 

More recently, the role of APoE, which is also implicated in atherosclerosis (narrowing of arteries due to plaque buildup), has been investigated from an alternate perspective.  Classical complement cascade (CCC) refers to an immunological mechanism that functions to activate phagocytosis, promote inflammation, and perforate cell membrane to eliminate invading microbes.  Intriguingly, the investigators at Ludwig-Maximilians-University (Germany) have found that CCC is activated in ApoE deficient mice.  Consistently, ApoE was shown to suppress CCC activity by binding to C1q protein, which initiates the CCC process (Yin et al., 2019).  To demonstrate the latter, they used proximity ligation assay (PLA) to detect C1q-ApoE complexes formed in cultured human cells, which was visualized by fluorescence microscopy. 

ErbB/HER family is comprised of 4 cell membrane-associated growth factor receptors necessary for normal mammary gland development.  However, Her-2 receptor is overexpressed in 20-30% of breast cancer and it prognosticates a poor outcome.  Recent works suggest that the dimerization of Her-3 (ErbB3) receptor with other HER family members may lead to the activation of Her-2.  Using proximal ligation assay, Her-3’s interaction with Her-1 (epidermal growth factor receptor, EGFR) was detected in breast cancer specimens (Karamouzis et al. 2015).

Bio-Synthesis, Inc. specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA).  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as conjugate to peptides.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology that we offer provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power was especially useful for diagnosis (ex. FISH using DNA probe).  More importantly, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides for clinical application.

https://www.biosyn.com/oligonucleotide-modification-services.aspx

https://www.biosyn.com/tew/fluorescent-labeling-of-oligonucleotides.aspx

https://www.biosyn.com/oligo-flourescent-labeling.aspx

References

Findeis MA. The role of amyloid beta peptide 42 in Alzheimer's disease.  Pharmacol Ther.  116:266-86. (2007). PMID: 17716740  DOI: 10.1016/j.pharmthera.2007.06.006

Hensey CE, Hong F, Durfee T, Qian YW, Lee EY, Lee WH.  Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization.  J Biol Chem. 269:1380-7 (1994).  PMID:8288605   https://www.jbc.org/content/269/2/1380.long

Holtzman DM, Herz J, Bu G. Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease.  Cold Spring Harb Perspect Med. 2:a006312 (2012).  PMID:22393530  doi: 10.1101/cshperspect.a006312.

Karamouzis MV, Dalagiorgou G, Georgopoulou U, Nonni A, Kontos M, Papavassiliou AG.  HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas". Oncotarget. 7: 5576–97 (2016).  PMC 4868707. PMID 26716646.   doi:10.18632/oncotarget.6762.

Macdonald-Obermann JL, Adak S, Landgraf R, Piwnica-Worms D, Pike LJ. Dynamic analysis of the epidermal growth factor (EGF) receptor-ErbB2-ErbB3 protein network by luciferase fragment complementation imaging.  J Biol Chem. 288:30773-84 (2013).  PMID: 24014028  doi: 10.1074/jbc.M113.489534. Epub 2013 Sep 6.

Perkins JR, Diboun I, Dessailly BH, Lees JG, Orengo C.  Transient protein-protein interactions: structural, functional, and network properties. Structure 18:1233-43 (2010) PMID: 2094701 doi: 10.1016/j.str.2010.08.007.

Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius K, Jarvius J, et al.  Direct Observation of Individual Endogenous Protein Complexes in Situ by Proximity Ligation.  Nat Methods 3, 995-1000  (2006). PMID: 17072308   DOI: 10.1038/nmeth947

Yin C, Ackermann S, Ma Z, Mohanta SK, Zhang C, Li Y, Nietzsche S, Westermann M, et al. ApoE attenuates unresolvable inflammation by complex formation with activated C1q.  Nat Med. 25:496-506 (2019).  PMID: 30692699  doi: 10.1038/s41591-018-0336-8.

Zatloukal B, Kufferath I, Thueringer A, Landegren U, Zatloukal K, Haybaeck J. Sensitivity and specificity of in situ proximity ligation for protein interaction analysis in a model of steatohepatitis with Mallory-Denk bodies.  PLoS One. 9(5):e96690 (2014).  PMID:24798445  doi: 10.1371/journal.pone.0096690. eCollection 2014.

Angiotensin-I-converting enzyme (ACE), also known as peptidyl-dipeptidase A or kininase II, was first isolated in 1956. ACE is  a chloride-dependent metalloenzyme. ACE cleaves a dipeptide from the carboxyl terminus of the decapeptide angiotensin I resulting in the potent vasopressor angiotensin II, a blood vessel constrictor.

In humans, two forms of the angiotensin-converting enzyme exist. The ubiquitous somatic ACE and the sperm-specific germinal ACE. The same gene encodes both proteins through transcription from alternative promoters. ACE regulates blood pressure as part of the renin-angiotensin-aldosterone and kallikrein-kinin systems as a physiological modulator of hematopoiesis. ACE cleaves angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II). Other peptide substrates are also cleaved by ACE. The vasodilator bradykinin, N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), and other bioactive peptides such as substance P, neurotensin, and enkephalin are included as well.

Angiotensin-converting enzyme 2 (ACE2) is a recently identified human homolog of ACE. ACE2 is a novel metallo-carboxy-peptidase with specificity, tissue distribution, and function different from those of ACE. Both, ACE2 and ACE are zinc metallopeptidases and angiotensin-converting enzymes existing in a membrane-associated and a secreted form. However, many differences exist between these two enzymes. For example, ACE2 contains a single zinc-binding catalytic domain, 42% identical to the human ACE active domain. ACE2 cleaves Ang I, Ang II, apelin-13, apelin-36, dynorphin A-(1–13), and des-Arg bradykinin. ACE2 appears to be important in cardiac function. ACE2 may play a role in the renin-angiotensin system by mediating cardiovascular and renal function.

Angiotensin-Converting Enzyme 2 is the Receptor for SARS-CoV S Protein

Researchers identified ACE 2 as the cellular receptor for SARS coronavirus (SARS-CoV) and the newly emerged coronavirus (SARS-CoV-2) that causes the epidemic COVID-19. The spike protein glycoprotein (S Protein) of the coronavirus is the key target for the development of vaccines, therapeutic antibodies as well as for diagnostics. Wrapp et al. have recently determined the structure of the 2019-nCoV S trimer structure at 3.5-angstroem-resolution using cryo-electron microscopy.

Figure 1: Different views of the Cryo-EM structure of the 209-nCoV spike protein [PBD ID 6VSB].

The spike protein of SARS coronavirus attaches the virus to the cellular receptor, ACE2. The receptor-binding domain (RBD) on the S protein mediates the interaction. The interaction between the S glycoprotein and ACE2 plays a critical role in SARS pathogenesis. Binding of S protein to ACE2 leads to the downregulation of the receptor resulting in the deregulation of the renin-angiotensin system and eventual lung injury.

Li et al. in 2005 determined the crystal structure at 2.0 Ångstroem resolution of the RBD bound with the peptidase domain of human ACE2. The structure of RBD is a scaffold for the design of coronavirus peptide vaccines.

Figure 2: Different views of the SARS coronavirus spike receptor-binding domain (RBD) in complex with the receptor ACE2. The interface between the two proteins illustrate residue that facilitate efficient cross-species infection and human-to-human transmission.

Since the spike protein mediates the entry of the coronavirus into cells, it is a valid target for the development of vaccines and antiviral agents such as viral inhibitor drugs. The S protein of SARS-CoV does not appear to be cleaved and is presumed to have two functional domains. The border between them has been suggested to be around amino acid 680. The receptor-binding domain (RBD) has been narrowed down to a 193 amino acid fragment (residues 318–510). This domain binds ACE2 with greater affinity than does a larger protein fragment representing the S1 domain (residues 12–672).  Several amino acid residues were found to be important for binding the S glycoprotein.

Han et al., in 2006, performed alanine scanning mutagenesis analyses of ACE2. Cells were infected with non-replicating SARS pseudoviruses to study effects of mutations on viral entry. The study found that charged amino acids between residues 22 and 57 are important for virus infection. Three general regions on ACE2 are important for binding the S glycoprotein: (1) residues K31 and Y41 on α-helix 1; (2) M82, Y83 and P84 on loop 2; and (3) K353, D355 and R357 on β-sheet 5.

ACE2-derived peptides that bind S glycoprotein with high affinity are predicted to block the interaction between ACE2 and the S protein thereby inhibiting virus infection. However, the longer peptides P4 and P5 were found to be more active with approximately 50 % inhibitory concentrations (IC50) of 50 μM and 6 μM, respectively. The peptide P6 was found to be much more potent than peptides P4 or P5 peptides.

Table 1: Peptides studied

P1  EEQAKTFLDK

P2          DKFNHEAED

P3  EEQAKTFLDKFNHEAEDLFY 

P4  EEQAKTFLDKFNHEAEDLFYQSS

P5 EEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEE

P6  EEQAKTFLDKFNHEAEDLFYQSSGLKGDFR

Han et al. also synthesized an artificial peptide P6 containing 31 amino acids. The research group joined the peptide fragments 22 to 44, and 351 to 357 to each other with a glycine. Peptide P6 inhibited SARS pseudovirus infection with an IC50 of approximately 0.1 μM. 60- and 500-fold lower than peptides P5 and P4, respectively.

The potent antiviral activity of P6 peptide suggested that its conformation may resemble the one in the ACE2 crystal structure when it is bound to S protein.

These findings suggest that the P6 peptide is a strong candidate for the design of therapeutic peptides that inhibit SARS-CoV infection and help prevent lethal lung failure. However, more research needs to be done before finding an efficient drug or vaccine for COV19.

Reference

Dong P. Han, Adam Penn-Nicholson, Michael W. Cho; Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor. Virology, Volume 350, Issue 1, 20 June 2006, Pages 15-25. [ScienceDirect]

Lili Huang, Daniel J. Sexton, Kirsten Skogerson, Mary Devlin, Rodger Smith, Indra Sanyal, Tom Parry, Rachel Kent, Jasmin Enright, Qi-long Wu, Greg Conley, Daniel DeOliveira, Lee Morganelli, Matthew Ducar, Charles R. Wescott and Robert C. Ladner; Novel Peptide Inhibitors of Angiotensin-converting Enzyme 2. 2003. The Journal of Biological Chemistry 278, 15532-15540. [JBC]

Li F, Li W, Farzan M, Harrison SC.; Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005 Sep 16;309(5742):1864-8. [PubMed]

Masuyer G, Schwager SL, Sturrock ED, Isaac RE, Acharya KR. Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides. Sci Rep. 2012;2:717. [PMC]

Riordan JF. Angiotensin-I-converting enzyme and its relatives. Genome Biol. 2003;4(8):225. doi: 10.1186/gb-2003-4-8-225. [PMC]

Xiaolong Tian, Cheng Li, Ailing Huang, Shuai Xia, Sicong Lu, Zhengli Shi, Lu Lu, Shibo Jiang, Zhenlin Yang, Yanling Wu & Tianlei Ying (2020) Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody, Emerging Microbes & Infections, 9:1, 382-385.

Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS.; Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020 Mar 13;367(6483):1260-1263. [PubMed]

Woodland, David. “Progress Toward a Vaccine for Middle-Eastern Respiratory Syndrome.” Viral Immunology, vol. 27, no. 10, Mary Ann Liebert, Inc., Dec. 2014, p. 483.

 Bio-Synthesis offers Primers, Probes and Standards to Researchers around the World to help combat the new Coronavirus SARS-CoV-2 (COVID19) and to speed up accurate diagnosis of the virus.

Available primer and probes

 
Others

References

CDC - US Centers for Disease Control and Prevention. (2020, February 4). Real-time RT-PCR panel for detection 2019-novel coronavirus.

Charite Virology - Corman et al. (2020, January 17). Diagnostic detection of 2019-nCoV by real-time RT-PCR.

CDC China - National Institute For Viral Disease Control and Prevention. (2020, January 21). Specific primers and probes for detection 2019 novel coronavirus.

HKU MedProtocol

World Health Organization (WHO)

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NAAE (N- (2-Aminoethly)-1 aziridineethanamine) is a potent angiotensin-converting enzyme two (2) inhibitor. Huentelman et al. in 2004 identified this inhibitor molecule during a molecular docking study.

Figure 1: Structure of N- (2-Aminoethly)-1 aziridineethanamine (NAAE).

Angiotensin-converting enzyme 2 is considered an important therapeutic target for cardiovascular diseases and severe acute respiratory syndrome (SARS and SARS-Cov-2 – COVID19) outbreaks.
Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor targeted by the SARS coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2), which is the cause for the epidemic COVID-19.

Yan et al. recently reported the structure of the full-length human ACE2 in complex with the receptor-binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2 at a resolution of 2.9 Å, but with a 3.5 Å resolution at the ACE2-RBD interface. The structure showed that the extracellular peptidase domain of ACE2 recognizes the RBD and that an ACE2 dimer can accommodate two S protein trimers. According to Yan et al., the structural model suggests a model for the interaction of the spike protein with ACE2 by simultaneous binding of two S protein trimers to one ACE2 dimer.

Angiotensin-converting enzyme (ACE) is a highly glycosylated, transmembrane protein occurring in two differently spliced forms with similar but not identical substrate specificity. ACE acts as a carboxypeptidase. ACE removes the C-terminal dipeptide from angiotensin I (DRVYIHPFHL) to form angiotensin II (DRVYIHPF). However, unlike ACE, ACE2 removes a single C-terminal amino acid from angiotensin II to generate angiotensin-(1-7; DRVYIHP), or, with less efficiency, from angiotensin I to form angiotensin-(1-9; DRVYIHPFH). Studies found that ACE2 is not affected by classical ACE inhibitors. There appears to be a close interplay between ACE, ACE2, and peptides such as apelin and neurotensin.

The infection strategy of a coronavirus is complex. The SARS-CoV spike glycoprotein recognizes ACE2 as a receptor on the cell surface. During entering the cytoplasm, the virus core particle containing the genomic RNA bound to the nucleoprotein is released. The 5'-two-thirds of the genomic RNA is translated by the host's ribosomes generating the virus replicase polyprotein. The replicase attaches to the 3'-end of the input genome and begins replication of a full-length anti-genome and synthesizes negative-strand subgenomic RNAs. These RNAs serve as templates for a synthesis for new genomic RNA and viral subgenomic mRNAs (sgRNAs). All coronavirus mRNAs have a conserved 5'-end and are 3'-co-terminal and polyadenylated. However, to clarify the exact mechanism, more research will be needed. The release of the virus occurs after processing and assembly of virus particles in the Golgi apparatus and rough endoplasmic reticulum. Also, coronaviruses appear to use nuclear factors for the replication process. For more detail, see Turner et al. 2004.

In the search for a molecule that inhibits the interaction between ACE2 and the coronavirus Spike protein, Huentelman et al. used a molecular docking study for the identification of the ACE2 inhibitor molecule NAAE.

Reference

Matthew J. Huentelman, Jasenka Zubcevic, Jose A. Hernández Prada, Xiaodong Xiao, Dimiter S. Dimitrov, Mohan K. Raizada, and David A. Ostrov ; Structure-Based Discovery of a Novel Angiotensin-Converting Enzyme 2 Inhibitor. Hypertension. 2004;44:903–906. 

Anthony J Turner, Julian A Hiscox, Nigel M Hooper; ACE2: from vasopeptidase to SARS virus receptor.  Trends in Pharmacological Sciences. 25,6, 291-294, 2004.

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Vaccine development based on messenger RNA (mRNA) is a promising new approach. mRNA based vaccine development is potentially very useful to produce vaccines against coronaviruses such as SARS-CoV or SARS-Cov-2(COVID-19). mRNAs used as vaccines are a promising alternative to conventional vaccines.

Vaccines prevent many illnesses and save lives every day. With the occurrence of newly emerged viruses such as the coronaviruses or the Hanta virus, there is a need for more rapid development and large-scale deployment of effective vaccines. Presently (as of March 2020), an investigational vaccine for the protection against COVID-19 is in clinical trial phase 1. The mRNA-based vaccine is called mRNA-1273. The hope is that we will have a working vaccine against COVID-19 very soon.

According to Linares-Fernández et al. mRNA-based vaccine enable the design and production of well-controlled on-demand transcript sequences that can be adapted to any pandemic crises. In vitro-transcribed (IVT) mRNA used for vaccine production needs to be highly homogeneous. This vaccine should also be free of DNA, dsRNA, or 5’-triphosphate transcripts in order to avoid any overstimulation of innate immunity.

mRNA is the intermediate molecule between the translation of protein-encoding DNA and the production of proteins by ribosomes in the cytoplasm. The high potency, the capacity for a rapid development, the potential low cost of manufacture, and the safe delivery into cells and tissue makes mRNAs ideal candidates for vaccine development. However, historically, the use of mRNA as vaccines has been prevented due to the inherent instability of RNA as well as inefficient in vivo delivery. Advancements made in recent RNA technologies now help overcoming these hurdles and enable mRNA to become a promising therapeutic tool. Currently, mRNA vaccines are investigated in basic and clinical research.

Earlier reports focused on cancer vaccination however more recent reports also showed that mRNAs have the potential to protect against a wide variety of infectious pathogens, such as coronavirus, influenza virus, Ebola virus, Zika virus, Streptococcus spp. and T. gondii, and potentially the newly emerged coronavirus SARS-Cov-2(COVID-19).

According to Pardi et al., mRNA-based vaccination is non-infectious, does not integrate the coded sequence into the genome, and does not cause an infection or is mutagenic. Normally, mRNA is degraded by cellular processes. Also, the in vivo half-life of mRNA can be regulated by using various modifications including bridged nucleic acids and delivery method. And, inherent immunogenicity can be downmodulated. A careful design is necessary for mRNA vaccines since mRNAs interact with pattern recognition receptors (PRRs) in complex ways.

Features of mRNAs for vaccine development

Messenger RNA translation and stability

• The 5’- and 3’-UTR elements flanking the coding sequence strongly influence the stability and translation of mRNA. Both effects are of critical concern for vaccine development.
• Regulatory sequences derived from viral or eukaryotic genes can greatly increase the half-life and expression of therapeutic mRNAs.
• mRNAs require a 5’-cap structure for efficient protein production.
• To synthetic mRNAs, various versions of 5’-caps can be added synthetically or enzymatically.
• Since the poly(A) tail is very important for regulating mRNAs, an optimal length of poly(A) needs to be added. This can be achieved either directly from the encoding DNA template or by using poly(A) polymerase.
• Also, codon usages impact protein translation. Replacing rare codons with frequently used synonymous codons having abundant cognate tRNA in the cytosol can increase protein production.
• Enrichment of G:C content apparently also increases steady-state mRNA levels in vitro and protein expression in vivo.

 
Reference

NIH-Clincal-Trial-COVID-19-Vaccine Begins

Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov. 2018 Apr;17(4):261-279. doi: 10.1038/nrd.2017.243. [PMC]

Sergio Linares-Fernández, Céline Lacroix, Jean-Yves Exposito, Bernard Verrier; Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response. Trend in Molecular Medicine Vol. 26, 311-323.

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Vaccine development based on messenger RNA (mRNA) is a promising new approach. mRNA based vaccine development is potentially very useful to produce vaccines against coronaviruses such as SARS-CoV or SARS-Cov-2(COVID-19). mRNAs used as vaccines are a promising alternative to conventional vaccines.

Vaccines prevent many illnesses and save lives every day. With the occurrence of newly emerged viruses such as the coronaviruses or the Hanta virus, there is a need for more rapid development and large-scale deployment of effective vaccines. Presently (as of March 2020), an investigational vaccine for the protection against COVID-19 is in clinical trial phase 1. The mRNA-based vaccine is called mRNA-1273. The hope is that we will have a working vaccine against COVID-19 very soon.

According to Linares-Fernández et al., mRNA-based vaccine enable the design and production of well-controlled on-demand transcript sequences that can be adapted to any pandemic crises. In vitro-transcribed (IVT) mRNA used for vaccine production needs to be highly homogeneous. This vaccine should also be free of DNA, dsRNA, or 5’-triphosphate transcripts in order to avoid any overstimulation of innate immunity.

mRNA is the intermediate molecule between the translation of protein-encoding DNA and the production of proteins by ribosomes in the cytoplasm. The high potency, the capacity for a rapid development, the potential low cost of manufacture, and the safe delivery into cells and tissue makes mRNAs ideal candidates for vaccine development. However, historically, the use of mRNA as vaccines has been prevented due to the inherent instability of RNA as well as inefficient in vivo delivery. Advancements made in recent RNA technologies now help overcoming these hurdles and enable mRNA to become a promising therapeutic tool. Currently, mRNA vaccines are investigated in basic and clinical research.

Earlier reports focused on cancer vaccination however more recent reports also showed that mRNAs have the potential to protect against a wide variety of infectious pathogens, such as coronavirus, influenza virus, Ebola virus, Zika virus, Streptococcus spp. and T. gondii, and potentially the newly emerged coronavirus SARS-Cov-2(COVID-19).

According to Pardi et al., mRNA-based vaccination is non-infectious, does not integrate the coded sequence into the genome, and does not cause an infection or is mutagenic. Normally, mRNA is degraded by cellular processes. Also, the in vivo half-life of mRNA can be regulated by using various modifications including bridged nucleic acids and delivery method. And, inherent immunogenicity can be downmodulated. A careful design is necessary for mRNA vaccines since pathogen-associated molecular patterns (PAMPs) of foreign RNA can be recognized through interactions with pattern recognition receptors (PRRs) in complex ways.

Features of mRNAs for vaccine development

Messenger RNA translation and stability

• The 5’- and 3’-UTR elements flanking the coding sequence strongly influence the stability and translation of mRNA. Both effects are of critical concern for vaccine development.
• Regulatory sequences derived from viral or eukaryotic genes can greatly increase the half-life and expression of therapeutic mRNAs.
• mRNAs require a 5’-cap structure for efficient protein production.
• To synthetic mRNAs, various versions of 5’-caps can be added synthetically or enzymatically.
• Since the poly(A) tail is very important for regulating mRNAs, an optimal length of poly(A) needs to be added. This can be achieved either directly from the encoding DNA template or by using poly(A) polymerase.
• Also, codon usages impact protein translation. Replacing rare codons with frequently used synonymous codons having abundant cognate tRNA in the cytosol can increase protein production.
• Enrichment of G:C content apparently also increases steady-state mRNA levels in vitro and protein expression in vivo.

Biological Function

Region optimization of mRNAs

Optimization of the whole mRNA molecule

Avoid binding sites of miRNAs present in target cells.

Uridine depletion to avoid recognition by the innate immune system.

Production at high temperature (50 °C) using a thermostable polymerase and/or low magnesium concentration to decrease levels of dsRNAs.

Purify mRNA using HPLC to decrease amounts of dsRNA.

Avoid highly stable and long secondary structures at could activate PRRs.

{Adapted from Linares-Fernandez et al., Pardi et al., and Schlake et al.}

Reference

Amarante-Mendes Gustavo P., Adjemian Sandy, Branco Laura Migliari, Zanetti Larissa C., Weinlich Ricardo, Bortoluci Karina R.;  Pattern Recognition Receptors and the Host Cell Death Molecular Machinery. Frontiers in Immunology 9,  2018, 2379. [Link] 

Sergio Linares-Fernández, Céline Lacroix, Jean-Yves Exposito, Bernard Verrier; Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response. Trend in Molecular Medicine Vol. 26, 311-323.

NIH-Clincal-Trial-COVID-19-Vaccine Begins

Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov. 2018 Apr;17(4):261-279. doi: 10.1038/nrd.2017.243. [PMC]

Pathogen Associated Molecular Pattern

Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ. Developing mRNA-vaccine technologies. RNA Biol. 2012 Nov;9(11):1319-30. doi: 10.4161/rna.22269. [PMC]

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The following graphic genome depictions of the following coronaviruses are shown:

Human SARS CoV-2 (COVID-19), Human coronavirus 229E, Human coronavirus OC43, Human coronavirus HKU1, Human coronavirus NL63, SARS-coronavirus, MERS-coronavirus.

Human SARS CoV-2

GCF_009858895.2 Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1.





Human coronavirus 229E


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• Respiratory viruses such as the new coronavirus SARS-CoV-2 are not very stable. Destroying the lipid coating of the shell will prevent infection by the virus.
• Respiratory viruses such as the new coronavirus SARS-CoV-2 when expelled into the air by coughing, breathing or speaking can settle on surfaces. There they can linger in an active state for days, protected by mucus.
• Presently, scientists are not sure how long SARS-CoV-2 can remain active on a surface.
• One study performed in a hospital found that coronaviruses can survive on hard surfaces like glass, metal, or plastic for up to 9 days (J. Hosp. Infect. 2020, DOI: 10.1016/j.jhin.2020.01.022).
• Another study found that SARS-CoV-2 remains stable on plastic and stainless steel for 2–3 days (N. Engl. J. Med. 2020, DOI: 10.1056/NEJMc2004973).
• The virus can be spread to anyone touching the surface and to whatever that person touches next.
• Enveloped viruses like SARS-CoV-2, which rely on a protective lipid coating, are the easiest to deactivate.
• Ways to burst the shell are:
• Alcohol-based products disintegrate protective lipids.
• Quaternary ammonium disinfectants, commonly used in the health-care and food-service industries, attack protein and lipid structures, preventing the typical mode of infection.

• Bleach and other potent oxidizers easily break down a virus’s essential components.


Reference

Chemical & Engineering News (CEN) 2020-03-23

How do we know disinfectants kill coronaviruses?

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 Bio-Synthesis Inc. is pleased to offer a large variety of RNA molecules (both synthetic and enzymatically derived) for a number of research applications, including COVID 19 testing and analysis !

We also have COVID19 RNA controls such as those currently being used by FDA approved laboratories for COVID molecular testing !

The coronavirus SARS-CoV in 2002 to 2003 caused an outbreak of severe acute respiratory syndrom (SARS). Similar to SARS-CoV, the new SARS-CoV-2 (COVID-19) virus causes a zoonotic infection of the respiratory system in humans. This new virus is related to the SARS-associated coronavirus (SARS-CoV), however it is not the same virus.

According to the CDC, SARS was first discovered in Asia in February 2003. The outbreak lasted approximately six months. SARS-CoV 2003 spread to more than two dozen countries in North America, South America, Europe, and Asia before it stopped in July 2003.

A characteristic feature of coronavirus genomes is that approximately two-thirds of their (+)-sense RNA genome code for overlapping replicase genes ORF 1a and ORF 1 ab. The other one-third encodes a set of subgenomic mRNAs required for accessory proteins and structural proteins.

According to Sawicki et al. (2007), the virus genome encodes structural proteins, as well as nonstructural proteins. Structural proteins are critical in viral RNA synthesis and referred to as replicase-transcriptase proteins. Non-structural proteins are thought as nonessential for virus replication in cell culture. These nonessential proteins appear to give the virus a selective advantage in vivo and are considered as niche-specific proteins. The niche-specific protein, nonstructural protein 2 (nsp2) together with the structural protein N, the nucleocapsid protein, take part in viral RNA synthesis.

Krichel et al. in 2020 studied the processing of the SARS-CoV pp1a/ab nsp7-10 region and reported that initially, ORF 1a and ORF 1ab are directly translated into either replicase poly protein pp1a (nsp1 to 11) or pp1ab (nsp1to 16), respectively. A ribosomal (-1)-frameshift is responsible for differential translation of ORF 1a and ORF 1ab. Figure 1 illustrates the organization of the SARS-CoV-2 genome as well as the structural relationships of the genome- and subgenome-length mRNAs.



Figure 1: Organization of the SARS-CoV-2 genome. The structural relationship of the genome- and subgenome-length mRNAs is illustrated. The proteolytic processing of the replicase polyprotein 1ab is shown in the left part of the figure. [Genome]

Replicase Polyprotein 1ab



Figure 2: Features the replicase polyprotein 1ab [P0C6X7.1].

The translated polyproteins undergo proteolytic processing into 11 or 16 individual nsp’s.
The nsp’s are part of the replication/transcription complex (RTC). The RTC is a membrane-anchored, highly dynamic protein-RNA complex needed for the replication processes. Biochemical studies that included subcellular fractionation, in-situ hybridization observed with electron microscopy combined immunofluorescence suggest that most, if not all, coronavirus nsp proteins are part of the RTC. The RTC synthesizes both genome- and subgenome-length RNA.

Two coronavirus proteases are known to facilitate processing of the polyprotein. A papain-like protease (PLpro; nsp3) encoded between nsp 1 to 4, and the main chymotrypsin-like protease (Mpro; 3CLpro, nsp5) encoded between nsp 4 to 11/16. These two proteases are essential.  Therefore they have already been heavily investigated. Scientist studied their structure and function for suitability as drug targets.

Maturation of the polyprotein via auto-processing of nsp 4-5 and nsp 5-6 regulates the activity of MPro protease, as well as concentration and substrate-induced self-assembly into an active dimeric unit. The freed MPro protease liberates nsp’s 6 to 16 from the polyprotein by targeting the nsp inter-domain junctions. These junctions contain primarily -LQꜜS- or -LQꜜA- amino acid residues at positions P2, P1 and P1’. However, it appears that only Q at P1 is needed.




Reference

Coronavirus 2019

SARS History

Krichel B, Falke S, Hilgenfeld R, Redecke L, Uetrecht C.; Processing of the SARS-CoV pp1a/ab nsp7-10 region. Biochem J. 2020 Mar 13;477(5):1009-1019. [PMC]

Replicase polyprotein 1ab

Sawicki SG, Sawicki DL, Siddell SG.; A contemporary view of coronavirus transcription. J Virol. 2007 Jan;81(1):20-9. doi: 10.1128/JVI.01358-06. Epub 2006 Aug 23. PMID: 16928755; PMCID: PMC1797243. [PMC]

Peptide libraries or pools allow the stimulation of SARS-CoV-2 (COVID-19) specific T cells to develop therapeutic vaccines or drugs. After successful stimulation, T cells can be detected and isolated for further research, as needed.

Combinatorial peptide libraries allow the characterization of critical features of B cell epitopes as well as MHC class I and class II binding epitopes, either natural or synthetic.

Peptide libraries can be synthesized in a completely random fashion or containing one or several defined sequences or positions within the peptide sequence. Also, combinatorial peptide libraries support the design of peptides useful as vaccines against infectious diseases such as COVID-19 as well as therapeutic vaccines against tumors.

Minimized epitopes provided as lipopeptides, as derived from library screens, are heat-stable, non-toxic, fully biodegradable, and can be used as such for T cell stimulation. Lipopeptides are known to activate antigen-presenting macrophages and cells to stimulate innate immunity via specific interactions with receptors of the Toll family.

Bio-Synthesis’s website offers Peptide Library Tools to help design peptide libraries useful for screening for highly active compounds such as antigenic peptides, or receptor ligands, antimicrobial compounds, and enzyme inhibitors.

Reference

Robert G. Urban & Roman M. Chicz; MHC Molecules. Expression, Assembly and Function. R.G. Landes Company, Austin, Texas, U.S.A. 1962. ISBN 0-412-10281-1. 

Karl-Heinz Wiesmüller, Burkhard Fleckenstein and Günther Jung; Peptide Vaccines and Peptide Libraries. Biological Chemistry Volume 382: Issue 4.

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Since the emergence of the COVID-19 outbreak, there is a great need to understand the molecular biology and immunogenicity of the SARS-CoV-2 virus to enable the development of potent vaccines. To help speed up vaccine development, Ahmed et al. identified a set of B cell and T cell epitopes. Epitopes identified are derived from structural proteins of SARS-CoV spike (S) and nucleocapsid (N) proteins and are identical to SARS-CoV-2 proteins.

For the T cell epitopes, the research group performed a population coverage analysis of the associated major histocompatibility complex (MHC) alleles. As a result the proposed set of epitopes are estimated to provide a broad coverage globally, including China. The assumption is that this screened set of epitopes helps to guide experimental efforts towards the development of vaccines against SARS-CoV-2. During the screening, a total of 120 whole-genome sequences of SARS-CoV-2 were downloaded on 21 February 2020 from the GISAID database and used for the analysis.

Coronaviruses are positive-sense single-stranded RNA viruses belonging to the family Coronaviridae.

The typical organization of the genome is as follows:

5'-leader-UTR-replicase-S(Spike)-E(Envelope)-M(Membrane)-N(Nucleocapsid)-3'-UTR-poly(A) tail.

The 3'-end of the virus genome contains accessory genes scattered between structural genes. Accessory proteins appear not to be needed for replication in tissue culture but to be important in viral pathogenesis. The synthesis of polypeptide 1ab (pp1ab) involves programmed ribosomal frameshifting during translation of open reading frame 1a (orf1a). Frame shifting results in a new reading frame that produces a trans-frame protein product. In coronaviruses, a fixed portion of the ribosomes translating orf1a change reading frame at a specific location now decoding information contained in orf1b. See structure of coronavius nCoV-2019-2020, now  SARS CoV-2.

Table 1: SARS-CoV-derived T cell epitopes that are identical in SARS-CoV-2 for the N protein.

Table 2: SARS-CoV-derived T cell epitopes that are identical in SARS-CoV-2 for the S protein.

Ahmed SF, Quadeer AA, McKay MR.;  Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses. 2020 Feb 25;12(3). [PubMed]

Immune Epitope Database and Analysis Resource. A database that catalogs experimental data on antibody and T cell epitopes from humans, non-human primates, as well as other animal species in the context of infectious disease, allergy, autoimmunity and transplantation. [Link]

 Bio-Synthesis Inc. is pleased to offer a large variety of oligonucleotides and peptides for a number of research applications, including COVID 19 testing, analysis and vaccine development!

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The beginning of the year 2020 is marked by the emergence of the COVID-19 coronavirus that causes respiratory diseases.  However, coronaviruses have been quite prevalent considering the high number of coronaviruses that are known to infect a variety of animals including hedgehog, camel, rat, mink, mouse and bird.  For humans, the earliest coronavirus capable of infecting children and adults was identified in 1960s (Kahn et al., 2005).  Of the 7 human coronaviruses that have been identified, 4 strains (OC43, HKU1, NL63, 229E) cause relatively harmless common cold.  The SARS–CoV and MERS-CoV coronaviruses cause more severe respiratory disorders with mortality rates of ~9% and >30%, respectively. 

In the case of COVID-19, individuals with weakened immunity show greater susceptibility.  These include individuals whose immunity is compromised due to cancer therapy (ex. chemotherapy), acquired immune deficiency syndrome (ex. HIV), or transplantation (ex. bone marrow, organ) requiring immune suppressing drugs.  Some report has estimated that the risk of COVID-19 infection may increase two-fold for cancer patients (Al-Shamsi et al., 2020).  Further, exacerbating medical conditions such as chronic lung disease with serious asthma, diabetes, chronic kidney patients requiring dialysis, and liver disorders also constitute risk factors.  Fatality rates differ considerably depending on the countries (Oke et al., 2020). 

As COVID-19 was recently identified, viral proteins encoded by its genome have not been fully characterized.  The following information is based on the genomic analysis of COVID-19, and prior knowledge regarding coronaviruses in general and its most closely related SARS coronavirus (Knoops et al., 2008).  Coronaviruses are medium-sized (~120 nm diameter) RNA viruses and their genome consists of positive sense (translated) single stranded RNA that are among the largest (26-32 kb).  Replication of coronavirus involves the generation of negative strand intermediates (Fehr et al., 2015).  This is mediated by RNA replicase, ‘RNA-dependent RNA polymerase’ (RdRP), which catalyzes RNA synthesis using RNA template.  In addition to generating full-length negative stranded RNA template (for replication), it may generate discontinuous shorter negative stranded templates (for gene expression).  The multiple subgenomic mRNAs produced are unique as they contain sequences found at both ends of the genome.  Exactly how the full-length RNA is synthesized (while generating a nested set of RNAs with common polyadenylated 3’-ends) is not completely understood.   


                    
 

For gene expression, COVID-19 virus utilizes both transcriptional and post-transcriptional mechanisms, and its genome may encode multiple open reading frames (ORFs) and other accessory gene products (Malik et al., 2020).  In the case of SARS virus, replicase gene is encoded by ORF-1a and 1b, with the latter being translated via ‘ribosomal frameshifting’ occurring near the 3’ end of ORF-1a RNA.  For COVID-19 virus, ORF-1 and -2 spanning 2/3 of 5’ terminal genome may encode polyproteins pp1a and pp1b, which undergo further cleavage by proteases to yield 11 and 16 distinct proteins, respectively, to enable virus replication and genome maintenance.  Like other coronaviruses, the 3’-proximal domain of the genome may encode spike (S), membrane protein (M), envelope protein (E) and nucleocapsid (N).

Coronaviruses are mutation-prone, and known to undergo genetic recombination (ex. when multiple viruses infect the same cell), which may contribute to evolutionary divergence.  Following the isolation of COVID-19 strain, its genomic sequence was compared with other known coronaviruses.  The COVID-19 sequence aligned closely with Bat-SARS-like coronavirus exhibiting 88.2% identity (Malik et al, 2020), which closely parallels 86.9% identity reported by Zhu et al., 2020.  Further, investigators at Chinese Academy of Sciences (China) uncovered that the full-length genome of COVID-19 exhbited 96.2% identity with bat coronavirus BatCoV RaTG13 isolated from intermediate horseshoe bat Rhinolophus affinis from Yunnan province (Zhou et al., 2020) located to the southwest of (ca. >600 miles) Wuhan city., shedding light on its potential origin.  Figure denotes the proposed taxonomic distribution based on the alignments.  However, further research is being done to determine if other host(s) may have played a role in the evolution/transmission of COVID-19 to humans.

Critical to preventing epidemic is the ability to diagnose the infected early on to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  Bio-Synthesis, Inc. also specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA).  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as conjugate to peptides.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology that we offer provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power was especially useful for diagnosis (ex. FISH using DNA probe).  More importantly, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides for clinical application.

https://www.biosyn.com/oligonucleotide-modification-services.aspx

https://www.biosyn.com/oligo-flourescent-labeling.aspx

https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx

https://www.biosyn.com/tew/Coronavirus-Diagnostic-Assay-022520.aspx

https://www.biosyn.com/covid-19.aspx

References

Al-Shamsi HO, Alhazzani W, Alhuraiji A, Coomes EA, Chemaly RF, Almuhanna M, et al.  A practical approach to the management of cancer patients  during the novel coronavirus disease 2019 (COVID-19) pandemic: an international collaborative group.  Oncologist. Apr 3. (2020) PMID: 32243668.  doi: 10.1634/theoncologist.2020-0213.

Fehr AR, Perlman S.  Coronaviruses: an overview of their replication and pathogenesis.  Methods Mol Biol. 1282:1-23 (2015).  PMID: 25720466   doi: 10.1007/978-1-4939-2438-7_1

Kahn JS, McIntosh K.  History and recent advances in coronavirus discovery.  Pediatr Infect Dis J.  24:S223-7 (2005)  PMID:16378050  DOI: 10.1097/01.inf.0000188166.17324.60   

Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, Mommaas AM, Snijder EJ.  SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.  PLoS Biol. 6:e226 (2008).  PMID: 18798692  doi: 10.1371/journal.pbio.0060226.

Malik YS, Sircar S, Bhat S, Sharun K, Dhama K, Dadar M, Tiwari R, Chaicumpa W.  Emerging novel coronavirus (2019-nCoV)-current scenario, evolutionary perspective based on genome analysis and recent developments.  Vet Q. 40:68-76 (2020).  PMID:32036774  doi: 10.1080/01652176.2020.1727993.

Oke J, Heneghan C.  Global Covid-19 Case Fatality Rates.  https://www.cebm.net/global-covid-19-case-fatality-rates/

Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, Si H-R, Zhu Y, Li B, Huang C-L. et al. Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv  2020.01.22.914952;  doi: https://doi.org/10.1101/2020.01.22.914952

Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R. et al.  A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med.  382:727-733 (2020)  doi:10.1056/NEJMoa2001017

Sequence analysis of SARS-coronavirus isolates performed by Chung et al. in 2005 revealed that specific genotypes predominated at different periods of the SARS 2002-2003 epidemic. The availability of genomic sequence information allows tracing the footprint of viral infections as well as monitoring viral evolution. However, direct sequencing analysis of large numbers of clinical samples is cumbersome and time-consuming. Hence in 2005, Chung et al. developed and reported a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination.

The fact that the genomic sequence of the causative agent, SARS-CoV, was characterized in 2003 allowed the development of primer and probes sets, including synthetic standards. Early studies focusing on the detection and diagnosis of SARS-CoV demonstrated that specific viral genotypes predominated at certain periods during the outbreak. The availability of sequence data on SARS-CoV made it possible to subclassify viral isolates into several major genotypes based on nucleotide variations.

Pavlović-Lažetić et al. in 2005 grouped viral genome polymorphism into two groups, one with a small number of SNVs and another with a large number of SNVs, including up to four subgroups concerning insertions and deletions. During a bioinformatic study, three nine-locus genotypes: TTTT/TTCGG, CGCC/TTCAT, and TGCC/TTCGT, with four subgenotypes, were found as well. Chung et al. used this information for the design of primer, probe, and synthetic standard sets.

Correctly selected and designed primer and probe sets enable accurate characterization and screening of SARS-coronavirus genotypes. The use of these tools allows studying epidemiological relationships as well between documented cases during an outbreak.

Table 1: Primers and probes

* probes for SNV 17564 are anti-sense sequences.

Recently the number of genomic sequences for SARS-CoV-2 has increased tremendously.  Utilizing this information now allows the design of primer, probe, and standard sets for the discrimination of SARS-CoV-2 strains similar to the ones listed in Table 1.

Adding bridged nucleic acids (BNAs) at selected positions within the oligonucleotide sequence of a probe allows designing primers and probes with enhanced sensitivity, increased base stacking, binding affinity, aqueous solubility, and nuclease resistance. Oligonucleotides modified with BNAs, for example, qPCR probe, exhibit improved duplex and triplex formation by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone. BNA based probes are especially useful for diagnostic tools such as FISH probes and others. Also, BNA oligonucleotides are less toxic than other modified nucleotides for clinical application.

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