Recently emerged technologies now allow the discovery of capped RNAs including NAD-RNA and other small RNA caps. For example, a recently developed method called CapQuant allowed the identification of capped-RNA and cap-like RNAs in bacteria, viruses, yeast, and human tissue.

Wang et al. in 2019 with the help of CapQuant discovered new cap structures in human and mouse tissues. The research group used isotope-dilution liquid chromatography tandem mass-spectrometry (LC-MS/MS) for quantitative analysis of RNA cap structures. A different approach combined mass spectrometric characterization with crystallography for the determination of equilibrium dissociation constants values for different N7-alkylated caps interacting with eukaryotic translation initiation factor eIF4E (Brown et al., Wang et al.).  

Figure 1: Topology of a typical eukaryotic mRNA molecule (Adapted from Farrell, 2017).

In cells, many genes are constantly transcribed by RNA polymerase II. RNA polymerase II is involved in the integration of associated nuclear events such as splicing and polyadenylation. Multiple quantities of heterogeneous nuclear RNA (hnRNA) transcripts are turned over in the nucleus. After apparent quality control in the nucleus in eukaryotic cells, mRNAs emerge from precursor hnRNAs through a series of modification reactions. Modifications include the formation of the 5’-cap, methylation of the cap, splicing, 3’-end processing, and often, polyadenylation. Nakazato et al. discovered the polyadenylation of bacterial mRNA transcripts in 1975 (Nakazato et al. 1975).

Since transcripts are produced at different rates from different loci, the position of a gene or allele in a chromosome, the classification of transcripts is based on their cytoplasmic prevalence or abundance. A typical eukaryotic mRNA molecule shares structural features with other mRNA molecules. However, the production of a functional mRNA is quite complex. Characteristic structural features of mRNAs include the 5’-cap, a 5’-untranslated region (5’-UTR) or leader sequence, the coding region, a 3’-UTR or trailer sequence, and a poly(A) tail. In general, mRNAs do not have long half-lives to allow the cell to be flexible enough to respond quickly to environmental changes. The eukaryotic 5’-cap identifies a transcript as an mRNA and stabilizes the 5’-end against attack by nucleases. Additionally, the poly(A) tail plays a role in the stability of mRNAs as well.

The conversion of an RNA transcript to cap 0 RNA requires three sequential enzymatic steps:

(i) Removal of the 5′ terminal γ-phosphate by RNA triphosphatase activity (TPase),

(ii) Transfer of a GMP group to the resultant diphosphate 5′ terminus by RNA guanylyltransferase activity (GTase), and the

(iii) Modification of the N7 amine of the guanosine cap by guanine-N7 methyltransferase activity (MTase).

In vitro transcription allows the addition of cap structures to RNA transcripts. 

Preparation of capped RNA

In 2003, Huang F. reported a method for the preparation of RNAs modified with small caps. The following small caps added to RNA molecules were coenzyme A (CoA), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD). All three coenzymes contained an adenosine group. This approach allows the preparation of coenzyme-linked RNA libraries for in vitro selection, coenzyme-coupled specific RNA sequences for other uses such as fluorescent labeling to detect specific RNA or DNA sequences, the investigation of RNA structures, and the study of RNA-RNA and RNA-protein interactions.

Adenosine derivatives such as ATP, 3’-Dephospho-coenzyme A (De-P-CoA), NAD, and FAD with the help of a transcription promoter sequence derived from T7 class II promoters initiate transcription. Using adenosine makes the preparation of adenosine-initiated RNA with free 5’-hydroxyl groups possible.

T7 RNA polymerase only requires the adenosine group for recognition and to initiate transcription. Therefore, other adenosine derivatives also allow the preparation of adenosine derivative-linked RNAs.

The conjugation of other biologically active molecules such as coenzymes S-adenosylcysteine, S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (SAM), the sugar-containing molecule adenosine 5’-diphosphoglucose (ADPG) and the signaling molecules diadenosine polyphosphates Ap(3)A and Ap(4)A to the 5'-end of RNAs is also possible.

T7 Class II promoter Sequence

5’-TAATACGACTCACTATTAGGAG--->  DNA template

   ATTATGCTGAGTCATAATCCTC--->

      + R-A, NTPs,T7 RNA polymerase

5’-R-AGGAG--->  RNA transcripts

  pppAGGAG--->

Figure 2: Adenosine derivative-initiated transcription under the T7 class II promoter (φ2.5).

In this approach, in addition to nucleoside 5′-triphosphates (NTPs), an adenosine derivative (R-A) is added to the transcription solution. R-A serves as the transcription initiator to produce R-A-RNA. Adenosine triphosphate (ATP) competes with R-A for transcription initiation, resulting in normal RNA with 5′-triphosphate, pppRNA.

Reference

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To date, numerous variants of COVID-19 have been documented.  Of significance are the specific mutations that confer greater infectivity potential, thus providing a vantage point for its transmission.  The genomic surveillance of viral sequences has identified mutations occurring at distinct genetic loci.  D614G initially identified in Germany and spread globally refers to a point mutation converting the residue #614 in the spike (S) protein of COVID-19 from aspartic acid to glycine (not located in receptor binding domain). Though this mutation may allow greater infectivity, the infected host was able to neutralize the variant (Hou et al., 2020;  Yurkovetskiy et al., 2021).

The D614G mutation (plus N501Y in the receptor binding domain) was present in the B.1.1.7 variant (harbors 23 mutations including 3 amino acid deletions plus 7 missense mutations in S protein), which rapidly expanded in the United Kingdom.  It is interesting to note that N501Y represents mutation in one of six residues (L455, F486, Q493, S494, N501 and Y505) implicated in binding to the ACE2 receptor.  Nevertheless, the immunity rendered by the mRNA vaccine (BNT162b2) was able to neutralize (albeit less effectively) the B.1.1.7 variant (Muik et al., 2021)

 This was followed by the emergence of the B.1.1.298 variant in Denmark (harbors 2 amino acid deletion and 4 missense mutations, ex. Y453F), which led to the destruction of 17 million minks to avoid cross-infectivity to humans, and the B.1.429variant in California (USA) containing 4 missense mutations in the spike protein, ex. L452R in the receptor binding domain).  Their impact on the vaccine efficacy has not been determined.

Of concern are P.1 (12 missense mutation) and P.2 (3 missense mutation) variants emerged in Brazil (derived from B.1.1.28 lineage) harboring the mutations E484K along with K417T and N501Y in the receptor binding domain of S protein, which may evade antibody-based immunity (Nonaka et al., 2021). 

Of greater concern is B.1.351 variant emerged in South Africa and has spread internationally (contains K417N, E484K, and N501Y plus other mutations in S protein along with other mutations outside the S gene) as it may escape neutralization by the antibodies (Wang et al, 2021).  

                    

In general, the lower the genetic diversity, the higher is the probability of developing a successful vaccine.  Hence, for mumps or measles virus displaying low degree of genetic diversity, vaccines are already available.  In contrast, for HIV-1 or hepatitis C virus exhibiting high level of genetic diversity, no vaccines are currently available.  In the case of COVID-19, its genetic diversity falls below that of mumps or measles.

 Nevertheless, to assess the impact of variants on vaccine-induced humoral (B cell based; antibody) immunity, the investigators at the Harvard University (USA) constructed pseudo-COVID-19 virus expressing the specific mutations found in the variants.  Next, they asked whether the serum from individuals vaccinated by mRNA vaccines (Pfizer–BioNTech or Moderna vaccine issued under Emergency Use Authorization by FDA) could neutralize the pseudovirus (Garcia-Beltran et al. 2021).  Of the 10 globally circulating COVID-19 variants examined, 5 variants (P.1, P.2, B.1.351(v1, v2, v3) were resistant to neutralization by the serum.  As these variants share only several mutated residues (K417N/T, E484K, and N501Y), it suggested that a small number of mutations might be sufficient to escape the vaccine-induced antibody response. 

 Despite the above, there has been reports (not published or independently reviewed) suggesting that current vaccines may still prevent disease progression in the B.1.351 infected individuals.  To investigate if the purported protection could result from cellular (cytotoxic T cell) response, the investigators at the Johns Hopkins University (USA) compared the site of mutations in the variants B.1.1.7, B.1.351, and B.1.1.248 (this Brazilian variant was later re-classified as B.1.1.28 that gave rise to P.1 and P.2) with epitopes recognized by T cells.  Intriguingly, of the 52 mutations, 51 did not disrupt the epitopes (Redd et al., 2021).  Though the experimental data for T cells response (against COVID-19 variants) was lacking, it raises a possibility that vaccine-induced cytotoxic T cells may be able to recognize the variants. 

The key to preventing epidemic is the ability to diagnose the infected early 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.  It 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 to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/tew/Mutations-in-the-SARS-CoV-2-Spike-Protein.aspx

References

Garcia-Beltran WF, Lam EC, et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity.  Cell. 2021 Mar 12:S0092-8674(21)00298-1.  PMID: 33743213

Hou YJ, Chiba S, Halfmann P, et al. SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo.  Science. 370:1464-1468 (2020).  PMID: 33184236

Muik A, Wallisch AK, et al. Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera.  Science  371:1152-1153 (2021).  PMID: 33514629

Nonaka CKV, Franco MM, et al. Genomic Evidence of SARS-CoV-2 Reinfection Involving E484K Spike Mutation, Brazil.  Emerg Infect Dis.  27:1522-1524 (2021).  PMID: 33605869

Rausch JW, Capoferri AA, et al.  Low genetic diversity may be an Achilles heel of SARS-CoV-2.   Proc Natl Acad Sci U S A. 117:24614-24616 (2020). PMID: 32958678

Redd AD, Nardin A, et al. CD8+ T cell responses in COVID-19 convalescent individuals target conserved epitopes from multiple prominent SARS-CoV-2 circulating variants.  medRxiv. 2021 Feb 12:2021.02.11.21251585.  PMID: 33594378

Wang P, Nair MS, et al. Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization.  bioRxiv. 2021 Jan 26:2021.01.25.428137.    PMID: 33532778

Yurkovetskiy L, Wang X, et al. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.  Cell.  183:739-751.e8 (2020).  PMID: 32991842

Recent improvements in instrumentation and methods now allow the production of capped RNA  in large quantities needed to study proteins and protein complexes that bind capped RNAs. Fuchs et al., in 2016, reported a method that allows the production of both cap-0 and cap-1 RNA in high amounts. Synthetic capped RNA enables structural and functional studies of proteins and enzymes in complex with capped RNA.

Eukaryotic messenger RNA (mRNA) contains cap structures. The 5’-guanine-N7-methyl cap is a significant feature of these mRNAs. The capping of mRNA is essential for most subsequent steps in the mRNA life cycle. Cap structures are involved in pre-mRNA splicing, pre-mRNA recognition, mRNA export, translation initiation, and stabilization of mRNAs via protection against degradation by 5’-3’-exonucleases and removal of the cap structure to prevent the decay of the transcript.

Also, many viruses can capture cellular cap structures to evade the host’s immune system.

Reference

Fuchs AL, Neu A, Sprangers R. A general method for rapid and cost-efficient large-scale production of 5' capped RNA. RNA. 2016 Sep;22(9):1454-66. doi: 10.1261/rna.056614.116. Epub 2016 Jul 1. [PMC]

Even before the era of COVID-19 pandemic, myocardial infarction has received much attention as it affects a significant number of people (~1 million cases in the U.S. and Europe annually; nearly 26 million people affected globally).  Myocardial infarction refers to the death of heart muscle cells brought about by the lack of oxygen supply, resulting in heart attack.  Hence, the occlusion of blood flow through the coronary arteries feeding the heart muscles by ruptured plaques (ex. buildup of cholesterol product) or blood clots could lead to the death of the supplied cardiac tissue.  The current methods to control blood pressure/improve heart pumping through drugs, or install pacemaker may not address the root cause of the disorder.

To facilitate heart muscle regeneration, numerous strategies have been deployed including administering of recombinant proteins (ex. erythropoietin to produce red blood cells), regenerative cardiovascular therapies (ex. transplantation of ex vivo amplified cells), etc. though the results have been less than satisfactory.  Among the problems faced were the limited long-term engraftment of cell based therapy, low gene transfer efficacy and the potential for genomic integration by gene (DNA) based therapeutics, immune response to viral delivery vectors, and short half-life of recombinant proteins within the tissue.  Against this backdrop, the 'modified RNA' (incorporating modified nucleotides to avoid innate immunity) has emerged as a potential alternative for treating cardiac disorders.


                    

Mechanistically, prior researches have uncovered that, in normal undamaged heart, heart muscle cells exhibit a turnover rate of ~1.3 - 4% annually, being replaced by the proliferation of pre-existing cardiomyocytes.  After the myocardial infarction, new cells arise from progenitors (stem cells) as well as the pre-existing cardiomyocytes (Malliaras et al., 2013).  Another line of investigation uncovered that following the myocardial infarction, epicardial cells (a sheet of cells covering heart) proliferate and differentiated into muscle cells that secrete paracrine factors, which promote blood vessel formation.  Among the secreted factors was VEGF-A (vascular endothelial growth factor A) (Zhou et al., 2011).

By nature, the expression pattern of modified RNA is 'pulse-like'.  This led K. Chien and his colleagues (Harvard University, USA) to hypothesize that modified RNA may be able to recapitulate the paracrine signals that are transient (as its effects are precisely timed and intended for a specific region).  To assess, modified RNA encoding VEGF-A was constructed incorporating 5-methylcytidine triphosphate and pseudouridine triphosphate, and delivered by myocardial injection at the time of myocardial infarction.  It resulted in the amplification of progenitor cells, followed by their mobilization into the myocardium and differentiating into cardiovascular types (to facilitate blood vessel formation), extending the survival in a mouse model (Zangi et al., 2013).

This has led AstraZeneca to collaborate with Moderna to further the translational research in 2013.  Of noteworthy is their finding that injecting a solution of naked mRNA (without protective coating) may be sufficient for its delivery into the heart in vivo.  By using mRNA, a prolonged expression of VEGF-A, which could precipitate side effects, could be avoided.  This has led to the current Phase 2 clinical trial testing the efficacy of VEGF-A encoding modified RNA in patients with decreased left ventricular function undergoing coronary artery bypass surgery (Anttila et al, 2020).  Likewise, the potential application of modified mRNA to treat cancer is being attempted.

The key to preventing epidemic is the ability to diagnose the infected early 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.  It 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 to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

References

Anttila V, Saraste A, et al. Synthetic mRNA Encoding VEGF-A in Patients Undergoing Coronary Artery Bypass Grafting: Design of a Phase 2a Clinical Trial.   Mol Ther Methods Clin Dev. 18:464-472 (2020).  PMID: 32728595

Malliaras K, Zhang Y, et al. Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart.  EMBO Mol Med. 5:191-209 (2013). PMID: 23255322

Zangi L, Chien KR, et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.  Nat Biotechnol. :898-907 (2013). PMID: 24013197

Zhou B, Honor LB, et al. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors.  J Clin Invest. 121:1894-904 (2011).  PMID: 21505261

Recently emerged technologies now allow the discovery of capped RNAs, including NAD-RNA and other small RNA caps. For example, a newly developed method called CapQuant allowed the identification of capped-RNA and cap-like RNAs in bacteria, viruses, yeast, and human tissue.

The combination of mass spectrometry and crystallography allowed Brown et al., in 2007, to determine equilibrium dissociation constants values for cap-binding proteins. The research group investigated different N7-alkylated caps interacting with eukaryotic translation initiation factor eIF4E.

In 2019, Wang et al. used CapQuant to discover new cap structures in human and mouse tissues. The research group used isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitative analysis of RNA cap structures. CapQuant allows transcriptome-wide qunatification of RNA-caps.

Figure 1: Outline of workflow for CapQuant method for mRNA capture. Legend: SIL, stable isotope label; NP1, Nuclease P1; TSS, Transcription Start Site. (Adapted from Wang et al.)

Also in 2020, Galloway et al. developed a method for the detection and quantification of mRNA cap structures present in cells called CAP-MAP.

Figure 2: Schematic outline of CapMap (Adapted from Galloway et al., 2020). To read more about this method click here.

Figure 3: Topology of a typical eukaryotic mRNA molecule (Adapted from Farrell, 2017).

In cells, many genes are constantly transcribed by RNA polymerase II. RNA polymerase II is involved in the integration of associated nuclear events such as splicing and polyadenylation. The nucleus turns over multiple quantities of heterogeneous nuclear RNA (hnRNA) transcripts.

After apparent quality control in the nucleus, mRNAs emerge from precursor hnRNAs through various modification reactions in eukaryotic cells. Modifications include the formation of the 5’-cap, methylation of the cap, splicing, 3’-end processing, and often, polyadenylation. Nakazato et al. discovered the polyadenylation of bacterial mRNA transcripts in 1975.

The production of transcripts occurs at different rates from different loci, the position of a gene or allele in a chromosome; therefore, transcripts are classified based on their cytoplasmic prevalence or abundance. A typical eukaryotic mRNA molecule shares structural features with other mRNA molecules. However, the production of a functional mRNA is quite complex. Characteristic structural features of mRNAs include the 5’-cap, a 5’-untranslated region (5’-UTR) or leader sequence, the coding region, a 3’-UTR or trailer sequence, and a poly(A) tail. In general, mRNAs do not have long half-lives to allow the cell to be flexible enough to respond quickly to environmental changes. The eukaryotic 5’-cap identifies a transcript as an mRNA and stabilizes the 5’-end against attack by nucleases. Additionally, the poly(A) tail plays a role in the stability of mRNAs as well.

The conversion of an RNA transcript to cap 0 RNA requires three sequential enzymatic steps

(i)      Removal of the 5′ terminal γ-phosphate by RNA triphosphatase activity (TPase),

(ii)     Transfer of a GMP group to the resultant diphosphate 5′ terminus by RNA guanylyltransferase activity (GTase), and the

(iii)     Modification of the N7 amine of the guanosine cap by guanine-N7 methyltransferase activity (MTase)

In vitro transcription allows the addition of cap structures to RNA transcripts.

Reference

Brown CJ, McNae I, Fischer PM, Walkinshaw MD; Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives. J Mol Biol (2007) 372 p.7-15. [PubMed]

Cap analysis

CAP-MAP.

CapQuant

Farrell, R.E. Jr.; RNA Methodologies. 5th Edition. Academic Press. 2017.

Alison Galloway, Abdelmadjid Atrih, Renata Grzela, Edward Darzynkiewicz, Michael A. J. Ferguson  and Victoria H. Cowling;  CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive approach to analysing mRNA cap structures. Open BiologyVolume 10, Issue 2. Published:26 February 2020. [Open Biology]

MS & X-ray crystallography 

Nakazato H, Venkatesan S, Edmonds M. Polyadenylic acid sequences in E. coli messenger RNA. Nature. 1975;256:144–146. [PubMed] 

Poly A

Wang J, Alvin Chew BL, Lai Y, Dong H, Xu L, Balamkundu S, Cai WM, Cui L, Liu CF, Fu XY, Lin Z, Shi PY, Lu TK, Luo D, Jaffrey SR, Dedon PC. Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA. Nucleic Acids Res. 2019 Nov 18;47(20):e130. [PMC]

Links to 5'-capped oligonucleotides

https://www.biosyn.com/capped-oligo.aspx

https://www.biosyn.com/faq/can-you-provide-5-capped-oligos.aspx

https://www.biosyn.com/tew/guanosine-triphosphate-oligo-capping.aspx

https://www.biosyn.com/TEW/The-5-Guanosine-triphosphate-Cap.aspx#!

https://www.biosyn.com/oligonucleotideproduct/n7-methylguanosine-triphosphate-terminal-cap.aspx#!

https://www.biosyn.com/tew/mRNA-Capping-and-Decapping-Enzymes.aspx

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" Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated and capped mRNA as well as long circular oligonucleotides".
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In eukaryotes, the 5'- terminal ends of cellular mRNAs contain an m7GpppN cap, in which N can be any nucleotide. The RNA helicase eIF4A and the scaffold protein eukaryotic translation initiation factor 4G (eIF4G) and the capping protein eIF4E are part of the complex that loads the mRNAs onto the 40 S ribosomal subunit, together with eIF3. eIF4E has a crucial role in the regulation of translation. Capping, splicing, and polyadenylation link mRNA metabolism mechanically to transcription. In addition, mRNA decay adds an additional layer of regulation to mRNA metabolism - both capping of the 5’-end and polyadenylation of the 3’-end increase mRNAs' stability.

Scientists found that RNA capping and decapping are critical features in eukaryotes. As recently discovered by Chen et al. via a method using high resolution liquid chromatography on-line mass spectrometry (LC-MS), in bacteria, the redox cofactor nicotinamide adenine dinucleotide (NAD) attaches to small regulatory RNAs in a cap-like manner. The enzyme Nudix hydrolase NudC acts as a NAD-decapping enzyme in vitro and in vivo. Cap-protein interactions can be studied and characterized with the help of LC-MS or in a combination of mass spectrometry and X-ray crystallography. For detailed structural studies of protein cap complexes, X-ray crystallography is the method of choice.

DNA ligases also require NAD as a cofactor. The superfamily of NAD⁺-dependent polynucleotide ligases act as repair enzyme by joining 3’-OH and 5’-PO₄ DNA or RNA ends. In 2017, Unciuleac et al. reported crystal structures of the Michaelis complexes of an ATP-dependent RNA ligase (bacteriophage T4 Rnl1) and an NAD⁺-dependent DNA ligase (Escherichia coli LigA). The solved structures illuminate the chemical and structural basis for lysine adenylylation, via distinctive two-metal (ATP) and one-metal (NAD+) mechanisms.

Figure 1: E. coli LigA (K115M) in complex with NAD⁺ (Unciuleac et al.; PDB ID 5TT5).

In 2016, Hoefer et al. solved the crystal structure of an Escherichia coli NAD-capped RNA hydrolase NudC substrate complex. The enzyme complex studied contained the substrates nicotinamide adenine dinucleotide (NAD) and the cleavage product nicotinamide mononucleotide (NMN). The structures revealed the catalytic residues lining the binding pocket and features of molecular recognition of substrate and product. NAD is a cofactor found in many enzymes, for example, glyceraldehyde phosphate dehydrogenase and other dehydrogenases, where it is bound by a protein domain called the Rossman fold.

The mRNA decapping enzyme NudC is a single-strand-specific RNA decapping enzyme with a strong preference for a purine as the first nucleotide. Biochemical experiments showed that NudC preferred NAD-RNA over NAD(H) by several orders of magnitude. These results suggest that NAD-RNA is its primary biological substrate. The study's findings indicate that NudC can bind a diverse cellular RNA population in an unspecific, most likely electrostatic manner.

Figure 2: NudC (NADH Pyrophosphatase) in complex with NAD (5IW4). Two images of the dimeric structure are shown here. NudC is single-strand specific and has a purine preference for the 5'-terminal nucleotide and strongly prefers NAD-linked RNA (NAD-RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner. 

The redox factor NAD is attached to small regulatory RNAs in bacteria as a cap. In Escherichia coli, the NAD cap stabilizes small regulatory RNAs in vitro against endonucleolytic processing by RNase E and against 5’-end modification by RNA pyrophosphohydrolase RppH. RNase E is involved in RNA decay, and RppH converts 5’-triphosphate RNA into 5’-monophosphate RNA triggering endonucleolytic processing. E. coli Nudix hydrolase NudC removes the NAD cap by hydrolyzing the pyrophosphate bond to produce nicotinamide mononucleotide (NMN), and 5’-monophosphate RNA. NudC is also known as NAD(H) pyrophosphohydrolase. NAD is found as a coenzyme in all living cells. NAD consists of two nucleotides joined together by their phosphate groups.{ https://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide }

Figure 3: Structures and models for the cofactors NADH and NAD+. The hydrogen carrying cofactor NADPH is a phosphorylated form of NADH, a derivative of the vitamin nicotinamide (B3, or the water soluble form of  niacin). https://en.wikipedia.org/wiki/Redox, https://en.wikipedia.org/wiki/Nicotinamide

Not much is known about RNA processing in E. coli; however, one possibility is that NAD capping may give the bacterium additional RNA protection by using a degradation pathway that is orthogonal to the RppH-triphosphate RNA pathway.

In vitro transcription allows the addition of cap structures to RNA transcripts. A method reported by Huang F. enables the preparation of capped RNAs containing small caps including coenzyme A (CoA), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD). All three coenzymes have an adenosine group. The method allows the preparation of coenzyme-linked RNA libraries useful for in vitro selection or to prepare coenzyme-coupled specific RNA sequences for other uses such as fluorescent labeling to detect specific RNA or DNA sequence, the investigation of RNA structures, and the study of RNA-RNA and RNA-protein interactions.
Adenosine derivatives such as ATP, 3’-Dephospho-coenzyme A (De-P-CoA), NAD, and FAD together with a transcription promoter sequence derived from T7 class II promoters initiate transcription. In the presence of adenosine, the preparation of adenosine-initiated RNA with free 5’-hydroxyl groups is possible. T7 RNA polymerase requires only the adenosine group to initiate transcription. Therefore, other adenosine derivatives also allow the preparation of adenosine derivative-linked RNA.

The method allows linking other biologically active molecules to the 5’-end of RNA as well including the coenzymes S-adenosylcysteine, S-adenosylhomocysteine (AdoHcy), and S-adenosylmethionine (SAM), the sugar-containing molecule adenosine 5’-diphosphoglucose (ADPG) and the signaling molecules diadenosine polyphosphates Ap(3)A and Ap(4)A.

As discovered in 1975, like eukaryotic mRNA, bacterial mRNA transcripts can also be polyadenylated.

Reference

Katherine A. Braun, Elton T. Young; Coupling mRNA Synthesis and Decay. Molecular and Cellular Biology Oct 2014, 34 (22) 4078-4087; DOI: 10.1128/MCB.00535-14.  https://mcb.asm.org/content/34/22/4078

Brown CJ, McNae I, Fischer PM, Walkinshaw MD; Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives. J Mol Biol (2007) 372 p.7-15. [PubMed];

https://www.biosyn.com/tew/Mass-spectrometry-combined-with-X-ray-crystallography-allows-the-characterization-of-cap-protein-interactions..aspx#!

Chen YG, Kowtoniuk WE, Agarwal I, Shen Y, Liu DR. LC/MS analysis of cellular RNA reveals NAD-linked RNA. Nat. Chem. Biol. 2009;5:879–881. [PMC] [PubMed] [Google Scholar] – A method to detect small RNA conjugates.

Höfer K, Li S, Abele F, Frindert J, Schlotthauer J, Grawenhoff J, Du J, Patel DJ, Jäschke A. Structure and function of the bacterial decapping enzyme NudC. Nat Chem Biol. 2016 Sep;12(9):730-4. doi: 10.1038/nchembio.2132. Epub 2016 Jul 18. PMID: 27428510; PMCID: PMC5003112. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003112/

Huang F. Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription. Nucleic Acids Res. 2003 Feb 1;31(3):e8. doi: 10.1093/nar/gng008.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC149220/

Metzler, D.E.; Biochemistry. The chemical reactions of living cells. Vol 1 and 2. 2nd edition. Academic Press 
https://www.elsevier.com/books/biochemistry/metzler/978-0-08-092471-7

Unciuleac MC, Goldgur Y, Shuman S. Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases. Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):2592-2597. doi: 10.1073/pnas.1619220114. Epub 2017 Feb 21. PMID: 28223499; PMCID: PMC5347617. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347617/

Winnacker, E-L.; From Genes to Clones. VCH.  https://www.amazon.com/Genes-Clones-Introduction-Gene-Technology/dp/3527261990.
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hRSV is a medium-sized enveloped, negative-sense RNA virus (as opposed to coronavirus with positive-sense RNA genome), which was first isolated from chimpanzee in 1955 and subsequently isolated in human infants.  It is a common causative agent for respiratory infection in infants/children and potentially the elderly or immune compromised individuals.  Similar to COVID-19, hRSV mediates fusion with the cell membrane using its fusion protein F, which is comprised of N-terminal F2 subunit and C-terminal F1 subunit.  In the pre-fusion conformation, the hydrophobic fusion peptide stays buried (Huang et al., 2019).  Upon activation, it undergoes unfolding to transform from a globular to linear conformation, allowing the fusion peptide to insert into the host cell membrane and form a bridge, which then undergoes further conformational change to fuse the viral and cell membranes (to allow the transfer of hRSV genome into the infected cell).

 Until recently, the structural information of hRSV's F protein was not available.  To resolve its structure through X-ray diffraction measurement, one must first obtain a sufficient amount of the protein to form a crystal.  The structure of pre-fusion F protein is quite unstable though it adopts a rigid post-fusion structure upon engaging the host cell membrane.  Its unstable structure has hampered the attempt to obtain a sufficient quantity via expressing the recombinant F protein in transfected cells.

To stabilize its 3D conformation, various adjustments or modifications had to be introduced to the F protein.  For the post-fusion conformation, it required removing the fusion peptide from the construct to achieve a moderate level of expression.  For the pre-fusion conformation, an antibody was added to trap the conformation or disulfide bonds were incorporated artificially to stabilize the structure.  Alternatively, Langedijk and colleagues at Janssen Infectious Diseases and Vaccines (The Netherlands) stabilized the refolding regions of F protein via the substitution of proline residues (Krarup et al., 2015).

                    

Likewise, through proline substitutions, the pre-fusion conformation of the spike (S) protein was stabilized to achieve expression in the case of MERS-CoV coronavirus (Wang et al., 2019).   MERS-CoV is a coronavirus that emerged in Saudi Arabia in 2012 exhibiting a significantly higher rate of mortality than SARS (or COVID-19) coronavirus.  For COVID-19, a similar technique was used to stabilize the pre-fusion conformation of the S protein by substituting two proline residues (in the C-terminal S2 fusion apparatus), which yielded a higher level of expression.  It allowed J. McLellan (University of Texas at Austin, USA), B. Graham (National Institute of Allergy and Infectious Diseases, USA) and their colleagues as well as D. Veesler and colleagues (University of Washington, USA) to obtain the cryo-electron microscopic image of COVID-19 spike protein (Wrapp et al., 2020; Walls et al., 2020).  An X-ray diffraction image of a complex of COVID-19 spike protein bound to an antibody was later obtained by I. Wilson and colleagues (Scripps Research Institute, USA) (Yuan et al., 2021).

As with the structural work, the mRNA vaccines targeting COVID-19 also require protein expression in vivo after the injection.  Hence, for vaccine development, the authors suggested that the stabilized structure of the pre-fusion COVID-19 S protein may "maintain the most neutralization sensitive epitopes when used as candidate vaccine antigens" (Wrapp et al., 2020).   Yet, based on the prior data obtained concerning the convalescent cases that have subsequently recovered, it appears those infected with COVID-19 were capable of mounting the antibody-based or cellular based immunity against the coronavirus (despite the 'unstable' pre-fusion S protein structure in vivo).  Assuming that the antibody generated using the stabilized pre-fusion S protein could recognize the unstable version of the same protein found in vivo,Moderna Therapeutics and Pfizer/BioNTech opted to utilize the mRNA sequence encoding the COVID-19 spike protein containing the two proline substitutions for the vaccine, which has been administered to the public recently (Polack et al., 2020; Keech et al., 2020).  The efficacy of the vaccine remains a grave concern for cancer patients as the pandemic increased their mortality 5-fold.

 The key to preventing epidemic is the ability to diagnose the infected early 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.  It 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 to conjugate to peptidesor antibodies. 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 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 is especially useful for diagnosis (ex. FISH using DNA probe). For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

References

Huang J, Diaz D, Mousa JJ.  Antibody Epitopes of Pneumovirus Fusion Proteins.  Front Immunol.  29;10:2778 (2019).  PMID: 31849961

Keech C, Albert G, et al. Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine.  N Engl J Med. 383:2320-2332 (2020).  PMID: 32877576

Krarup A, Truan D, et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism.  Nat Commun. 6:8143 (2015).  PMID: 26333350

Polack FP, Thomas SJ, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine.  N Engl J Med. 383:2603-2615 (2020).  PMID: 33301246

Walls AC, Veesler D, et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.    Cell. 181:281-292.e6 (2020).  PMID: 32155444

Wang N, Graham BS, McLellan JS et al. Structural Definition of a Neutralization-Sensitive Epitope on the MERS-CoV S1-NTD.   Cell Rep. 28:3395-3405.e6 (2019). PMID: 31553909

Wrapp D, Wang N, et al.  Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.  Science. 367:1260-1263 (2020).   PMID: 32075877

Yuan M, Wilson IA, et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV.   Science.  368(:630-633 (2020.  PMID: 32245784

Nucleoside phosphoramidite monomers containing two protected hydroxyl functional groups utilized as building blocks allow the synthesis of branched oligonucleotides. During the biosynthesis of eukaryotic messenger RNA, branched oligoribonucleotides are naturally found as 'lariat structures' in splicing intermediates.

Branched DNA (bDNA) allows the design and synthesis of signal amplification assays to detect nucleic acid sequences in hybridization assays quantitatively. Examples are assays for the detection of early viral infection states in plasma. Sensitive assays are needed to measure small amounts of viral RNA as this is the case for Human Immunodeficiency Virus Type 1 (HIV-1) or SARS-CoV and SARS-CoV-2 (COVID-19) infections.

In addition, the incorporation of branched nucleotide monomers into DNA or RNA also allows the construction of complex DNA and RNA structures such as DNA origami or complex nanostructures based on nucleic acid sequences. Well-positioned branching monomers enable the synthesis of well-defined DNA or RNA structures such as oligonucleotide-based hydrogels or nanoparticles.

Today, bDNA, developed in 1989 by Horn and Urdea, is widely used in clinical and research laboratories for quantitative detection of specific nucleic acid sequences. Assays utilizing bDNA have a wide dynamic range and allow reliable detection of a few target molecules in various samples.

Unlike in PCR, in a bDNA assay, only the signal is amplified, lowering the risk for detecting contaminations. Also, a minor sequence variation in probe-binding regions of the targets does not compromise assay performance, resulting in a more robust quantitation across genotypes. At the lower end of the assay's dynamic range, reproducibility is usually superior to PCR assays.

The synthesis of branched DNA oligonucleotides is possible using protected phosphoramidites with two protected hydroxyl functional groups as the branching monomers. To synthesize branched oligonucleotides used as signal-amplifying multimers, Horn and Urdea utilized standard phosphoramidite chemistry to synthesize a primary oligonucleotide on controlled pore glass (CPG) supports. The scientists used two primary hydroxyl functional groups for the synthesis of branched oligonucleotides on a solid phase. The use of dimethoxytrityl protection or 0-levulinate protection enables the incorporation of the branching points into the oligonucleotide. The synthesis of protected phosphoramidites started from 4-(1,2,4-triazole)-1-(β,-D-5-O- dimethoxytrityl-3-O-t-butyldimethylsilyl-2-deoxyribofuranosyl)-5-methyl-2(1H)- pyrimidinone followed by triazole displacement with 6-aminohexanol.

The researchers used two strategies for the synthesis of bDNAs.

1. The first synthesis strategy utilizing dimethoxytrityl protection for both primary hydroxyl functions resulted in "fork" structures.

2. The second strategy is employing levulinate protection resulted in "comb" structures.

Figure 1:  Synthesis strategies for branched DNA (bDNA): (left) Fork structures. (right) Comb structures.

The simultaneous deprotection of the two hydroxyl functions in the branching nucleotides allowed the simultaneous condensation of two additional branching monomers or of two identical nucleoside phosphoramidites to the first oligonucleotide sequence. Deprotection of the functional groups produced two reactive sites that allowed further synthesis. The incorporation of multiple branching monomers permitted the addition of several secondary sequences directly to the primary sequence. After successfully synthesizing the primary sequence, the researchers condensed the oligomer with nucleotide phosphoramidite derivatives that possessed two protected hydroxyl functions.

Fork structure synthesis

The synthesis of the fork structure required synthesis of the primary sequence followed by condensation of one branching nucleotide, two thymidines used as molecular spacers, and two more branching nucleotides, followed by the synthesis of the secondary sequence.

Comb structure synthesis

Using O-levulinate to protect the N4-(6-hydroxylhexyl) functional group allows the synthesis of comb structures. Selective removal of the levulinyl group created reactive groups on the branching nucleotides available for condensing the second sequence or additional branching nucleotides. Two thymidines function as spacers.

Deprotection of the functional groups produced two reactive sites needed for further synthesis. The incorporation of multiple branching monomers allowed the addition of several secondary sequences directly to the primary sequence. After successful synthesis, the researchers condensed the oligomer with nucleotide phosphoramidite derivatives that possessed two protected hydroxyl functions.

The first-generation signal-amplifying assay used large, branched single-stranded polydeoxyribonucleotides (bDNA) combined with a labeled short hybridization probe to amplify targeted nucleic acid sequences.

In subsequent years, Collins et al. developed quantitative hybridization assays based on branched DNA signal amplification further.

Figure 3: Illustration of the basic first generation bDNA assays showing assay component (Adapted from Collins et al. 1997).

Figure 4: Illustration of the second and third generation bDNA assays showing assay component. This assay approach utilizes preamplifier oligonucleotides (Adapted from Collins et al. 1997).

Reference

https://www.glenresearch.com/10-1018.html ,  5-Me-dC Brancher Phosphoramidite

Siti Noor Fathilah Ahmad Ariffin. Mini Review Branched DNA: A Novel Technique for Molecular Diagnostics in Bone Studies. Research Updates in Medical Sciences (RUMeS) 2013, Volume 1; Issue 1 page 27-29.

Collins ML, Zayati C, Detmer JJ, Daly B, Kolberg JA, Cha TA, Irvine BD, Tucker J, Urdea MS. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem. 1995 Mar 20;226(1):120–129.

Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C, Horn T, Ahle D, Detmer J, Shen LP, Kolberg J, Bushnell S, Urdea MS, Ho DD. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res. 1997 Aug 1;25(15):2979-84. doi: 10.1093/nar/25.15.2979. PMID: 9224596; PMCID: PMC146852.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC146852/

Horn T, Urdea M (1989) Forks and combs and DNA: the synthesis of branched oligonucleotides. Nucleic Acids Res 17: 6959–6967. [PMC]

T. Horn, C.A. Chang, and M.S. Urdea, Nucleic Acids Res, 1997, 25, 4842-4849.

Kern D, Collins M, Fultz T, Detmer J, Hamren S, Peterkin JJ, Sheridan P, Urdea M, White R, Yeghiazarian T, Todd J. An enhanced-sensitivity branched-DNA assay for quantification of human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol. 1996 Dec;34(12):3196-202. doi: 10.1128/jcm.34.12.3196-3202.1996. [PMC]

Urdea, M. Branched DNA Signal Amplification. Nat Biotechnol 12, 926–928 (1994). [nature biotechnology] 

For a considerable period, immunotherapy employing cytokines such as interleukin-2 or alpha-interferon has been lagging behind other standard treatments, i.e. surgery, chemotherapy and radiotherapy for cancer.  It became less prioritized with the event of 'targeted therapy' as the latter inhibiting oncogenic signaling pathways received greater attention.  However, within the last decade, there has been a great resurgence of interest in immunotherapy due to its noticeable impact on melanoma.

Prior to the recent revival of interest, immunotherapy has been experimenting with the potential of triggering a quasi-autoimmune reaction against cancer.  Though the anticancer vaccines are intended to target tumor specific antigen generated through mutation, it is still part of one's own physiological repertoire.  Thus, there is a risk of triggering autoimmunity against other unintended (normal) targets in the process of destroying cancer.  To avoid such mishap, the immune system has installed a monitoring system called 'immune checkpoint'.  (Note: the checkpoint is distinct from 'DNA damage checkpoint' which monitors damaged DNA to allow time for repair before replicating)  (Sharma et al., 2021).

The activity of host immune system consists of both the antibody-based response and the cellular response.  For the latter, the target protein is proteolytically cleaved into short peptides within the cytosol, followed by its presentation by the major (or minor) histocompatibility complex (MHC) for the recognition by T cell receptors.  In addition to MHC molecules, the antigen presenting process requires co-stimulation by other factors, which include proteins such as B7-1 (CD80) present on dendritic cells and others.  The co-stimulatory molecule B7-1 present on antigen presenting cells is recognized by CD28 present on T cells during the MHC-to-TCR interaction.  In addition to the above, the immune system also consists of Tregs (regulatory T cells) which function to suppress the activation and proliferation of effector T cells.  CTLA-4 (expressed in Treg cells or upregulated in activated T cells) can bind to B7 to turn off the activation.  

PD-1 (programmed cell death protein-1, CD279) is a member of the immunoglobulin superfamily and is expressed on T or B cells, which was discovered by T. Honjo  of Kyoto University (Japan; Nobel prize 2018).  As an 'immune checkpoint' protein, PD-1 functions to suppress immune response via facilitating the apoptosis of antigen-recognizing T cells or inhibiting the death of Treg cells.  PD-1 (expressed in T or B cells) is a receptor for the ligand PD-L1 or PD-L2 (member of B7 family), whose interaction with PD-1 negatively regulates the immune response of cytotoxic lymphocytes (i.e. CD8+ T cells) (Robert, 2020).

Hence, disrupting the interaction between PD-1 to PD-1L (expressed by tumor cells) has become the major focus of pharmaceutical industries to enhance the immunological response to tumors.  This has led to the development of FDA-approved antibodies recognizing PD-1 (Inivolumab) or CTLA4 (Ipilimumab) [in addition to 3 PD-L1 inhibitors] following the demonstration of their therapeutic efficacy by J. Allison of Univ. of Texas M. D. Anderson Cancer Center (USA; Nobel prize 2018).  Despite the media hype, the therapeutic efficacy of 'immune checkpoint blockade' drugs is limited to ~20% of melanoma patients (plus small cell lung cancer, head and neck cancer and others potentially), which is difficult to explain based on the mechanism.  The puzzle remains a key unresolved issue along with the side effects it causes (ex. thyroiditis requiring hormone therapy). (Bardhan et al. 2016).

                         

All currently FDA approved immune checkpoint inhibitors are monoclonal antibodies.  As such, their limited ability to penetrate solid tumors represents a critical disadvantage for solid tumor therapy (Deng et al. 2016).  Another issue (not unexpected) is the reduction of T cells expressing PD-1 or PD-L1 by the anti-PD-1 antibody-based drugs, which compromises the efficacy of immunotherapy.  The decrease in T cells occurs due to the recognition of antibody-coated T cells by NK (Natural Killer) immune cells, which release perforin and proteolytic enzymes (granzymes) to kill the target cells (Brahmer et al., 2010).  Another mechanism through which antibody-coated cells can be lysed is via 'complement mediated cytotoxicity'; however, this path can be bypassed for type 4 IgG antibodies, which have been used to develop the 'immune checkpoint inhibitors'.

To remedy the situation, peptides capable of disrupting the PD-1 and PD-L1 interaction are increasingly being sought.  Unlike the antibodies, small synthetic peptides can penetrate solid tumors effectively, exhibit little immunogenicity, conjugate to tumor-targeting agents or encapsulate in nanoparticles readily, manufacture easily, and disrupt the protein-to-interaction (unlike the chemical drugs).  To this end, the investigators at the University of Missouri (USA) isolated a peptide that blocks the PD-1/PD-L1 interaction by screening a random-peptide displaying phage library (Liu et al., 2019).

Of relevance is the continuing modification of the peptides displayed by the bacteriophages to improve the phage display technology.   These include the peptides (whose termini could be bound to a scaffold) developed by the Bicycle Therapeutics (United Kingdom).  The peptide could be designed to encode multiple sequences in tandem, which could then bind to the different facets of the same scaffold.   The resultant peptide may engage multiple therapeutic targets simultaneously.

The key to preventing epidemic is the ability to diagnose the infected early 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.  It 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 to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

References

Bardhan K, Anagnostou T, et al. The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation.  Front Immunol.  7:550 (2016).  PMID: 28018338

Brahmer JR, Drake CG, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.  J Clin Oncol. 28:3167-75 (2010). PMID: 20516446

Deng R, Bumbaca D, et al. Preclinical pharmacokinetics, pharmacodynamics, tissue distribution, and tumor penetration of anti-PD-L1 monoclonal antibody, an immune checkpoint inhibitor.   MAbs. 8:593-603 (2016).   PMID: 26918260

Liu H, Zhao Z, et al.  Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy. J Immunother Cancer.  7:270 (2019).  PMID: 31640814

Robert C. A decade of immune-checkpoint inhibitors in cancer therapy.   Nat Commun. 11:3801 (2020)  PMID: 32732879

Sharma P, Siddiqui BA, et al. The Next Decade of Immune Checkpoint Therapy.  Cancer Discov.  11:838-857 (2021).  PMID: 33811120

The tryptophan-cage or "Trp-cage" is thought to be the smallest fully protein-like folding motif. The Trp-cage mini proteins are small, folded peptides with 18 to 20 residues in length. The 20 residue Trp-cage mini protein adopts a stable folded structure with well-defined secondary structure elements containing a hydrophobic core arranged around a single central Trp residue. The peptide can fold spontaneously into a stable 3D structure within ~4 µs. Qui et al. utilized laser temperature jump spectroscopy to measure the folding speed of the peptide. The observed fast-folding speed exceeded contact-order predictions and approached diffusional “speed limits” for protein folding.

The amino acid sequence of a protein determines the protein's final conformation or folding. A protein fold usually refers to a protein's native three-dimensional structure. The proper function of a protein in its native environment requires a correctly folded protein.

Since folding mechanisms of proteins are still not well understood, the Trp-cage mini protein is a model peptide for protein folding studies using bio-physical and bioinformatical methods such as laser temperature jump spectroscopy, differential scanning calorimetry, circular dichroism spectroscopy, and molecular dynamics simulations. 

Structural models of Trp-cage mini proteins.

 The software Chimera from UCSF was used for the illustrations of the structural models.

Alignment of Trp-cage peptide sequences

Additional References

1. Qiu L, Pabit SA, Roitberg AE, Hagen SJ; (2002) Smaller and faster: the 20-residue Trp-cage protein folds in 4 micros. J. Am. Chem. Soc. 124: 12952–12953. [PubMed]

2. Streicher WW, Makhatadze GI (2007) Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy. Biochemistry. 46: 2876–2880. [PubMed]

3. Kannan S, Zacharias M (2014) Role of Tryptophan Side Chain Dynamics on the Trp-Cage Mini-Protein Folding Studied by Molecular Dynamics Simulations. PLoS ONE 9(2): e88383. https://doi.org/10.1371/journal.pone.0088383. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088383 

4. Jaenicke R. Protein stability and protein folding. Ciba Found Symp. 1991;161:206-16; discussion 217-21. PMID: 1814693. https://pubmed.ncbi.nlm.nih.gov/1814693/

5. Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33-45. doi: 10.1038/355033a0. PMID: 1731198.  https://pubmed.ncbi.nlm.nih.gov/1731198/

6. Lilie H, Lang K, Rudolph R, Buchner J. Prolyl isomerases catalyze antibody folding in vitro. Protein Sci. 1993 Sep;2(9):1490-6. doi: 10.1002/pro.5560020913. PMID: 8104614; PMCID: PMC2142458. https://pubmed.ncbi.nlm.nih.gov/8104614/

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The mechanism of aging has been studied from multiple angles using various animal models (albeit largely not understood).  The accumulation of genetic mutations has been implicated as a major cause of aging.  These spontaneously occurring mutations are thought to occur during the process of DNA replication or due to faulty DNA repair.  In the case of germ-line tissues (for reproduction), short-lived organisms such as flies (Drosophila) or mice exhibit higher mutation rates than long-lived mammals like the humans.  A similar phenomenon was observed in somatic tissues, which showed an increase in mutation frequency with age in mice, resulting in large genomic rearrangements (Vjig et al., 2002).  As such, molecules such as reactive oxygen species (ROS), which can crosslink DNA, or reducing sugars (ex. glucose, fructose), which form covalent links with structural proteins like collagen (stiffens blood vessels), are implicated in aging.

An alternate view portrays a more "programmed" mechanism of aging.   For instance, at the genetic level, specific methylation changes in DNA may occur with aging; alternatively, the length of telomeres may shorten as cells undergo senescence. 

Throughout the history, the ideal of reaching immortality has been a subject of intense fascination.  In the 1980s, researchers found that a single-gene mutation could increase the lifespan of nematode worm (C. elegans) significantly (Klass, 1983; Friedman, 1988).  This has led to the whole genome RNA interference screening (using siRNAs), which resulted in identifying >200 genes whose silencing (individually) increases lifespan (25-30% in flies, ~40% in mice) (Vjig et al., 2008).  Interestingly, many of the genes suppressed function in biochemical pathways that regulate cell growth (insulin or insulin-like growth factor-1 signaling), energy metabolism (ex. mitochondrial electron transport), etc., mimicking calorie-restriction.

                    

Over the years, pharmaceutical industries as well as the academic institutions have been actively engaged in finding novel therapeutics in natural product resources as diverse as the deep-sea environment or Amazon rainforest.  During 1960s, an expedition to Easter Island (i.e. Rapa Nui island located in South Pacific Ocean >2000 miles west of Chile) resulted in the discovery of an anti-fungal drug 'rapamycin' (named after the island) in 1972, which arrests cell cycle progression at G1 phase.  As it inhibits T cell proliferation, rapamycin has been approved by FDA (U. S. Food & Drug Administration) as an immunosuppressant (to suppress immune rejection of transplanted organs or coronary stents).

Subsequent research has uncovered that rapamycin forms a complex with FKBP12 protein, which in turn inhibits mTOR.   mTOR is a protein kinase that represents a core component of mTOR complexes 1 and 2.  It regulates various processes critical to cancer development such as cell proliferation, cell movement, etc.  The mTOR kinase integrates upstream signals (ex. nutrients, growth factors, energy level) with downstream events such as gene transcription or protein synthesis to impact autophagy (degradation of defective cell organelles), metabolism, growth, etc. that are critical for cell survival.  Subsequently, rapamycin (enterically delivered via feeding) was shown to extend the lifespan of worms, flies, and mice in the laboratory, generating an enormous amount of interest pharmaceutically (albeit FDA does not approve drugs that merely impede aging) (Robida‐Stubbs et al., 2012; Bjedov et al., 2010; Harrison et al., 2009; Livi et al., 2013). 

For vaccine induced immune response against pathogens like COVID-19 coronavirus or cancer, 'memory cells' are thought to play a critical role.  Immunologically, inhibition of mTOR by rapamycin led to converting activated CD8⁺ T cells into memory cells.  Nonetheless, as rapamycin is associated with immunosuppression, investigators at the University of Miami (USA) have developed an aptamer-guided siRNA targeting RAPTOR (a subunit of mTOR complex 1) and showed that it performs superior to rapamycin in mounting antitumor immunity against melanoma in a mouse model (Berezhnoy et al., 2014).

The key to preventing epidemic is the ability to diagnose the infected early 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.  It 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 to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

References

Bjedov I, Toivonen JM, et al. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metabolism. 2010; 11:35‐46. PMID:  20074526

Berezhnoy A, Gilboa E, et al. Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity.  J Clin Invest. 124:188-97 (2014).  PMID: 24292708

Friedman DB, Johnson TE. A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility.  Genetics.  118:75-86 (1988). PMID: 8608934

Harrison DE, Strong R, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009; 460:392‐395.  PMID:  19587680  

Hua H, Kong Q, et al.  Targeting mTOR for cancer therapy.  J Hematol Oncol.  12:71 (2019).  PMID: 31277692

Klass MR.  A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results.   Mech Ageing Dev. 22:279-86 (1983).  PMID: 6632998

Livi CB, Sharp ZD, et al. Rapamycin extends life span of Rb1+/- mice by inhibiting neuroendocrine tumors.  Aging (Albany NY).  5:100-10 (2013).  PMID: 23454836

Robida‐Stubbs S, Glover‐Cutter K, , et al.  TOR signaling and rapamycin influence longevity by regulating SKN‐1/Nrf and DAF‐16/FoxO.  Cell Metabolism. 15:713‐724 (2002).  PMID: 22560223

Vijg J, Dollé ME. Large genome rearrangements as a primary cause of aging.   Mech Ageing Dev.  123:907-15 (2002). PMID: 12044939

Vijg J, Campisi J.  Puzzles, promises and a cure for ageing.   Nature.  454:1065-71 (2008).  PMID: 18756247

For the U.S., Center for Disease Control (CDC) has posted that there are no "Variant of High Consequence" that defies current diagnostic capability or evades the vaccine-induced immunity or the EUA (Emergency Use Authorization) approved therapeutics. 

For "Variants of Concern", it listed COVID-19 strains Alpha (B.1.1.7 first identified in U.K.), Beta (B.1.351 first identified in S. Africa), Gamma (P.1 first identified in Brazil/Japan), and Delta (B.1.617.2 first identified in India) that exhibit increased transmissibility and disease severity, interference with diagnostic test target, and significant reduction in neutralization by antibody-based therapeutics or vaccines.

For the "Variants of Interest", Iota (B.1.526 first identified in NY, U.S.), Epsilon (B.1.427 or B.1.429 first identified in Calif., U.S.), Kappa (B.1.617.1 or B.1.617.3 first identified in India), Zeta (P.2 first identified in Brazil), and Eta (B.1.525 first identified in NIgeria/U.K.) were listed with the potential impact on the above parameters.  As for the latter, CDC further defined it as "variant with specific genetic markers that have been associated with changes to receptor binding" (https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html ).

In the latest update provided by Moderna Inc. (research article preprint posted June 28, 2021 in bioRxiv), the efficacy of the originally devised mRNA-1273 vaccine against newly emerged COVID-19 variants was described (Choi et al., 2021).  In the report, the ability of the sera containing antibodies from mRNA-1273 vaccinated individuals (1 week following 2^nd dose) to neutralize the coronavirus was assessed.  For the neutralization assay, the authors constructed multiple recombinant 'pseudoviruses' with each virus expressing the spike protein of different COVID-19 variants (instead of utilizing the true COVID-19 coronaviruses).

                    

The pseudovirus was derived from Vesicular stomatitis virus (VSV) that has been engineered to lack its envelope glycoprotein G (mediates fusion with the host cell's plasma membrane), which cannot replicate in culture.   VSV is a negative stranded RNA virus that normally infects various animals as well as humans, which can be maintained in a biosafety level 2 containment facilities.  These properties have been exploited to allow VSV (lacking its G protein) to express the glycoprotein of high-risk viruses that require biosafety level 3 or 4 containment (COVID-19 may require level 3 facility) (Whitt, 2010).  The recombinant VSV utilized in the assay has been engineered to expresses the spike protein of COVID-19 (also expresses the luciferase reporter gene (Choi et al., 2021).  The construction of such pseudovirus has been previously described, allowing it to be used in biosafety level 1 facility (Zettl et al., 2020).  Thus, by assaying the level of fluorescence emitted by the infected cells, the extent of neutralization afforded by the anti-COVID-19 antibody present in vaccinated individual's serum can be readily determined.

The neutralization results obtained with the variants were compared against the result obtained with D614G (changes residue 614 from Asp to Gly) mutant.  The COVID-19 variant with D614G mutation (in spike protein) is currently the most widely circulating strain globally (Choi et al., 2021).  Among the data reported, the vaccine's efficacy was reduced by 8.0 and 8.4 fold against A.VOI.V2 (1^st detected in Angola, Africa) and B.1.351-v3 (Beta) variants, respectively.  Against P.1 (Gamma) and B.1.617.2 (Kappa) variants, its efficacy was reduced by 3.2 and 2.1 fold, respectively.  A 4.2 fold reduction was observed against B.1.525 (Eta) variant (1^st detected in Nigeria, Africa).  To counter the drop in efficacy, Moderna Inc. is currently testing the potential of using mRNA-1273.351 (targets the spike protein of Beta variant) as a booster for those who have been previously vaccinated.  The mRNA-1273.211 multivalent booster vaccine is comprised of both the original mRNA-1273 vaccine and mRNA-1273.351 (50:50 mix) (research article preprint posted April 13, 2021 in bioRxiv) (Wu et al., 2021). Nevertheless, for the immunosuppressed individuals (ex. cancer patients undergoing chemotherapy), other forms of therapy than vaccines may be necessary.

The key to preventing epidemic is the ability to diagnose the infected early 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.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

References

Choi A, Koch M, et al.  Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants.  bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.449914; this version posted June 28, 2021.

Whitt MA.  Generation of VSV pseudotypes using recombinant deltaG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines.   J Virol Methods. 169:365-74. (2010).  PMID: 20709108

Wu K, Choi A, et al.  Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice.  ioRxiv preprint doi: https://doi.org/10.1101/2021.04.13.439482; this version posted April 13, 2021.

Zettl F, Meister TL, et al.  Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes.  Vaccines (Basel). 8:386 (2020).  PMID: 32679691

Biological processes are naturally regulated; however, scientists have developed and utilized chemical tools to investigate and control cellular processes. For example, small-molecule probes allow to perturb and control cellular processes, providing an understanding of biological function. Photoactive compounds such as caged or photo-switchable molecules enable activation or deactivation of targeted biochemical pathways after photo-activation.

Naturally, some higher organisms respond to light via photoreceptor proteins which mediate their growth after light stimulation. For example, these signal molecules help plants determine the direction of the light sources. Activated genes lead to a change in hormone level gradients allowing a plant to grow toward the light. Phytochromes are photoreceptor molecules present in plants, bacteria, and fungi, regulating the organism’s germination as a response to light. The photoreceptor phytochrome controls the transcription of its genes via negative feedback.

The use of synthetic photolabile compounds enables the regulation of biological or chemical processes.

Caged ATP

Caged ATP [NPE-caged ATP; P3-(1-(2-nitrophenyl)ethyladenosine 5’-triphosphate]  is a nucleotide analog containing a blocking group at the terminal phosphate group, the γ-phosphate. The presence of the blocking group renters the molecule biologically inactive. Flash photolysis of the blocking or caging group with UV light illumination at around 360 nm rapidly releases the caging group, releasing the free nucleotide locally.

The photolysis of “caged ATP” generates ATP in situ. McCray et al., in 1980, reported that the pulsed laser energy utilized correlates with the amount of ATP formation during photolysis of caged ATP. The research group characterized the kinetics of ATP-induced dissociation of actomyosin using photo-released ATP. The photolyzed of caged ATP occurred at a concentration of 2.5 mM using a single 30-nanosecond laser pulse at 347 nm from a frequency-doubled ruby laser of 25 mJ energy. This photoreaction generated 500 μM ATP.

Figure 1: Laser flash photolysis of caged ATP (McCray et al. 1980). A 347-nm laser pulse released the active nucleotide.

Optochemical control of oligonucleotides

The wavelength of many fluorescent functional groups falls within the UV range, typically 360-366 nm, and thus is orthogonal to all commonly used fluorescent proteins. Other UV light used: 365 nm and 532 nm.

Caged nucleotides

Caged nucleotides are nucleotide analog containing a blocking group at the terminal phosphate group, the γ-phosphate. The presence of the blocking group renters the molecule biologically inactive. Flash photolysis of the blocking or caging group with UV light illumination at around 360 nm rapidly releases the caging group which in turn releases the free nucleotide at the site of illumination.

Table 1:  Photoactivation UV Wavelengths

a From Invitrogen (Molecular Probes). c From Calbiochem. c From Tocris. ε, extinction coefficient; Φ, quantum yield. ND, not determined.

(Adapted from: Ellis-Davies GC. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods. 2007 Aug;4(8):619-28. doi: 10.1038/nmeth1072. PMID: 17664946; PMCID: PMC4207253)

Applications of caged molecules

Applications of photocleavable oligos:  300 to 350 nm UV light.

Caged nucleobases for opto-chemical control of DNA functions

Caged Oligonucleotides: 360 to 440 nm

Chemical structures of caged nucleobases

Caged cirRNA: Photolysis conditions: 350 nm, 30 mW/cm²

Design of caging molecules or functional groups

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Presently, more than 150 distinct RNA modifications have been identified (McCowan et al., 2020), which includes 111 for transfer RNAs (tRNAs), 33 for ribosomal RNAs (rRNAs), 11 for noncoding RNAs.  The type of RNA modification could be simple (ex. methylations, thiolation, hydroxylation), complex (ex. ring closure, acylation, glycosylation, aminoacylation) or unique (ex. incorporate selenium).

As for the mRNA, 17 modifications have been identified thus far   (McCowan et al., 2020).  The lesser number of modifications found for mRNA reflects the difficulty of mapping the modified base position precisely and the lack of information regarding the identity of the interacting proteins (Chen et al., 2020).  The well known modifications include methylation [ex. N6-methyladenosine (m6A), N1-methyladenosine (m1A), 2-O-dimethyladenosine (m6Am), 5-methylcytosine (m5C)] and isomerization [ex. pseudouridine (Ψ)] (Visser et al., 2020).  Among them, m6A represents the most abundant type as it accounts for 0.2-0.5% of total adenosine nucleotides in cellular mRNA (2 or 3 sites per transcript). Initially, however, the prevalence of m6A was not readily accepted, in part, due to the inability to detect, which was greatly improved via the immunoblotting technique (Meyer et al., 2014).

Each type of mRNA modification may vary in terms of distribution as well as functionality.  In the case of m6A, which affects ~30% of brain transcripts (also tRNA, rRNA, noncoding RNA), it is commonly added internally near the stop codon or in the 3-UTR (untranslated region).  Though it does not affect the specificity of base pairing, the identity of the associating protein (i.e. "reader") dictates the fate of the modified mRNA (ex. YTHDC1 promotes translation, YTHDF2 enhances degradation).  In contrast, m6A_m (modifies ~0.016% of all adenines in humans) occurs at the first nucleotide (following m7G cap) at the mRNA terminus and may increase stability to enhance expression.  The m1A modification, affecting ~20% of transcripts, occurs primarily in the 5'-UTR near the translational initiation site and may increase translation.   The m5C addition, which occurs in tRNA, rRNA and mRNA, may interact with the ALYREF protein to promote nuclear export.  Pseudouridine, which occurs in noncoding RNA as well as mRNA, may function to weaken the RNA-to-protein interaction (ex. PUF protein) or stabilize the structure of RNA (Vissers et al., 2020).

                    

Mechanistically, multiple means of modifying RNA have been identified. As for the "writer", the modification may be achieved enzymatically, i.e. methyl transferase complex (comprised of METTL3, METTL14, HAKAI, KIAA1429, RBM15/B) for m6A modification, DNMT2 or NSUN2 for m5C modification, PUS1 or PUS7 for isomerization of uridine to pseudouridine (Vissers et al., 2020). The A to I (inosine) deamination is mediated by ADAR (double-stranded RNA-specific adenosine deaminase) (Nishikura, 2019) whereas C to U deamination is mediated by the PPR (pentatricopeptide repeat) protein (Takenaka et al., 2014).

For 2'-O-methylation (rRNA) or the pseudouridine conversion, the modification may be mediated by a distinct complex [consisting of snoRNA (small nucleolar RNA targeting a specific mRNA sequence) and 4 proteins (ex. fibrillarin/Nop1p, NOP56, NOP58, SNU13 for the 'C/D box' snoRNA)].  For the capped mRNA, 2'-O-methylation of first and second nucleotide is catalyzed by CMTR1 (hMTR1) (Belanger et al, 2010) and CMTR2 (hMTR2), respectively (Werner et al, 2011).    Similar (but catalytically distinct) methyl transferases are encoded by various viruses (Smietanski et al., 2014).

RNA editing also includes deletion or insertion, and, in the case of negative stranded RNA genome containing paramyxovirus (ex. measles, mumps virus), stuttering of RNA polymerase at a discrete site could lead to G insertion, causing a frameshifting of the open reading frame (Hausemann et al., 1999).  In the case of mitochondrial mRNA, the U insertion requires aligning of mRNA to the template 'guide RNA', whose associating proteins then endonucleolytically excise the mRNA to insert U before re-joining (Simpson et al., 1995; Blum et al., 1999).

The role of the mRNA cap structure in avoiding host immune response has been well recognized.  In the case of COVID-19 coronavirus, its virion contains 5'-capped genomic RNA with 3'-poly-A tail, which is transcribed into negative strand to serve as a template for generating additional copies of its genome as well as multiple shorter sub-genomic RNAs for protein production.  The latter is modified by (1) RNA/NTP triphosphatase (Nsp13; to hydrolyze first phosphate), (2) guanylyltransferase enzyme (Nsp12) to transfer G to form Gppp-RNA, (3) Nsp10/14 to methylate the G at N7 using S-adenosylmethionine, (4) Nsp10/16 for 2'-O-methylation of the first ensuing nucleotide, which is critical to deter recognition by RIG-1 or MDA5 to incite interferon response, whose 3D structure was determined using fixed-target serial synchrotron crystallography (Wilamowski et al., 2021; Basu et al., 2021).

The key to preventing epidemic is the ability to diagnose the infected early 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.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/tew/Messenger-RNA-turnover-and-their-half-live.aspx#!

https://www.biosyn.com/tew/Pseudouridine,-an-abundant-post-transcriptional-RNA-modification.aspx

References

Basu S, Mak T, et al. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.  Biochem J. 478:2481-2497 (2021). PMID: 34198328

Benne, R., J. Van den Burg, et al. Transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell 46:819–826 (1986).  PMID: 3019552

Bélanger F, Stepinski J, et al. Characterization of hMTr1, a human Cap1 2'-O-ribose methyltransferase.    J Biol Chem. 285:33037-33044 (2010).   PMID: 20713356

Blum B, Bakalara N, et al.  A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell. 60:189-98 (1990).  PMID: 1688737

Chen LQ, Zhao WS, et al.  Mapping and editing of nucleic acid modifications.   Comput Struct Biotechnol J.  18:661-667 (2020).  PMID: 32257049

Hausmann S, Law FM, et al. Regulation of parathyroid hormone/parathyroid hormone-related protein receptor expression by osteoblast-deposited extracellular matrix in a human osteoblast-like cell line.  J Cell Physiol. 165:164-71 (1995).  PMID: 7559797

McCown PJ, Ruszkowska A, et al.  Naturally occurring modified ribonucleosides. Wiley Interdiscip Rev RNA. 11:e1595 (2020).   PMID: 32301288

Meyer KD, Jaffrey SR. The dynamic epitranscriptome: N6-methyladenosine and gene expression control.  Nat Rev Mol Cell Biol. 15:313-26 (2014).  PMID: 24713629

Nishikura K. Functions and regulation of RNA editing by ADAR deaminases.  Annu Rev Biochem.  79:321-49 (2010).    PMID: 20192758

Simpson L, Thiemann OH.  Sense from nonsense: RNA editing in mitochondria of kinetoplastid protozoa and slime molds. Cell. 81: 837–40 (1995).   PMID:  7781060

Smietanski M, Werner M, et al. Structural analysis of human 2'-O-ribose methyltransferases involved in mRNA cap structure formation.  Nat Commun. 5:3004 (2014).  PMID: 24402442

Takenaka M, Verbitskiy D, et al. RNA editing in plant mitochondria-connecting RNA target sequences and acting proteins.  Mitochondrion. Pt B:191-7 (2014).  PMID: 24732437

Vissers C, Sinha A, et al.  The epitranscriptome in stem cell biology and neural development.   Neurobiol Dis. 146:105139 (2020).   PMID: 33065280

Werner M, Purta E, et al. 2'-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family.   Nucleic Acids Res. 39:4756-68 (2011).   PMID: 21310715

Wilamowski M, Sherrell DA, et al. 2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography.  Proc Natl Acad Sci U S A. 118:e2100170118 (2021).   PMID: 33972410

Specific DNA probes allow the detection and discrimination of specific nucleic acid target sequences. In the early years of probe development, DNA probe development utilized material from natural sources or cloning DNA. However, synthetic DNA hybridization probes are now more commonly used for diagnostics.

Extensive research showed that using specific oligonucleotides allows sensitive detection and distinguishing of various virus types, including Hepatitis B virus, HSV type 1 and HSV type 2 virus, HIV viruses, and coronaviruses such as SARS-CoV and SARS-CoV-2 in clinical isolates.

Also, specific modification of synthetic oligonucleotides enables the design and synthesis of non-radioactive in situ hybridization probes. Biotin-labeled probes are one example. Over three-fourths of men infected with human immunodeficiency virus (HIV) also have at least one herpes virus detected in their semen. In this case, the cytomegalovirus (CMV) is most prevalent. The presence of CMV is associated with higher T-cell immune activation and with HIV disease progression in treated and untreated individuals.

HIV is known to have a significant genetic variability; therefore, achieving accurate quantification by real-time PCR may require primers and probes specific for each viral variant. An HIV database is available to download sequences for particular variants. 

HIV Genome

Structural Model of the HIV Particle

All primer and probes can be biotinylated or labeled as needed, for example with fluorophores to create hybridization probes.

 Table 1: Primer and Probes for HIV-1 Diagnostics using RT-PCR

.

Reference

Althaus CF, Gianella S, Rieder P, et al. Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance. J Virol Methods. 2010;165:151–60. [Pdf]

Christopherson, C., Kidane, Y., Conway, B., Krowka, J., Sheppard, H., Kwok, S., 2000. PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells. J.Clin.Microbiol. 38, 630-634. [PMC]

Fischer, M., Joos, B., Hirschel, B., Bleiber, G., Weber, R., Günthard, H.F., 2004. Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef. J Infect Dis 190, 1979-88. [Pdf]

HIV database

Kaiser, P., Joos, B., Niederoest, B., Weber, R., Günthard, H.F., Fischer, M., Study, T.S.H.C., 2007. Productive Human Immunodeficiency Virus 1 Infection in peripheral Blood Predominantely Takes Place in CD4/CD8 double negative T Lymphocytes. J Virol 81, 9693-9706. [PMC]

Lewin, S.R., Vesanen, M., Kostrikis, L., Hurley, A., Duran, M., Zhang, L., Ho, D.D., Markowitz, M., 1999. Use of Real-Time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J.Virol. 73, 6099-6103. [PMC]

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

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Like other drugs, oligonucleotide drugs will have to pass through multiple barriers before reaching their cellular targets in the human body. For oligonucleotide-based therapies to work in the central nervous system, therapeutic antisense oligonucleotides (ASOs), interfering RNAs (RNAi), or silencing RNA (siRNA), need to cross the blood-brain barrier (BBB). The term blood-brain barrier (BBB) describes the unique properties of the microvasculature of the central nervous system (See Daneman and Prat for a review).

The blood-brain barrier consists of a system of blood vessels of the central nervous system (CNS) with unique properties that allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. The blood-brain barrier maintains brain homeostasis and prevents foreign molecules from entering the brain.

Oligonucleotide-based molecules potentially allow the design and synthesis of therapeutics for the treatment or cure of many diseases. Conjugation to hydrophobic molecules such as cholesterol and tocopherol and modifications of nucleic acids promise to enhance their delivery into cells or tissue and increase the stability of therapeutic oligonucleotides.

Most medicines approved by the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and other regulatory authorities do not significally accumulate in the brain since these medicines do not cross the blood-brain barrier.

Potentially, oligonucleotide-based agents can treat or cure almost any disease and promise to be critical therapeutic drug classes of the future. Bio-conjugated oligonucleotides are now emerging from basic research and are successfully translated to the clinic.

In 2015, Nishina et al. developed a system for the delivery of short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. Nishina et al. identified a new BNA-DNA gapmer structure in which drug delivery system molecules are bound to ASOs with unlocked nucleic acid (UNA) sequences. The study found an α-tocopherol–conjugated siRNA is effective and safe for RNA interference–mediated gene silencing in vivo.

The research group extended the 5′-end of an ASO sequence by using 5′-α-tocopherol–conjugated to 4- to 7-mer UNAs for improved performance. In the liver of mice, the intravenous injection with the α-tocopherol–conjugated chimeric ASO achieved a more potent silencing than ASO alone. The UNA wing was cleaved or degraded inside the cells resulting in the release of α-tocopherol from the 13-mer gapmer ASO. This reaction resulted in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism in vivo.

In 2021, Nagata et al. showed that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS in mice and rats after subcutaneous or intravenous administration. After administration, the HDOs distribute throughout the brain, spinal cord, and peripheral tissues. The specifically selected HDOs suppressed the expression of four target genes by up to 90% in the CNS. In contrast, single-stranded ASOs conjugated to cholesterol showed limited activity. The research group observed gene knockout in major CNS cell types with the most significant effect in neurons and microglial cells. Subcutaneous delivery or dividing intravenous injections limited side effects. This study showed that cholesterol-conjugated HDOs crossed the blood-brain barrier more effectively making them candidates for future CNS drugs.

How does cholesterol help transport molecules such as oligonucleotides between cells?

Cholesterol transport occurs among organelles. Cholesterol is delivered to lysosomes by low-density plasma lipoprotein (LDL) via receptor-mediated endocytosis. Each LDL particle contains approximately 500 molecules of free cholesterol and about 1,500 molecules of esterified cholesterol. Acid lipase hydrolyzes esterified cholesterol in the lumen of the lysosome. The free cholesterol exits the lysosomal compartment to reach the plasma membrane and the endoplasmic reticulum (ER) to perform its role. The exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its iso-octyl sidechain buried and its 3ß-hydroxyl exposed.

Figure 1: Cholesterol, chemical structure and molecular model.
(Drugbank: DB04540)

In 2009, Kwon et al. solved the structure of the N-terminal domain of NPC1. The two proteins, NTD of NPC1, were thought to interact either with the NPC1's membrane domain or with the membrane domain of a neighboring NPC1 molecule. Several years later, Chu et al., in 2015, reported a dynamic membrane contact between peroxisomes and lysosomes. Lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on the peroxisomal membrane mediates the interaction. The presence of LDL-cholesterol enhances these contacts. As a result, lysosomes transport cholesterol to peroxisomes. These findings established the role of peroxisomes in intracellular cholesterol transport.

Figure 2:  The structure of N-terminal domain of NPC1 shows the distinct subdomains for binding and transfer of cholesterol (3GKI. Kwon et al. 2009).

How does α-tocopherol help transport molecules such as oligonucleotides between cells?

Vitamins are essential for the growth, reproduction, and functioning of the human body. Intracellular carrier proteins solubilize and transport the fat-soluble vitamins A, D, E, and K. Vitamin E comprises a chromanol ring and an aliphatic side chain. In liver cells, the α-Tocopherol transfer protein (α-TTP) recognizes tocopherol by the three methyl groups on the chromanol ring, specifically, the methyl group at position 5, the hydroxyl group on the chromanol ring, and the orientation of the phytyl side chain. The protein α-TTP transfers α-tocopherol between membranes through direct protein-membrane interactions.

The interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans.

Figure 3: Vitamin E homologs. Tocopherols and tocotrienols, each have four isomers (α, β, γ and δ).

Figure 4: Structure Of Human Alpha-Tocopherol Transfer Protein [1OIZ] in complex with a fragment of Triton X-100 [PDB ID 1OIZ].

In humans, the liver protein α-Tocopherol transfer protein (α-TTP) selectively retains tocopherols and tocotrienols from dietary vitamin E. It mediates their transfer to nascent plasma very-low-density lipoprotein (VLFL). The crystal structure of alpha-TTP revealed two conformations. Tocopherol is bound such that a mobile helical surface segment hides the hydrophobic binding pocket. However, detergents are bound in an open conformation. This conformation may represent the membrane-bound form (Meier et al. 2003). 

Bioconjugation chemistry is crucial for the design and synthesis of many therapeutic oligonucleotides.  These molecules enable mechanistic studies at the molecular interphase of biological targets to develop agents for molecular medicine. An example for a conjugated oligonucleotide useful for photoregulatory studies is shown in figure 5.

Figure 5: Example of a phtocleavable cholesterol-conjugated oligonucleotide. This molecule type can be used for the design and synthesis of caged siRNA useful for photoregulation approaches.

Reference

Benizri S, Gissot A, Martin A, Vialet B, Grinstaff MW, Barthélémy P. Bioconjugated Oligonucleotides: Recent Developments and Therapeutic Applications. Bioconjug Chem. 2019 Feb 20;30(2):366-383. [PMC]

Chu BB, Liao YC, Qi W, Xie C, Du X, Wang J, Yang H, Miao HH, Li BL, Song BL. Cholesterol transport through lysosome-peroxisome membrane contacts. Cell. 2015 Apr 9;161(2):291-306. doi: 10.1016/j.cell.2015.02.019. Erratum in: Cell. 2021 Jan 7;184(1):289. [Cell]

Daneman R, Prat A. The blood-brain barrier. Cold Spring Harb Perspect Biol. 2015 Jan 5;7(1):a020412. [PMC]

Kono, N. and Arai, H. (2015), Intracellular Transport of Fat-Soluble Vitamins A and E. Traffic, 16: 19-34. https://doi.org/10.1111/tra.12231

Kwon HJ, Abi-Mosleh L, Wang ML, Deisenhofer J, Goldstein JL, Brown MS, Infante RE. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell. 2009 Jun 26;137(7):1213-24. [PMC]

Meier R, Tomizaki T, Schulze-Briese C, Baumann U, Stocker A. The molecular basis of vitamin E retention: structure of human alpha-tocopherol transfer protein. J Mol Biol. 2003 Aug 15;331(3):725-34. doi: 10.1016/s0022-2836(03)00724-1. PMID: 12899840.

Nagata T, Dwyer CA, Yoshida-Tanaka K, Ihara K, Ohyagi M, Kaburagi H, Miyata H, Ebihara S, Yoshioka K, Ishii T, Miyata K, Miyata K, Powers B, Igari T, Yamamoto S, Arimura N, Hirabayashi H, Uchihara T, Hara RI, Wada T, Bennett CF, Seth PP, Rigo F, Yokota T. Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS. Nat Biotechnol. 2021 Aug 12. doi: 10.1038/s41587-021-00972-x. Epub ahead of print. [PubMed]

Nishina K, Piao W, Yoshida-Tanaka K, Sujino Y, Nishina T, Yamamoto T, Nitta K, Yoshioka K, Kuwahara H, Yasuhara H, Baba T, Ono F, Miyata K, Miyake K, Seth PP, Low A, Yoshida M, Bennett CF, Kataoka K, Mizusawa H, Obika S, Yokota T. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing. Nat Commun. 2015 Aug 10;6:7969. [PMC]

Yang, J., Chen, C., and Tang, X. (2018) Cholesterol-modified caged siRNA for photoregulating exogenous gene expression. Bioconjug. Chem. 29, 1010-1015. [ACS] 

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Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
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 For therapy, the attractiveness of siRNAs lies in their ability for a repeated utilization, thus enabling the degradation of multiple mRNA copies by a given siRNA molecule.  Briefly, the mechanism of RNA interference entails (1) processing of long double stranded RNA by DICER and (2) the cleavage of targeted mRNA by RISC (RNA-induced silencing complex).  Human DICER is a multi-domain protein comprised of helicase, double stranded RNA binding domain, RNAse III, and PAZ (Piwi/Ago/Zwille) (Paturi et al., 2021), which functions in gene regulation, antiviral defense and development.  Human DICER belongs to a RNase III (class IV) family and its substrate selection/cleavage activity is modulated by 2 double strand RNA binding proteins: PACT and TRBP (HIV-1 TAR RNA-binding protein) that interact with its helicase domain.  (Chendrimada et al, 2005; Hasse et al, 2005;  Taylor et al., 2013).

 To generate microRNA (miRNA), nascent transcript (i.e. primary RNA, 'pri-miRNA') is initially processed by DROSHA (nucleus) into 70 bp 'pre-miRNA' with a stem-loop structure and 2 nucleotide 3' overhang, which is then cleaved by DICER (cytoplasm) to generate mature microRNA.  Wheareas dsRNA with 2 nucleotide 3' overhang (pre-miRNA) is recognized by its PAZ domain, dsRNAs with blunt terminii are recognized by its helicase domain (requires ATP to unwind) for the subsequent cleavage by its RNase III domain.  For human DICER, the distance between the PAZ and RNase III domains may determine the length (21 bp) of siRNA or miRNA generated (Lau et al., 2012; MacRae et al., 2006).

In Drosophila, the characteristics of 21-23 bp dsRNA products generated (i.e. 5'-monophosphates and 3' hydroxyls at 5' ends; 2 nucleotides overhang at 3' ends; staggered cuts) indicated that DICER belongs to the RNase III family (as determined by Tuschl and colleagues, Max Planck Institute, Germany)  (Elbashir et al., 2000).

The RISC complex is comprised of DICER, TRBP, and Argonaut 2 (RNase that cleaves mRNA).    In vivo, dsRNA generated by DICER appears to be fed directionally to RISC for the cleavage of target mRNA (hence may select the guide strand) due to proximity; however, synthetic siRNAs may engage RISC in either orientation (Elbashir et al., 2000). 


                    

Previously it was thought that the 5' phosphate may not be essential for silencing; however, a recent report suggests that its presence may be necessary as it contacts a domain in Argonaut 2 of RISC (Roberts et al., 2020).

For 3' overhang, 2-nucleotide was most potent (in silencing) while 4-6 nucleotide was inactive.  Blunt ends or 1-nucleotide 5' overhangs were inconsistent in activity (Elbashir et al., 2000).   Replacing 2 nucleotide 3' overhang with deoxy-form had no effect though complete replacement of all bases with 2'-deoxy or 2'-O-methyl group blocked silencing (nevertheless, for Givlaari, every nucleotide is chemically modified with 2'-F or 2'-O-methyl group).   A commonr industry practice is to use 2 dT residues for 3' overhang (for ease of synthesis, cost, nuclease stability)' however, it may reduce maximum silencing potential (if does not match target sequence or hybridize with a strand containing non-dT overhang) in Drosophila (Boutla et al., 2003) and some suggested that the ribose form may be more potent in humans (Hohjoh, 2002).  Further, the deoxy form of 3' overhang (as well as the sequence of siRNA) may affect the "duration" of silencing negatively whereas 2'-O-methyl group has no effect (Strapp et al., 2010).  Structurally, Patel and colleagues (Sloan Kettering Cancer Center, USA) determined that the PAZ domain (of human Argonaut protein eIF2c1) makes extensive contacts with the guide strand (anchors 2-nucleotide 3' overhang by turning it from the duplex into a protein pocket) and a minimal contact wth the complementary strand (5'-terminal residue) of a siRNA-like molecule (Ma et al., 2004).

As for the backbone, while the use phosphorothioate linkage may confer nuclease resistance, the risk of developing thrombocytopenia (bleeding disorder due to lesser clotting) has been described (Frazier et al., 2015).  The incorporation of sulfur generates chiral centers with distinct potency depending on the specific sterioisomer (Roberts et al., 2020).

For sugar modification, 2ʹ-O-methoxyethyl and 2ʹ-Fluoro are used to provide nuclease resistance or to enhance binding efficacy.   The use of 2ʹ-O-methoxyethyl may  help to avoid inciting innate immuity mediated by TLR (toll like receptors), RIG-1, or PKR system.

Regarding the dose, for Onpattro, ~0.3 mg per kilogram of body mass (or 30 mg for >100 kg individuals) of current GMP grade siRNA was administered intravenously per patient (once every 3 weeks for 18 months for the clinical trial).  Delivery vectors have utilized nanoparticles or N-acetylgalactosamine with the latter targeting asialoglycoprotein receptor, which is highly expressed in the liver cells.  However, for delivering to non-liver tissues, conjugation to peptides or nanoparticles modified with peptides targeting specific tissues is increasingly being sought (Roberts et al., 2020).

These advances led to the approval of the siRNA drug Oxlumo (Lumasiran; Alnylam Pharmaceuticals) to treat primary hyperoxaluria type 1 by FDA in 2020.  Its chemical modifications are patterned after Givlaari.  Hyperoxaluria type 1, which causes kidney stones, is caused by mutant alanine-glyoxylate aminotransferase in the peroxisome of liver cells.  This causes its substrate (glyoxylate) to accumulate, resulting in its production of oxalate, which is catalyzed by glycolate oxidase (it also catalyzes conversion of glycolate to glyoxylate), whose mRNA is targeted by Oxlumo for degradation.  Despite targeting liver, Oxlumo is administered subcutaneously (6 mg/kg body weight monthly for 3 doses, followed by 3 mg/kg monthly for maintenance therapy; annual list price 493,000$). 

The key to preventing epidemic is the ability to diagnose the infected early 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.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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References

Boutla A, Delidakis C, et al. Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos.  Oligonucleotides.  13:295-301 (2003).  PMID: 15000820

Chendrimada TP, Gregory RI, et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.  Nature. 436:740-4 (2005).  PMID: 15973356

Frazier KS. Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist's perspective.  Toxicol Pathol. 2015 Jan;43(1):78-89.  PMID: 25385330

Haase AD, Jaskiewicz L, et al. RBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing.  EMBO Rep. 6:961-7 (2005). PMID: 16142218

Hohjoh H. RNA interference (RNA(i)) induction with various types of synthetic oligonucleotide duplexes in cultured human cells.  FEBS Lett.  521:195-9 (2002).  PMID: 12096714

Lau PW, Guiley KZ, et al. The molecular architecture of human Dicer.  Nat Struct Mol Biol. 19:436-40 (2012).  PMID: 22426548

Ma JB, Patel DJ, et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain.  Nature. 429:318-322 (2004).  PMID: 15152257

Macrae IJ, Doudna JA, et al. Structural basis for double-stranded RNA processing by Dicer.  Science. 311:195-8 (2006).  PMID: 16410517

Paturi S, Deshmukh MV. A Glimpse of "Dicer Biology" Through the Structural and Functional Perspective.  Front Mol Biosci. 8:643657 (2021).  PMID: 34026825

Roberts TC, Langer R, et al. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 19:673-694 (2020).  PMID: 32782413

Strapps WR, Pickering V, et al. The siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing.  Nucleic Acids Res. 38:4788-97 (2010).  PMID: 20360048

Taylor DW, Ma E, et al. Substrate-specific structural rearrangements of human Dicer.  Nat Struct Mol Biol. 20:662-70 (2013).  PMID: 23624860

 For the ongoing pandemic, the mRNA vaccines targeting the spike protein of the COVID-19 coronavirus were introduced in a timely manner to curtail its progression.  This preventive measure relies on the ability of the injected mRNA to mount both the humoral and cellular immunity to preempt the oncoming infection.  The administration of the mRNA vaccines, which began in the early months of 2021 led to a significant decline in the rate of ensuing COVID-19 infections (though it tends to decline during warmer months).  Nevertheless, targeting a single molecular entity (spike protein) of a virus comprised of nearly 29 proteins could be challenging--especially, in lieu of the higher mutation rates of RNA viruses.  Hence, the ability of the vaccine to counter the emerging COVID-19 variants is continually being assessed.  Supporting the therapeutic value of the mRNA vaccines is the finding that the rate of infection /hospitalization was significantly reduced amongst the vaccinated.

For a considerable period, the ability to synthesize nucleic acids has played a pivotal role in biological and medical researches.  It began with the ability to synthesize short oligonucleotides as they could be used in multiple biological applications such as mutagenesis, molecular cloning, DNA sequencing, etc. that involve hybridization.  Equally significant is the ability to utilize oligonucleotides for various medical applications including PCR-based assays for diagnosing the disease onset, testing for genetic counseling, monitoring altered gene expression via microarrays, etc.  For translational medicine, the potential use of oligonucleotides for therapeutic purposes is increasingly being recognized.  This has resulted in numerous ongoing clinical trials and the FDA approval of multiple siRNAs, antisense oligonucleotides, and gapmers.  Continuing with the advance is the application of oligonucleotides for gene synthesis as part of the genome construction endeavor at the DNA level.

At the RNA level, the therapeutic potential of the synthetic nucleic acids was extended to mRNAs, which are significantly longer than oligonucleotides.  The earlier attempts were to intravenously or intradermally inject melanoma patients with dendritic cells, which have been electroporated with mRNA vaccine encoding cancer immunogens (Wilgenhof et al., 2013).   In 2021, the medical utility of the mRNA vaccine technology was tested against the newly emerged COVID-19 coronavirus.  To target the spike protein of COVID-19, it required synthesizing mRNAs of >4 kilobases in length.  To achieve this length, biopharmaceutical industries resorted to the in vitro transcription technology. 

                    

The general design of the template used to produce mRNA vaccines utilized the following format: 5'-cap (to increase translation, stability; to decrease immunogenicity, degradation), 5'-UTR (to promote interaction with ribosome), codon-optimized spike protein coding sequence (to augment translation), 3'-UTR (to enhance stability), and poly-A tail (to increase stability, translation).  The mRNA was engineered to incorporate modified nucleic acids to avoid immunogenicity or degradation, ex. N1-methyl pseudouridine (Nance et al. 2021).  As such, critical to its development was the the employing of a RNA polymerase that could accommodate nucleotides with non-natural bases.

Generally, there are 3 strategies for the production of RNA with modified nucleotides (Milisavljevič et al., 2018).  First approach is to exploit the catalysis of RNA polymerase to incorporate modified NTPs (as in the case of T7 polymerase).  The second method is to modify post-transcriptionally using enzymes (ex. using methyl transferase) after the RNA synthesis.  Third option is through chemical modification of the RNA using bioothogonal (ex. click) chemistry (George et al.., 2017; Holstein et al., 2016; George et al., 2020).

The ability of RNA polymerase to incorporate modified nucleotides was recognized as early as 1963, when the ability of HeLa cell derived RNA polymerase to incorporate pseudouridine (incorporated adjacent to purine nucleotides preferentially) was observed (Goldberg et al, 1963).  Later, the ability of T7 polymerase to incorporate modified NTPs was described, so long as it does not perturb base-pairing (Milligan et al., 1989).  Subsequently, it was found that T7 RNA polymerase permits a wide variety of modifications to nucleotides including biotin (affinity labeling), alkyne/azido groups (click chemistry), 5-vinylU (for further chemical modification), 5-iodoU (for cross-coupling modification), amino acid-like side chain (aptamer selection), diazirin (crosslinking), and fluorophore (Milisavljevič et al., 2018).  T7 RNA polymerase could also tolerate sugar modifications (ex. 2'-O-carbamoyl uridine) (Masaki et al., 2016; Huang et al., 1997)

Several advances preceded the production of the mRNA vaccine for COVID-19.  The bulk synthesis of modified mRNA relied on the cloning/sequencing of T7 bacteriophage gene encoding RNA polymerase by W. Studier and colleagues (Brookhaven National Laboratory, USA) in 1983 (Davaloo et al., 1984).  Further attempts to mutagenize T7 RNA polymerase to obtain variants that could incorporate modified nucleotides (ex. 2'-C-branched uridine) also took place (Siegmund et al, 2012; Pavey et al., 2004).  Critical was the finding by Kariko and colleagues (University of Pennsylvania, USA) in 2005 that nucleotide modifications such as pseudouridine, thiouridine, and 5-methylcytidine decrease immunostimulation by TLRs (toll-like receptors), as it provided foundation for applying mRNA for anti-COVID-19 vaccine (or stem cell technology, cancer therapy potentially) (Nance et al., 2021; Kariko et al., 2005).  The topics of further interest include the mRNA design (ex. the effect of ubiquitous incorporation of N1-methyl pseudouridine on 5'-UTR or 3'-UTR functionality), the impact on diagnosis (Rt-PCR), and recombination.

The key to preventing epidemic is the ability to diagnose the infected early 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.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/tew/Messenger-RNA-turnover-and-their-half-live.aspx#!

https://www.biosyn.com/tew/Pseudouridine,-an-abundant-post-transcriptional-RNA-modification.aspx

https://www.biosyn.com/tew/the-potential-use-of-dna-or-mrna-based-vaccines-incorporating-modified-nucleotides-to-suppress-cancer-or-covid-19-pandemic.aspx

References

Davanloo P, Studier FW, et al. Cloning and expression of the gene for bacteriophage T7 RNA polymerase.  Proc Natl Acad Sci U S A. 81:2035-9 (1984).  PMID: 6371808

George JT, Srivatsan SG. Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry.  Methods. 120:28-38 (2017).  PMID: 28215631

George JT , Srivatsan SG .  Bioorthogonal chemistry-based RNA labeling technologies: evolution and current state.  Chem Commun (Camb).  56:12307-12318 (2020).   PMID: 33026365

Goldberg IH, Rabinowitz M.  Comparative utilization of pseudouridine triphosphate and uridine triphosphate by ribonucleic acid polymerase.    J Biol Chem. 238:1793-800 (1963).  PMID: 13948670

Holstein JM, Rentmeister A.  Current covalent modification methods for detecting RNA in fixed and living cells.  Methods. 98:18-25 (2016).  PMID: 26615954

Huang Y, Eckstein F, et al.  Mechanism of ribose 2'-group discrimination by an RNA polymerase.  Biochemistry. 1997 36:8231-42.  PMID: 9204868

Karikó K,  Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 23:165-75 (2005).  PMID: 16111635

Masaki Y, Ito H, et al. Enzymatic synthesis and reverse transcription of RNAs incorporating 2'-O-carbamoyl uridine triphosphate.  Chem Commun (Camb). 52:12889-12892 (2016).  PMID: 27738673

Milisavljevič N, Perlíková P, et al.   Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates. Org Biomol Chem. 16:5800-5807 (2018). PMID: 30063056

Milligan JF, Uhlenbeck OC.  Synthesis of small RNAs using T7 RNA polymerase.  Methods Enzymol. 1989;180:51-62.  PMID: 2482430

Nance KD, Meier JL.  Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines.  ACS Cent Sci.  7:748-756 (2021). PMID: 34075344

Pavey JB, Lawrence AJ, et al.  Synthesis and transcription studies on 5'-triphosphates derived from 2'-C-branched-uridines: 2'-homouridine-5'-triphosphate is a substrate for T7 RNA polymerase.  Org Biomol Chem. 2:869-75 (2004).  PMID: 15007416

Siegmund V, Santner T, et al.  Screening mutant libraries of T7 RNA polymerase for candidates with increased acceptance of 2'-modified nucleotides.  Chem Commun (Camb). 48:9870-2 (2012).  PMID: 22932771

Wilgenhof S, Van Nuffel AMT, et al. A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients.  Ann Oncol. 24: 2686-2693 (2013).  PMID: 23904461

The advances in molecular biology have enabled a greater understanding of the mechanism underlying cognitive processes and the corresponding disorders.  Transmitting the signals by the nervous system involves a complex interplay between adjacent neurons.  

Prior to the excitation, the membrane of the neurons are kept in a polarized state (-70 miliVolts).  In the synapsis (space between adjacent neurons), the pre-synaptic neuron releases neurotransmitters.  For instance, in the case of sensory neurons, external signals such as light or sound are converted into the release of neurotransmitter.    The binding of the neurotransmitters to the receptors present in the post-synaptic neuron causes it to become depolarized through the action of voltage-gated ion channels, which allows the influx of ions such as sodium into the cell interior.  The rise in potential due to depolarization is followed by the the closure of the sodium channels and the opening of potassium channels to allow outward efflux of potassium ions to repolarize the cell membrane.  This process (i.e. axon potential) involves the sequential activation of ion channels to propagate the signal along the axon, resulting in the nerve conduction with a speed as high as 150 m/s (meter per second) for myelinated neurons.

 The diverse types of neurotransmitters include neuropeptides (ex. somatostatin), amino acids [ex. glutamate, GABA (gamma-aminobutyric acid)], gas (ex. nitric oxide), trace amines (ex. tyramine, tryptamine) in addition to the more commonly known molecules such as acetylcholine.  Among them are monoamines such as serotonin, epinephrine, norepinephrine, dopamine, and histamine.  The neurotransmitter serotonin is involved in a number of important cognitive processes including mood, sleep, thermoregulation, cognition, appetite, learning, vasoconstriction (blood pressure), memory, etc.

 The biosynthesis of serotonin involves converting L-tryptophan to 5-hydroxy-L-tryptophan, followed by its decarboxylation to yield serotonin. Upon excitation, serotonin is released into synapsis, where it binds to the serotonin receptor (categorized into 7 families) to activate the post-synaptic neuron. The released serotonin can be cleared from synapsis via serotonin reuptake pump.   Serotonin is degraded by monoamine oxidase (MAO), which also degrades other monoamine neurotransmitters such as norepinephrine and dopamine.  Following the discovery of two forms of MAO in 1968, the question remained as to whether the MAO A and B isoenzymes represent two distinct proteins or a single enzyme that has been differentially modified post-translationally (Johnston, 1968).  The enigma was solved by J. Shih and colleagues (University of Southern California, USA), who determined that they represent two separate polypeptides through the molecular cloning and sequencing of cDNAs encoding human MAO A and B (Bach et al., 1988).

                    

Lower levels of serotonin are associated with depression, and preventing the reuptake of serotonin from synapsis by the adjacent neurons, or suppressing the degradation of serotonin by inhibiting monoamine oxidase represent current antidepressant pharmacological strategies.  However, the use of non-selective MAO inhibitors (blocks both MAO A and B) can be accompanied by an adverse side effect known as 'cheese reaction', which occurs if administered while consuming fermented food (ex. wine, cheese), processed meat, bean products (ex. soy sauce) or chocolate that are rich in tyramine.  While both MAO A and B are expressed in the central nervous system, MAO A is also expressed in gastrointestinal tissues.  The inhibition of MAO A (plus absence of MAO B) in intestine by the drugs allows tyramine (undegraded) to enter circulation and activate medulla to release norepinephrine, which increases blood pressure suddenly via vasoconstriction.

 The identification of the MAO A gene enabled the generation of a 'knockout' mouse lacking the MAO A, which was done in collaboration with Seif and colleagues [Centre National de la Recherche Scientifique (CNRS), France] (Cases et al., 1995).  The MAO A deficiency led to an increased level of serotonin in the brain of mouse pups.  Intriguingly, adult male mice lacking MAO A exhibited enhanced aggression.  The result is reminiscent of Brunner syndrome (Brunner, 1993), which described abnormal aggression by males from a Dutch family carrying mutated (point deletion) MAO A gene (located in X chromosome) and lacking MAO A completely.  Nevertheless, it is thought that multiple factors including genes and environment may play critical role in the development of complex brain functions that underlie human behavior.

 To characterize the above phenomenon, the genes whose expression was altered in MAO A knockout mice were identified.  It entailed the generation of fluor-labeled probes using cDNAs reverse transcribed from mRNAs isolated from the brain of MAO A mutant mouse pups to hybridize to the Affymetrix GeneChip microarrays (Chen et al., 2017).  The differentially expressed genes identified were involved in various neural activities including cognitive function, neurodevelopment, neurotransmission, and apoptosis.

 Several lines of data suggest that MAO A may also play a role in prostate carcinogenesis.  An elevated expression of MAO A was observed in advanced stage prostate cancer (True et al., 2006, Wu et al., 2014).  The promoter of MAO A gene is regulated by androgen (male hormone) (Ou et al., 2006).  In the PTEN knockout mouse model of invasive prostate cancer, inactivating MAO A gene decreased the incidence of invasive cancer (Liao et al., 2018).  Mechanistically, inhibiting MAO A activity using drugs suppressed the signaling by androgen receptor (Gaur et al., 2019).  These results have led to a clinical trial (Phase II) assessing the efficacy of phenelzine (inhibits MAO A and B) in biochemically recurrent prostate cancer (BRPC) (Gross et al, 2021).  BRPC refers to the cases, in which the rise in PSA (prostate specific antigen) level is detected despite the inability to image metastasized cancer.

The key to preventing epidemic is the ability to diagnose the infected early 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.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies.  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 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 is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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

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

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

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/tew/diagnosing-the-androgen-receptor-splicing-variant-arv7-a-biomarker-of-resistance-to-anti-hormone-therapy-for-advanced-stage-metastatic-prostate-cancer.aspx

References

Bach AW, Shih JC, et al. cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties.  Proc Natl Acad Sci U S A.  85:4934-8 (1988).  PMID: 3387449

Brunner HG, Nelen M, et al. Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A.  Science. 262:578-80 (1993).  PMID: 8211186

Cases O, Shih JC, et al.  Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA.  Science.  268:1763-6 (1995).  PMID: 7792602

Chen K, Shih JC, et al.  Altered gene expression in early postnatal monoamine oxidase A knockout mice.   Brain Res.  1669:18-26 (2017).  PMID: 28535982

Gaur S, Shih JC, et al.  Effect of Monoamine oxidase A (MAOA) inhibitors on androgen-sensitive and castration-resistant prostate cancer cells.  Prostate. 79:667-677 (2019).  PMID: 30693539

Gross ME, Shih JC, et al. Phase 2 trial of monoamine oxidase inhibitor phenelzine in biochemical recurrent prostate cancer.  Prostate Cancer Prostatic Dis.  24:61-68 (2021).  PMID: 32123315

Johnston JP.  Some observations upon a new inhibitor of monoamine oxidase in brain tissue.  Biochem Pharmacol.  17:1285-97 (1968). PMID: 5659776

Liao CP, Gross ME, Shih JC, et al. Loss of MAOA in epithelia inhibits adenocarcinoma development, cell proliferation and cancer stem cells in prostate.  Oncogene.   37:5175-5190 (2018).  PMID: 29844571

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During the past decades, multiple laboratories developed messenger RNA (mRNA) vaccines. However, the recent COVID-19 pandemic has made them now a clinical reality. Current events have shown that mRNA vaccines can rapidly and safely protect patients from infectious diseases. In addition, synthetic mRNA enables the development of a broader range of therapeutic drugs as well. However, additional research is required to optimize the design of mRNA-based vaccines, their intercellular delivery, and their uses beyond SARS-CoV-2 prevention.

Modern synthesis methods and protocols now allow the production of large-scale mRNA-based products. With advances made in "in vitro transcription" (IVT) technology and the development of delivery vehicles, mRNA can now be packaged in lipid delivery vehicles to penetrate cell membranes more easily. A standard method of cell delivery is packaging mRNA into polymeric- and lipid-based nanoparticles. All these improvements have increased the demand for synthetic mRNA as therapeutics for human diseases.

Potential mRNA drugs include vaccines against infectious agents and cancers, cancer therapy, the treatment of genetic disorders, regenerative therapeutics, and their use in immunotherapies.

Biosynthesis Inc. offers affordable custom synthesis of mRNA (up to several kbs), including modifications. Our in-house enzymes generate very high cap-1 efficiency, ensuring the highest translation level with a minimum generation of immunogenicity. Furthermore, Biosynthesis Inc. provides synthetic mRNA in amounts ranging from micrograms to grams.

Our service includes:

• Design, synthesis, and sequence verification of mRNA templates using default pUC vector.

• Adding cap-1 (m7GmrN) cap and polyA tail to increase mRNA stability and translation efficiency.

• Reduces host cell immune response by incorporating modified nucleotides (5mCTP and ψUTP).

• DNase treatment to remove DNA template.

• Purification using oligoT agarose bead.

• Wide range of synthesis scales (from ug to multi-gram).

• Fully traceable ISO compliance documentation system.

QC:

• PAGE gel to confirm the size and purity of mRNA.

• ESI-MS mass spectrometry to confirm cap-1 efficiency (optional).