Priming the innate immune system could prepare our immune system for a future viral attack by SARS-CoV-2, the cause of COVID-19, and prevent infection and even clear the infection faster. A viral infection can induce memory T cells in humans, influencing the severity of disease after reinfection.

The term "trained immunity" refers to the long-term functional reprogramming of innate immune cells. Cells of the innate immune system can remember earlier encounters with pathogens. It is now well established that macrophages, monocytes, and natural killer cells show enhanced responsiveness when reencountering a pathogen.

A host's immune responses are divided into innate immune responses and adaptive immune responses. The innate immune system reacts rapidly, whereas the adaptive immune system is slower but developing a more specific immunological memory. Observations from specific mammalian models of vaccination showed that protection from reinfection could occur independently of T and B lymphocytes. Hence, innate immunity can display adaptive characteristics after challenged with a pathogen or their products. This type of immunological memory is known as "trained immunity" or "innate immune memory."

Recently, Kolodny et al. suggested that the innate immune system's priming could be a way to prepare a human's immune system for future viral attacks such as the infection by SARS-CoV-2, the cause of COVID-19.

How can this be achieved?  The administration of a standard vaccine or any reagent that elicits a broad anti-viral innate immune response could do the job.

Several points will need to be considered when selecting this approach:

• Ensure that the priming does not evoke an autoimmune response.
• Select and test priming agents that do not trigger adverse effects.
  
• Select agents that reduce the likelihood of immune system dysregulation and hyper-inflammation.
  

However, to achieve this, carefully designed clinical trials may be needed to determine this approach's risks and opportunities.

Reference

Cirovic B, de Bree LCJ, Groh L, Blok BA, Chan J, van der Velden WJFM, Bremmers MEJ, van Crevel R, Händler K, Picelli S, Schulte-Schrepping J, Klee K, Oosting M, Koeken VACM, van Ingen J, Li Y, Benn CS, Schultze JL, Joosten LAB, Curtis N, Netea MG, Schlitzer A. BCG Vaccination in Humans Elicits Trained Immunity via the Hematopoietic Progenitor Compartment. Cell Host Microbe. 2020 Aug 12;28(2):322-334. [PMC]

Kolodny O, Berger M, Feldman MW, Ram Y. A new perspective for mitigation of SARS-CoV-2 infection: priming the innate immune system for viral attack. Open Biol. (2020) 10:200138.

Mihai G. Netea, Leo A. B. Joosten, Eicke Latz, Kingston H. G. Mills, Gioacchino Natoli,Hendrik G. Stunnenberg, Luke A. J. O’Neill, Ramnik J. Xavier; Trained immunity: A program of innate immune memory in health and disease Science  22 Apr 2016: Vol. 352, Issue 6284, aaf1098. [sciencemag] [Figures and Table]

---...---

Oligonucleotides are relatively stable molecules. However, to avoid degradation and loss, it is essential to store oligonucleotides under the right conditions.

Handling, stability, and storage of oligonucleotides

DNA oligonucleotides free of DNAse are relatively stable in deionized water. However, in an aqueous solution, slow acid-catalyzed depurination can occur. Because of its chemical structure, RNA is less stable than DNA. Exposure of RNA to small amounts of RNases will impact the stability of RNA oligonucleotides.

Since RNases are prevalent in many standard laboratory conditions, use RNAse free water and buffers for RNA storage and handling. RNA is most stable when stored as an ethanol precipitate at -80°C.

Long term storage

For long term storage, the temperature is most important. Hence, it is best to store oligonucleotides in solid form or a solution frozen at -20°C or -80°C.   

For storage at four (4) °C, resuspend oligonucleotides in TE buffer. Oligonucleotides resuspended in the correct buffer are more stable at room temperature than dried oligonucleotides.

In general, store oligonucleotides in the dark and avoid exposure to UV light.

Store oligonucleotides modified with photolabile group frozen in the dark at -20°C, or lower and avoid light exposure. 

Store RNA oligonucleotides as ethanol precipitate at -80°C.

Short term storage

Oligonucleotides stored at 37 °C or 98.6°F are stable up to 6 weeks when kept dry, resuspended in water or TE buffer.  

Oligonucleotides stored in water at room temperature are usually stable for up to 6 weeks.

Aliquoting oligonucleotides

Divide oligonucleotide solutions after receiving into aliquots and store in several tubes or vials in the freezer at -20°C. 

If possible, select aliquot volumes between 50 to 250 μl and label them carefully.

Freeze-dried or lyophilized oligonucleotides 

Liquid nitrogen or a similar approach allows the freezing of oligonucleotides in aqueous solutions. A high vacuum lyophilizer freeze-dries the samples. This process removes water by sublimation and results in a fluffy white powder. However, because of the water-loving nature (hydrophilicity) of nucleic acids, a freeze-dried solid oligonucleotide can contain approximately 40% of water by weight. Therefore, quantitation of the amount of oligonucleotide present needs to be done after dissolving in water by measuring the absorbance.

Freeze-drying allows storing oligonucleotides for long periods.

---...---
 

Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, base, sugar and internucleotide linkages.
Bio-Synthesis specialize in complex oligonucleotide modifications using phosphodiester backbone, purine and pyrimidine heterocyclic bases, and sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

Contact us for more info

Ribose 2’-O-methylation is a molecular signature of self and non-self.

Messenger RNAs (mRNAs) of higher eukaryotes have 5’-cap structures with ribose 2’-O-methylation. The mRNA can occur methylated at the 2’-O position of the first and second nucleotide upstream of the 5’-cap, known as cap 1 and cap 2. Uncapped RNA molecules, for example, nascent viral transcripts, may be detected as "non-self" by host cells. This event triggers an antiviral innate immune response by producing interferons.

Figure 1: RNA cap structure: The Cap 0 structure consists of a guanosine residue, methylated in the N-7 position bound to the terminal 5’-end nucleotide of the mRNA via a 5’-5' triphosphate bridge. Subsequent 2’-O-methylation in the ribose of the first, or both the first and the second, transcribed mRNA nucleotides generates cap1 or cap2, respectively. Adapted from Belenage et al.

{ Bélanger, F.; Stepinski, J.; Darzynkiewicz, E.; Pelletier, J. Characterization of hMTr1, a human Cap1 2’-O-ribose methyltransferase. J. Biol. Chem. 2010, 285, 33037–33044 [PMC];  Dilyana G. Dimitrova, Laure Teysset and Clément Carré; RNA 2’-O-Methylation (Nm) Modification in Human Diseases. Genes 2019, 10, 117 [PMC] }

Züst et al., in 2011, demonstrated that human and mouse coronavirus mutants that do not have a 2’-O-methyltransferase induced a higher type I interferon expression and were sensitive to type I interferon. The study showed that the 2′-O-methylation of mRNA provides a molecular signature with a dual role in the interaction with host innate immune responses.

2’-O-Methylation of mRNA protects viral RNA from recognition by melanoma differentiation-associated protein 5 (Mda5). Mda5 functions as a pattern recognition receptor and detects double-stranded RNA (dsRNA). The presence of the modification prevents Mda5-dependent production of type I interferon in virus-infected cells.

2’-O-Methylation of viral mRNA allows the virus to evade interferon-mediated restriction of viral replication. 2’-O-methylation of viral mRNA is vital in innate immune responses in host cells. The methylation status in DNA allows distinction between self and non-self-nucleic acids since methylated CpG dinucleotide motifs in DNA activate Toll-like receptor 9. When Mda5 detects the absence of 2’-O-methylation in the mRNA, it initiates an immune response. A functioning innate immune response in the host relies on the reliable detection of pathogens. This immune response allows the host to limit pathogen replication and spread.

The researcher studied coronaviruses because coronaviruses encode their own 5′ mRNA cap-methylation machinery. This feature allows the investigation of recombinant viruses' phenotype with mutant 2′-O-methyltransferase proteins. The study showed that 2′-O-methyltransferase activity is associated with the viral nonstructural protein nsp16. The nsp16 protein is highly conserved among coronaviruses and an integral subunit of the viral replicase-transcriptase complexes located at virus-induced double-membraned vesicles in the host cell cytoplasm.

Züst et al. produced a mutant by substituting alanine for the aspartic acid at position 129 of the highly conserved catalytic KDKE tetrad of nsp16 (HCoV-D129A). The substitution result was a completely deactivated 2′-O-methyltransferase activity in recombinant, bacteria-expressed nsp16 proteins of feline coronavirus and severe acute respiratory syndrome coronavirus. However, using the vaccinia virus 2′-O-methyltransferase VP39 in vitro again methylated poly(A)-containing RNA from HCoV-D129A-infected cells.

{ Züst R, Cervantes-Barragan L, Habjan M, Maier R, Neuman BW, Ziebuhr J, et al. (February 2011). "Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5". Nature Immunology. 12 (2): 137–43. doi:10.1038/ni.1979. PMC 3182538. PMID 21217758. [Nature] }

Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, base, sugar and internucleotide linkages including messenger RNA.
Bio-Synthesis specialize in complex oligonucleotide modifications using phosphodiester backbone, purine and pyrimidine heterocyclic bases, and sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

---...---

The spike (S) protein of the virus mediates receptor binding and membrane fusion. The sequence specificity also defines the range of the hosts and the specificity of the virus. Also, the S protein is the target for a variety of neutralizing antibodies and vaccine design.

Gene recombination or mutations of the receptor-binding domain (RBD) appears to allow transmission between different hosts. Some of these mutations can lead to a higher mortality rate of the infected hosts. Some observed mutations are in the reference sequence for the spike protein. Korber et al. developed a analysis pipeline to track mutations in the SARS-CoV-2 spike protein. However, the clinical evidence of all these mutations still needs to be verified.

Schematic of the coronavirus spike (S) protein domain structure

The location of receptor-binding subunit S1, the membrane-fusion subunit S2, the transmembrane anchor (TM), the intercellular tail (IC) are indicated. The location of the S1 N-terminal domain (S1-NTD), the S1 C-terminal domain (S1-CTD), the fusion peptide (FP), and the heptade repeat regions N and C (HR-N and HR-C) are indicated as well.

The following image shows the annotated sequence for SARS-CoV-2 spike protein mutations from the coronavirus associated with COVID-19 originating in Wuhan of Hubei province in China. Source: ORIGIN   YP_009724390, 1273 a, surface glycoprotein [Severe acute respiratory syndrome coronavirus 2].

     N-term.  S1

   1 MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHSTQDLFLPFFSS1

     ---------- ---------- ---------- ---------- ---------- ---------- 
  61 NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV
     ---------- ---------Y ---------- -------F-- ---------- ----------
                      S:D80Y               S:S98F
 121 NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE

     ---------- ---------- ---------- ---------- ---------- ----------
 181 GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT
     ---------- ---------- ---------- ---------- -V-------- ----------
                                                 20A.EU1
 241 LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK
     ---------- ---------- ---------- ---------- ---------- ----------

 301 CTLKSFTVEK GIYQTSNFRVQPTESIVRFP NITNLCPFGEVFNATRFASVYAWNRKRISNRBD

     ---------- ---------- ---------- ------SL-- IL-S---S-- -S-D----N-

 361 CVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD

     ------F--S -T----LN-- ---LA----- --P-I----- ----VQ-IE- ---R--N---
     ---------- -S-----R-- ---------- ---------- ---------- ---E------
     ---------- ---------- ---------- ---------- ---------- ---A------

 421 YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC
     ---------- ----SR--KK ---RFV---- -RF--L-R-- ---------- QV---SNIS-
                                                                20A.EU2
     ---------- ---------- -----S---- -----E-Q-- ---------- ------R---
     ---------- ---------- ---------- ---------- ---------- ------I---
                     ACE2 BINDING
 481 NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHA PATVCGPKKS TNLVKNKCVN
     DSA-SS---S ---P----R- --F-C--H-APA-------- ---------- ----------
     --I------- ---------- ---------SRS-------- ---------- ----------

Reference

Antiviral Peptides SARS-CoVs

Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG, Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res. 2020 Apr;176:104742. [PMC]

Coronavirus Genomes

COVID Vaccine Peptides

Du L, He Y, Zhou Y, et al. The spike protein of SARS-CoV--a target for vaccine and therapeutic development. Nature reviews. Microbiology. 2009 Mar;7(3):226-236. [PMC]

Emma B. Hodcroft, Moira Zuber, Sarah Nadeau, Iñaki Comas, Fernando González Candelas, SeqCOVID-SPAIN consortium, Tanja Stadler, Richard A. Neher; Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020. medRxiv 2020.10.25.20219063.
 
Isabel, S., Graña-Miraglia, L., Gutierrez, J.M. et al. Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide. Sci Rep 10, 14031 (2020). [pdf]

B Korber, WM Fischer, S Gnanakaran, H Yoon, J Theiler, W Abfalterer, B Foley, EE Giorgi, T Bhattacharya, MD Parker, DG Partridge, CM Evans, TM Freeman, TI de Silva, on behalf of the Sheffield COVID-19 Genomics Group, CC LaBranche, DC Montefiori; Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. bioRxiv 2020.04.29.069054. [pdf]

Lai AL, Millet JK, Daniel S, Freed JH, Whittaker GR. The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner. J Mol Biol. 2017 Dec 8;429(24):3875-3892. [PMC]

Shen S, Tan TH, Tan YJ. Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV. Methods Mol Biol. 2007;379:127-35. [PMC]

Structure and sequence of SARS-CoV-2

Tang T, Bidon M, Jaimes JA, Whittaker GR, Daniel S. Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral Res. 2020 Jun;178:104792. doi: 10.1016/j.antiviral.2020.104792. [PMC]

Therapeutic Peptides

Therapeutic strageties for vaccination

Xiao X, Dimitrov DS. The SARS-CoV S glycoprotein. Cell Mol Life Sci. 2004 Oct;61(19-20):2428-30. [PMC]

---...---

In this regard, nucleic acid-based vaccines could be ideal as they can be prepared in a short period of time without requiring an extensive knowledge regarding the biology of the virus.  There are two types of the vaccines: DNA or mRNA--with the former being easier to prepare with less concern about the stability/degradation and lower costs.  Upon internalization, the DNA vaccine must travel to the nucleus to be transcribed to mRNA (a process which may pose the risk of potential integration into the genomic DNA), which must then be exported to the cytoplasm to express antigen. Thus far, a limited success has been achieved with DNA vaccines in clinical trials presumably due to the insufficient 'strength' of the immunity induced (Kallen et al., 2014).

In contrast, mRNA vaccines only need to translocate across the cell membrane to allow the translation of the encoded antigen in the cytoplasm.  The issue with low stability may be resolved through lyophilization as the lyophylized mRNA vaccines was reported to stay undegraded for a considerable duration at 25-40^oC.  As for the cost, mRNA vaccines could be made available at prices comparable to other vaccines based on protein, DNA, peptide, etc.  The genetic target of mRNA vaccine could be switched from one to another easily and its production could be adapted to meet the GMP manufacturing standard.

                    

The early works demonstrated that direct injection of DNA or RNA expression vectors into muscle could allow protein expression in mice (Wolff et al., 1990).  This has inspired the development of vaccines comprised of mRNA-encoded antigens that could be delivered ex vivo or injected intradermally (underneath the skin).  The 'ex vivo' approach refers to the process of excising tissues, transfecting with a vaccine construct (ex. via eletroporation) outside the body, and then returning it to the individual.  The initial trial involved treating advanced-stage melanoma patients with dendritic cells electroporated with mRNA vaccines encoding MAAs (melanoma associated antigens), i.e. MAGE-A3, tyrosinase, MAGE-C2, gp100 (Wilgenhof et al. 2011).

Critical to this undertaking was the recognition of the role of dendritic cells in antigen presentation, the ability to expand dendritic cells in vitro, and the identification of tumor-associated antigens (Van Nuffel et al., 2010).  The commonly used approach is to characterize the 'mutanome' (mutated gene sequences) via deep sequencing to identify mutated sequences that are highly immunogenic as mRNA vaccine.  The antigen encoded could be chimeric as adding a signal peptide to N-terminus or an endosomal trafficking signal to C-terminus could improve antigenic presentation by MHC molecules (Kreiter et al., 2008).  Further, the maturation status of dendritic cells could affect the uptake efficiency of mRNA vaccines {Kuhn et al., 2010).

Several types of mRNA vaccines currently exist.  The 'non-replicating mRNA' vaccine consists of the coding sequence (of the antigen) that is 5'-capped, 5' and 3' untranslated regions, and poly-A sequence. The use of T7 polymerase to generate mRNA transcript using linearized plasmid may generate double stranded RNAs due to self-priming (Triana-Alonso et al., 1996).  The mRNA designed by Moderna Therapeutics (USA) incorporated modified nucleotides to avoid activating interferon-associated genes, and added 2 proline residues to stabilize the spike protein.  While a lipid-based nanoparticle was used to protect mRNAs, delivery vector remains a significant issue.  A phase I clinical trial (doses from 25 ug to 250 ug) showed that it induced antigen-binding and neutralizing antibodies (plus activating T cells) while causing side effects (ex. chills, fatigue, headache, muscle pain) which augmented after the second dose (Jackson et al., 2020). Phase III study (to determine if it can protect against COVID-19) is still ongoing.  In contrast, the 'self-amplifying mRNA' vaccine is generated using alphavirus whose structural gene is replaced with an antigen of interest while retaining the genes mediating viral replication. Being a positive stranded RNA virus, it generates double stranded RNA as intermediates, which could potentially trigger innate immunity (ex. cytokine release) (Vogel et al., 2018). Alternatively, the construct may contain another cistron expressing replicase to amplify the mRNA vaccine (Jackson et al., 2020).    

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.  For medicinal chemistry, it specializes in peptide synthesis, characterization, modification, purification to generate various peptide-based building blocks as well as pharmaceutical intermediates—in addition to peptide libraries, peptide arrays, peptidomimetics.   Antibody purification, characterization/quantification, modification and labeling are also offered.  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 conjugate to peptides.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology 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/mRNA-vaccines-and-innate-immunity.aspx#!

https://www.biosyn.com/tew/Messenger-RNA-(mRNA)-for-Vaccine-Development-Against-Coronavirus.aspx

https://www.biosyn.com/tew/Ribose-2’-O-methylation,-“self-and-non-self,”-and-Coronaviruses.aspx

References

Callaway E.  Why Oxford's positive COVID vaccine results are puzzling scientists.   Nature.  Nov 23 (2020).  PMID: 33230278

Jackson NAC, Kester KE, et al. The promise of mRNA vaccines: a biotech and industrial perspective.  NPJ Vaccines. 5:11 (2020). PMID: 32047656

Kallen KJ, Theß A.Kallen KJ, et al. A development that may evolve into a revolution in medicine: mRNA as the basis for novel, nucleotide-based vaccines and drugs.  Ther Adv Vaccines. 2:10-31 (2014).  PMID: 24757523

Kreiter S, Castle JC, et al. Targeting the tumor mutanome for personalized vaccination therapy.  Oncoimmunology.  1:768-769 (2012). PMID: 22934277

Kuhn AN, Diken M, et al. Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther. 17:961-71 (2010).  PMID: 20410931

Triana-Alonso FJ, Dabrowski M, et al. Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase.  J Biol Chem 270:6298-307 (1995).  PMID: 7534310

Van Nuffel AM, et al. Immunotherapy of cancer with dendritic cells loaded with tumor antigens and activated through mRNA electroporation. Methods Mol Biol 629:405-52 (2010).  PMID: 20387165

Vogel AB, Lambert L, Kinnear et al.  Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses.  Mol Ther.  26:446-455 (2018). PMID: 29275847

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

Wolff JA, Malone RW, Williams P, et al.  Direct gene transfer into mouse muscle in vivo.    Science 2471465-8 (1990). PMID: 1690918

Zhang C, Maruggi G, et al.  Advances in mRNA Vaccines for Infectious Diseases.  Front Immunol. 10:594 (2019). PMID: 30972078

Primers for influenza viruses, parainfluenza viruses, adenoviruses, coronaviruses, orthohantaviruses, respiratory syncytial virus and human metapneumovirus, and rhinoviruses.

Several viruses can infect the lower and upper respiratory tract of us humans. The respiratory system allows humans to breathe. The respiratory system includes several organs and structures needed to exchange gases such as oxygen (in) and carbon dioxide (out). Parts of the respiratory system are the nose and nasal cavity, the sinuses, mouth, throat, voice box, windpipe, diaphragm, and the lungs.

Many common viral infections target the upper respiratory system causing severe symptoms in infants, the elderly, and patients with lung or heart problems. 

The list of common respiratory viruses includes the epidemic influenza viruses A, B, C, avian influenza viruses, parainfluenza viruses 1–4, adenoviruses, coronaviruses, orthohantaviruses, and respiratory syncytial virus and human metapneumovirus, as well as rhinoviruses. 

Symptoms of different respiratory infections, also known as clinical presentation, caused by various viral pathogens, can be very similar. Hence, the correct diagnosis is quite tricky. A rapid virological method will allow a specific and sensitive diagnosis at an early stage of the infection. Significant advances in modern molecular technics have enabled the speedy and sensitive detection of viral pathogens. Polymerase chain reaction (PCR) based methods are now considered as the gold standard of viral assays. For many RNA viruses, including respiratory viruses, multiplex reverse transcription (RT)-PCR assay-based diagnosis allows rapid, sensitive, and specific detection.

Coiras et al., in 2004, developed a multiplex RT-nested PCR assay for the detection and identification of several respiratory viruses.These include the human parainfluenza viruses types 1, 2, 3, and 4AB, the coronaviruses type 229E and OC43, and generic human enteroviruses and rhinoviruses. The researchers designed primers selecting sequences from the conserved regions of haemagglutinin genes, the conserved regions of coronavirus spike protein genes, and the polyprotein gene of rhinoviruses and enteroviruses, between the 5’-non-coding region (5’-NCR) and VP4/VP2 regions. Table 1 lists GenBank accession numbers of the viral sequences,  sequences, and properties of all primers studied.

Table 1: Primers for Respiratory Viruses including Human Parainfluenza Viruses (Parainf.), Coronaviruses, Enteroviruses (Enterov.), and Rhinoviruses (Rhinov.) Used in the First Round Multiplex RT-PCR and in the Following Nested PCR (Adapted from Coiras et al.).

^a1, forward;2,reverse in first-round RT-PCR. ^b3, forward;4,reverse in nested PCR. ^cPrimer located up-stream from coding region for haemagglutinin gene. ^dGene position referred to Poliovirus1strain Sabin (Accession no. V01150). Note: All rhinoviruses have a deletion of approximately 116 bp as regards enteroviruses.

Van de Pol et al. used primers for real-time PCR diagnostic of respiratory viruses from patients admitted with respiratory symptoms. Diagnostic of specific respiratory viruses allows clinicians to initiate optimal patient management and initiate adequate (future) use of antiviral therapy and optimal infection control.

Table 2: Primers and probes for real-time PCR detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses (Adapted from van de Pol et al.).

 ^aFAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binding.

Reference

M.T. Coiras, J.C. Aguilar, M.L. García, I. Casas, and P. Pérez-Breňa; Simultaneous Detection of Fourteen Respiratory Viruses in Clinical Specimens by Two Multiplex Reverse Transcription Nested-PCR Assays. Journal of Medical Virology 72:484–495 (2004). [PMC]

Infectious Diseases

The Respiratory System

Alma C. van de Pol, Anton M. van Loon, Tom F. W. Wolfs, Nicolaas J. G. Jansen, Monique Nijhuis, Els Klein Breteler,1 Rob Schuurman, and John W. A. Rossen;  Increased Detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses with Real-Time PCR in Samples from Patients with Respiratory Symptoms. JOURNAL OF CLINICAL MICROBIOLOGY, July 2007, p. 2260–2262. [PMC]

---...---

The following contains a list of definitions for terms commonly used in a laboratory.

      Table 1:  Some Definitions of Chemical and Biochemical Terms used in Laboratories

---...---

 Paralleling the situation with breast cancer, prostate cancer may occur in secretory glands and is regulated by hormones--i.e. the male hormone testosterone.  Most of adenocarcinoma (accounts for 95-99% of prostate cancers) occur in acinar (secreting cells) than ductal (tubular cells) part. Recent researches suggest that the conversion of prostate gland cells into a cancerous state may entail mutation in genes such as ETS (transcription factor) in early stage, and in PTEN (occur in ~70% of patients), RB (decreases survivability 3-fold), p53 (tumor suppressor), ATM, CHK2 (DNA damage checkpoint), MMR, BRCA1/2 (DNA repair), etc. in later stages involving metastasis (Arora et al., 2018).

 A commonly used biomarker in the initial screening of prostate cancer is PSA (prostate specific antigen).  It represents a serine protease secreted by the prostate gland, which is normally present at a low level in the blood.  PSA may exist in either a 'free' state or in complex with serum proteins (ex. alpha 1-antichymotrypsin).  The elevation in the PSA level is a risk factor for prostate cancer though certain subtypes (ductal prostate cancer, neuroendocrine tumor, small cell carcinoma, etc.) may occur in the absence of such increase.  Other conditions like prostatitis (inflammation) or benign hyperplasia may increase the PSA level, resulting in false positives.  Thus, further diagnosis via the histological analysis of biopsied specimens is necessary to confirm positivity.

 The staging of prostate cancer (from T1 to T4) incorporates Gleason scoring system (2 to 10 with higher score representing aggressive cancer with poor outcome based on histopathological examination of the tumor).  At T2 stage, the tumor volume is larger than at T1; however, tumors are confined to within the prostate in both stages.  In T3 stage, tumors have invaded to nearby organs such as bladder; in T4, it has metastasized to distant organs such as the bone or nearby lymph nodes, which may be incurable despite the treatment.  The onset of 'biochemically recurrent' prostate cancer is indicated by the rise in PSA level despite the inability to monitor them through imaging--ex. metastatic prostate cancer.  T1 stage prostate cancer may remain stable for a considerable period but treatment options include prostectomy (surgically removing prostate), radiotherapy, etc. 

 As with breast cancer, anti-hormone therapy represents a significant part of the current treatment strategies for prostate cancer.  The testosterone level could be lowered (~95%) through further surgery or via treatment with drugs that block upstream signaling events occurring at the level of the brain (ex. lutenizing hormone releasing hormone agonist, or its receptor antagonist).  Despite the lowering of testosterone level, cancer may still grow (He et al., 2020).

               

Mechanistically, testosterone freely crosses the cell membrane to interact with its receptor (a nuclear receptor called 'androgen receptor') in the cytoplasm.  Binding of the ligand to the receptor activates (alters conformation) and dissociates from Hsp protein to enter the nucleus, where it transcriptionally activates >200 genes (including the PSA gene) by binding to the enhancer of the target gene's promoter (Jin et al., 2013).

Testosterone could also be produced by adrenal gland (situated above kidney) or prostate tumor tissue.  Other anti-hormone therapeutics (androgen-deprivation therapy: ADT) target events concerning testosterone directly.  Analogous to breast cancer chemopreventive therapeutics targeting estrogen biosynthesis pathway, abiraterone acetate is converted to its metabolite abiraterone, which inhibits the biosynthesis of testosterone.  Another strategy is to counter testosterone's ability to activate its cognate receptor, i. e. 'androgen receptor'.  Enzaluatemide is a novel anti-androgen drug, which is used to treat 'metastatic CRPC' (median survival of 9-13 months).  In addition to its antagonizing activity, the latter may prevent binding of the receptor to DNA or co-activators. 

Eventually, most prostate cancers become insensitive to anti-hormone therapy.  Diverse types of mutations have been reported: for instance, switching the antagonist drug to agonist, or rendering the androgen receptor responsive to irrelevant hormones, etc.  It may also occur through mutations that allow the receptor to be constitutively active without binding to testosterone.  Among the splicing mutants, ARv7, which lacks the ligand-binding domain, was the only variant detected at the protein level (Wadosky et al., 2017).  Though the ARv7 mutation is present in ~1% of early stage cancer, nearly 75% of metastatic prostate cancers harbor the splicing mutation (Zhang et al., 2020).  The landmark study by investigators at the Johns Hopkins School of Medicine (USA) demonstrated the association between ARv7 and the resistance to anti-hormone therapy (Antonarakis et al., 2014).  Further clinical study showed that ARv7-positive patients who received Taxane therapy (ex. Taxol derivative) had better outcome than those who received anti-hormone therapy (abiraterone or enzalutamide) (Antonarakis, 2015).  Finally, another mechanism through which the insensitivity to anti-hormone therapy could occur is through its ability to grow independent of testosterone.

As a result, ARv7 has emerged as an important therapy biomarker for advanced stage prostate cancer.  To detect ARv7 mRNA, RT-PCR could be performed on circulating tumor cells (CTCs) isolated from blood, which is more accessible than tumor biopsies--ex. Qiagen Adna Test ProstateCancerPanel AR-V7 Test.  Alternatively, CTCs could be assayed for the presence of ARv7 protein in the cell nucleus--ex. Oncotype DX AR-V7 Nucleus Detect Test.  As CTCs are rare, detecting the ARv7 mRNA in blood, urine, saliva or other liquid biopsies as circulating cell-free nucleic is being investigated extensively (Boerrigter et al., 2019).  One disadvantage of using tumor biopsies is that it may yield heterogeneous results depending on the specific loci being assayed.  In light of persisting Covid-19 pandemics, a point-of-care (POC) diagnostic method capable of detecting ARv7 would be desirable.

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.  For medicinal chemistry, it specializes in peptide synthesis, characterization, modification, purification to generate various peptide-based building blocks as well as pharmaceutical intermediates—in addition to peptide libraries, peptide arrays, peptidomimetics.   Antibody purification, characterization/quantification, modification and labeling are also offered.  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 conjugate to peptides.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology 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

References

Antonarakis ES, Lu C, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer.  N Engl J Med 371:1028-38 (2014).  PMID: 25184630

Antonarakis ES, Lu C, Luber B, et al. Androgen receptor splice variant 7 and efficacy of taxane chemotherapy in patients with metastatic castration-resistant prostate cancer. JAMA Oncol 1:582-91 (2015).  PMID: 26181238

Arora K, Barbieri CE. Molecular Subtypes of Prostate Cancer.   Curr Oncol Rep. 1;20:58 (2018). PMID: 29858674

Boerrigter E, Groen LN, et al. Clinical utility of emerging biomarkers in prostate cancer liquid biopsies.  Expert Rev Mol Diagn. 20:219-230 (2020).  PMID: 31577907

He L, Fang H, et al. Metastatic castration-resistant prostate cancer: Academic insights and perspectives through bibliometric analysis.  Medicine 99:e19760 (2020).  PMID: 32282738

Jin HJ, Kim J, et al. Androgen receptor genomic regulation.  Transl Androl Urol. 2:157-177 (2013). PMID: 25237629

Wadosky KM, Koochekpour S.  Androgen receptor splice variants and prostate cancer: From bench to bedside.  Oncotarget  8:18550-18576 (2017).  PMID: 28077788

Zhang T, Karsh LI, et al.  Androgen Receptor Splice Variant, AR-V7, as a Biomarker of Resistance to Androgen Axis-Targeted Therapies in Advanced Prostate Cancer.    Clin Genitourin Cancer 18:1-10 (2020).   PMID: 31653572

Vaccine development based on messenger RNA (mRNA) is a promising new very useful vaccination approach the production of vaccines against coronaviruses such as SARS-CoV or SARS-Cov-2 (COVID-19) as well as other viruses. mRNAs based vaccines are a promising alternative to conventional vaccines.

The stability of messenger RNAs (mRNAs) influences gene expression in all organisms. In mammals, the abundance of a particular cellular mRNA can fluctuate significantly as the mRNA's half-life changes without any change in transcription. Jess Ross, in a paper published in 1995, discussed the following three questions:

1.  Which sequences in mRNAs determine their half-lives?
2.  Which enzymes degrade mRNAs?
3.  Which factors regulate mRNA stability?

Many mRNAs appear to be degraded by a multistep pathway such that the later step depends on earlier events, for example. The shortening of poly(A) often precedes decay of the mRNA body. For example, the poly(A) tracts of globin mRNAs in young, newly formed developing red blood cells, just entering the circulation, are longer than those in older reticulocytes. Hence, the measured degradation or decay constants (kds) for globin mRNAs can differ in the two reticulocyte populations. As a result, mRNA decay kinetics do not necessarily follow the ideal situations or models. The quantity of full-length polyadenylated and deadenylated mRNA as measured by hybridization assays depends on the mRNA population's shortening rate and age. The cell type can influence the variables and mRNA studied. Sharova et al., in 2009, studied the half-lives of gene expression using whole-genome microarrays in pluripotent and differentiating mouse embrionic stem cells and found that decay rates of mRNAs vary substantially between genes.

Figure 1: A modified model of Francis Cricks “Central Dogma of Molecular Biology.” Schwanhausser et al. (2011) determined concentrations and degradation rates for 45000 mRNAs and proteins. Their mathematical model using these data describes the cellular dynamics governing protein production. Their analysis showed that ‘transcription is only half the story’, and translational control is as important in determining the final concentrations of proteins.

Factors determining mRNA stability and half-lives

Cis Determinants of mRNA stability

Poly(A):  Poly(A) protects mRNAs from rapid degradation.

(i)  Deadenylation is the first step in mRNA decay.
(ii)  A poly(A)-poly(A)-binding protein (PABP) complex at the mRNA 3’-end protects mRNAs from rapid destruction in vitro.

3’-untranslated regions (UTRs): UTRs influence the half-lives of mRNAs.

 (i)  Histone mRNA 3’-terminal stem-loop: The 3’-UTRs of histone mRNAs that lack poly(A) affect the processing rates at which the RNA in the nucleus, transported, translated, and degraded. The cell cycle controls these mRNAs. Histone mRNAs (hmRNAs) accumulate to high levels only in S-phase cells. hmRNAs are degraded rapidly at the end of the S phase or when DNA replication is inhibited in S-phase cells. The cis-element at 3'-end on histone mRNAs is responsible for the regulation of histone mRNA degradation.

 (ii) AU-rich elements (AUREs):  mRNAs containing an AURE and/or an oligo(U) region at a 3’-UTR tend to be unstable. Placing an AURE from the 39-UTR of an unstable mRNA within the 39-UTR of β-globin mRNA, from one encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), the chimeric transcript decays with a half-life of less than 30 min. The 3' untranslated region (UTR) of many messenger RNAs (mRNAs) coding for proto-oncogenes, nuclear transcription factors, and cytokines contain AUREs. ARUEs determine RNA stability in mammalian cells. For example, AUUUA sequences facilitate the degradation of the mRNA body.

 (iii) Iron-responsive element (IRE): Transferrin receptor and ferritin encoding mRNAs are regulated post-transcriptionally depending on intracellular iron concentration. The transferrin receptor imports iron into cells. Ferritin is a major iron storage protein in cells.  IRE binds an irons-regulatory protein, which depending on its location within the mRNA has different effects. The interaction transferrin receptor and ferritin mRNAs generate iron homeostasis. IREs regulate the half-life of the mRNAs and their translation.

 (iv) Long-range stem-loop of insulin-like growth factor II (IGF-II):  The insulin-like growth factor II (IGF-II) is necessary for prenatal growth, but its role in adult stem cell physiology is mostly unknown. Ziegler et al., in 2019, demonstrated that IGF-II is critical for multiple adult stem cell niches.
Several sequences in 3’-UTRs influence mRNA stability. 3’-UTRs can also influence mRNA half-lives indirectly, for example, by affecting translation or mRNA localization.

Messenger RNA (mRNA) coding region

The coding region of mRNAs can also determine the half-life of mRNAs.

 (i)  Mutations in the coding region of mRNAs from c-fos, c-myc, and tubulin can change the half-life. Also, sequences containing protein-binding sites influence the stability of mRNAs and their half-lives.

 (ii)  Truncated mRNAs lacking most of their 3’-UTRs have half-lives of 1 to 2 hours.

 (iii)  The introduction of nonsense mutations in the 5’-portion of the coding region destabilizes mRNAs.

5’-Untranslated region, mRNA cap and mRNA localization

5’-UTRs can affect mRNA stability. The introduction of translation-inhibiting stem-loops in the 5’-UTR can change mRNA half-lives several-fold. mRNA half-lives are influences by the length of the UTR, by reciprocal translocations. Longer 5’-UTRs appear to stabilize mRNAs. mRNA without caps are less stable than capped mRNAs, as observed in oocytes and cell-free mRNA decay reactions. Segments affecting the localization of the mRNA can change half-lives as well.

Effector proteins

Recent developments in mapping and quantification of RNA modifications demonstrated that modified residues are present in almost all cellular RNA types. Next-generation sequencing (NGS) technologies allowed transcriptome-wide RNA analysis, including modification mapping revealing the presence of significant mRNA modifications in the transcriptome of eukaryotic cells. The identified modifications are N⁶-methyladenosine (m⁶A), N⁶,2'-O-dimethyladenosine (m⁶Am), 8-oxo-7,8-dihyroguanosine (8-oxoG), pseudouridine (ψ), 5-methylcytidine (m⁵C), 5-hydroxylmethylcytidine, inosine, and N1-methyladenosine (m¹A). Several RNA modifications are now known to regulate mRNA stability. Similar to DNA, RNA can undergo various changes that play a role in cellular and biological processes. The new field studying these modification events in the cell is called "epitranscriptomics."

Effector proteins determine the fate of modified transcripts in a coordinated fashion. These are:

 (i)  RNA-modifying enzymes that transfer specific chemical groups to a target position on an RNA molecule,
      called "Writer Proteins."

 (ii)  RNA-binding proteins (RBPs) that specifically recognize modified nucleotides, called "Reader Proteins."

 (iii)  Proteins that remove specific chemical groups form modified nucleotides, called "Eraser Proteins."

Eraser proteins convert modified RNA back to un-modified RNA. Also, endogenous or exogenous chemicals can damage RNA molecules and create modified RNA without involving proteins. Some modifications are reversible, while others are irreversible.

RNA modifications can affect many molecular processes, including transcription, pre-mRNA splicing, RNA export, mRNA translation, and RNA degradation. The regulation of mRNA stability appears to be a crucial step in regulating gene expression. Molecular events involving RNA modifications help to shape the cellular transcriptome and proteome.

Table 1: Chemical structures of RNA modifications affecting mRNA stability.

 
Chemical structures

Chemical structure for N⁶-methyladenosine (m6A), N⁶,2'-O-dimethyladenosine (m⁶Am), 8-Oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (ψ), 5-methylcytidine (m⁵C), and N⁴-acetylcytidine (ac⁴C) are shown below.

---...---


Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, base, sugar and internucleotide linkages including messenger RNA.
Bio-Synthesis specialize in complex oligonucleotide modifications using phosphodiester backbone, purine and pyrimidine heterocyclic bases, and sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

Exogenous mRNA allows the development of mRNA-based therapeutics to treat a variety of diseases such as genetic disorders, cancer, infectious diseases, cardiovascular diseases, and vaccine development. Recent advances in chemical modifications of mRNA structures to synthesize modified mRNAs improved the stability and translational efficiency within cells when delivering exogenous mRNAs.

Recent examples for the use of exogenous mRNAs are the new vaccines against COVID-19 from Moderna, the COVID-19 vaccine (mRNA-1273), which is an mRNA vaccine against COVID-19 encoding for a prefusion stabilized form of the Spike (S) protein, and the Pfizer-BioNTech COVID-19 vaccine, also encoding the S protein of the virus.

Regulation of cellular gene expression occurs at the post-transcriptional level. Messenger RNA carries genetic information from the nucleus to the cytoplasm needed for the production of functional proteins. For a cell to function well, normal levels of mRNAs need to be maintained. mRNA is a single-stranded RNA transcribed from a DNA template sequence. mRNAs contain untranslated regions that control translation, degradation, and localization of the mRNA’s coding sequence.

RNA sequence regions of mRNAs include stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites, and various cis-acting elements that interact with RNA-binding proteins.

Typically, a mature eukaryotic mRNA consists of a 5’-methylguanosine (m7G) cap, a 5’-untranslated region (UTR), a coding region starting with the AUG codon, a 3’-UTR, and a polyadenylated A (poly(A)) tail. The Kozak sequence, GCCACCAUGG, in the 5’-UTR around the initiating methionine allows for sufficient ribosomal binding to initiate translation.

The 3’-UTR is also essential in regulating mRNAs' translation and stability and may contain miRNA binding sites. The poly(A) tail is a critical component for mRNA translation and degradation. Degradation of mRNAs starts from the 5'-cap structure if the poly(A) tail is shorter than 12 adenosine residues.

The stability and translation efficiency of exogenous mRNAs can be enhanced by many methods, including manipulations of UTRs, codon optimization, chemical modification, and the elongation of the poly(A) tail.

Transcription Control.

A variety of factors control or mediate transcription. Factors mediating transcription are transcription factors, RNA polymerase, a variety of cis-acting elements located in the DNA. Cis-acting elements include promotors, enhancers, silencers, and locus-control elements organized in a modular structure regulating pre-mRNA production.

Pre-mRNAs undergo several processing steps to become functional mRNAs. These are:

(i)    Removal of introns.
(ii)   Addition of a 7-methyl-guanylate (m7G) cap structure at the 5’-end of the first exon.
(ii)   Addition of 100 to 250 adenine residues (the poly(A) tail) at the 3’-end of the last exon.
(iV)  Cleavage of the primary transcript by endonucleolytic processing of the primary transcript.
9v)  mRNA editing.

In eukaryotes, the result is a mature mRNA with a modular structure consisting of a 5'-untranslated region (5'-UTR), a coding region made up of triplet codons that each encode an amino acid, and a 3'-untranslated region (3'-UTR). Figure 1 shows these and other features of mRNAs.

Figure 1: Model of a generic structure of a eukaryotic mRNA. Some post-transcriptional regulatory elements that affect gene expression are illustrated (Adapted from Mignone et al., 2020).

Special UTRs

Modified amino acid selenocysteine at UGA codons mediated by a conserved stem-loop structure in the 3’-UTR.

Nucleotide patterns or motifs located in the 5’-UTRs and 3’-UTRs can interact with specific RNA-binding proteins.

Interactions of sequence elements located in the UTRs and specific complementary non-coding RNAs play vital roles in regulation.

Repetitive elements such as CUG repeats in the 5’-UTR can bind to CUG-binding proteins. The transcription factor C/EBPβ is an example.

Also, the nuclear history of an mRNA can affect its fate in the cytoplasm.

Genomic sequence comparison revealed that the average length of 5’-UTRs ranges in size between 100 to 200 nucleotides and that it is roughly constant over diverse taxonomic classes.

The average length of 3’-UTRs can vary between 200 to 800 nucleotides between species.

Sometimes a single nucleotide in the 5’-UTR can initiate translation.

The G+C content in 5’-UTR sequences is greater than that of 3’-UTR sequences.

Genes localized in large GC-rich regions of a chromosome have shorter 5’-UTRs and 3’-UTRs than genes located in the GC-poor areas or isochores. Isochores are large DNA regions containing a high degree of guanine and cytosine (G-C and C-G).

Eukaryotic mRNAs contain several types of repeat sequences in the untranlated region such as short interspersed elements (SINEs; Alu elements), long interspersed elements (LINEs), minisatellites and microsatellites.

In humans, repeats are found in approximately 12% of 5’-UTRs and 36% of 3’-UTRs.

Reference

Chemical and enzymatic synthesis of mRNA and modified mRNA

COVID-19 Vaccines: Moderna, COVID-19 vaccine (mRNA-1273), and  Pfizer-BioNTech COVID-19 vaccine

Li B, Zhang X, Dong Y. Nanoscale platforms for messenger RNA delivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2019 Mar;11(2):e1530. [PMC]

Mignone F, Gissi C, Liuni S, Pesole G. Untranslated regions of mRNAs. Genome Biol. 2002;3(3):REVIEWS0004. doi: 10.1186/gb-2002-3-3-reviews0004. Epub 2002 Feb 28. [PMC]

mRNA modifications

Pichon X, Wilson LA, Stoneley M, Bastide A, King HA, Somers J, Willis AE. RNA binding protein/RNA element interactions and the control of translation. Curr Protein Pept Sci. 2012 Jun;13(4):294-304. [PMC]

RNA capping: 5’-methylguanosine (m7G) cap

Nishat Sultana, Yoav Hadas, Mohammad Tofael Kabir Sharkar, Keerat Kaur, Ajit Magadum, Ann Anu Kurian, Nadia Hossain, Bremy Alburquerque, Sakib Ahmed, Elena Chepurko, and Lior Zangi; Optimization of 5' Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury. Molecular Therapy: Methods & Clinical Development Vol. 17 June 2020. [link]

---...---

Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, base, sugar and internucleotide linkages including messenger RNA.
Bio-Synthesis specialize in complex oligonucleotide modifications using phosphodiester backbone, purine and pyrimidine heterocyclic bases, and sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.
Bio-Synthesis also provides synthetic capped RNA oligonucleotides.

The 5’-cap of eukaryotic mRNA is required for cellular function. The 5’-cap consist of an inverted 7-methylguanosine connected to the rest of the eukaryotic mRNA via a 5’-5’-triphosphate bridge (m7G cap or cap0). The cap, called cap0, acts as a quality control for correct mRNA processing. Cap0 contributes to the stability of eukaryotic mRNA, splicing, nuclear export, initiation of translation, and mRNA decay. Direct interaction molecules of the cap are the cap binding complex (CBC) in the nucleus needed for nuclear export and the eukaryotic translation initiation factor 4E (eIF4E) in the cytoplasm needed for cap-dependent translation.

Capped RNA is a marker for the innate immune system to distinguish triphosphorylated viral RNAs from cellular RNAs. The cytosolic receptor RIG-I mediates the antiviral response when activated by short single and double-stranded tri-phosphorylated RNAs and RNAs lacking the 2’-OH methylation at the first nucleotide (cap1). In addition to cap0 and cap1, other cap structures exist. Additional methyl groups can often be found at the second nucleotide (cap2).

Uncapped transcripts do not represent eukaryotic mRNAs therefore the preparation of correctly capped RNA is essential for synthetic mRNA used for the development of mRNA-based vaccines or RNA therapeutics. Altering the cap structure potentially increases mRNA stability and translational efficiency.

RNA polymerase II transcripts containing a protective 5',5'-triphosphate-linked 7-methylguanosine (m7G) cap can be decapped by enzymes like DCP2 initiating RNA decay. In the 5′–3′ mRNA decay pathway, poly(A) is shorted followed by cleavage of the mRNA cap. This reaction exposes the mRNA body to 5′–3′ exonucleases.

The capping of siRNAs increases their stability.

Wei et al., in 2013, used 1,2-bis(maleimido)ethane (BME) for the capping of hairpin RNAs to produce a capped siRNA called RhpRNA with a good serum and thermal stability. Teh protein Dicer was able to cleave these capped structures. RNA interference (RNAi) experiments showed that RhpRNA was highly efficient at RNAi with an IC50 value of 6 pM.

Adding a peptidyl cap to oligonucleotides stabilizes oligonucleotide base pairs.

Egetenmeyer & Richert, in 2011, identified a phosphodiester‐linked sequence of the residues of L‐prolinol, glycine, and oxolinic acid, dubbed ogOA, as a 5′‐cap that stabilizes any of the four canonical base pairs investigated, with ΔT_m values of up to +13.1 °C for an oligonucleotide octamer.

NAD capping and Decapping

As reviewed by Megerditch Kiledjian, in 2018, eukaryotic cells contain nicotinamide adenine dinucleotide (NAD⁺)-capped RNAs. The NAD⁺ cap is added by transcriptional initiation with NAD⁺ in the place of ATP, and also by a novel NAD⁺ capping mechanism. The 5′-end NAD⁺ cap promotes the rapid decay of RNA. The decay appears to be mediated at least in part by the DXO family of proteins.

Recent reports showed that Saccharomyces cerevisiae and mammalian cells also contain mRNAs carrying a novel nicotinamide adenine dinucleotide (NAD⁺) cap at their 5′-end. The presence of an NAD⁺ cap on mRNA appears to be a new mechanism for controlling gene expression through nucleotide metabolite-directed mRNA turnover. The NAD⁺ cap targets RNA for rapid decay in mammalian cells through the DXO decapping enzyme by removing intact NAD⁺ from RNA in a process called ‘deNADding’. The DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities as reviewed recently by Doamekpor et al. in 2020.

Figure 1: Structural model of the complex of mouse DXO wih 3’-FADP. In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. According to Doamekpor et al., FAD caps are also present on short RNAs (with less than ∼200 nucleotides) in human cells. The absence of DXO stabilizes the RNA (Doamekpor et al. 2020).

Wu et al., in 2019, reported the structure of human NUDT12 and showed that it is a cytosolic NAD-RNA decapping enzyme. Each monomer of the active NUDT12 homodimers contributes to the two functional catalytic pockets. The ∼600-kDa dodecamer complex between bleomycin hydrolase (BLMH) and NUDT12 revealed that BLMH localizes NUDT12 to a few discrete cytoplasmic granules that are distinct from P-bodies. These two proteins, when artificially tethered to a reporter RNA, downregulate gene expression in vivo. The loss of Nudt12 resulted in a significant upregulation of circadian clock transcripts in the mouse liver. The study revealed a physiological role for NUDT12 in the cytosolic surveillance of NAD-RNAs.

Figure 2: Crystal structure of the catalytic domain of human peroxisomal NADH pyrophosphatase NUDT12 in complex with 7-methyl-guanosine-5'-triphosphate.

Reference

Doamekpor, Selom, Grudzien-Nogalska, Ewa, Mlynarska-Cieslak, Agnieszka, Kowalska, Joanna, Kiledjian, Megerditch, Tong, Liang; 2020/05/06. DXO/Rai1 enzymes remove 5'-end FAD and dephospho-CoA caps on RNAs. Nucleic acids research 48, 10.1093/nar/gkaa297.. PDB ID 6WUF. [PMC]

Egetenmeyer, S.; Richert,; A 5′‐Cap for DNA Probes Binding RNA Target Strands. C. Chem. – Eur. J. 2011, 17, 11813–11827. [PubMed]

Megerditch Kiledjian; Eukaryotic RNA 5′-End NAD+ Capping and DeNADding. TICB; Volume 28, Issue 6, June 2018, Pages 454-464. [PMC]

Wei, L.; Cao, L.; Xi, Z. Highly Potent and Stable Capped siRNAs with Picomolar Activity for RNA Interference.  Angew. Chem., Int. Ed. 2013, 52, 6501–6503.

Wu H, Li L, Chen KM, Homolka D, Gos P, Fleury-Olela F, McCarthy AA, Pillai RS. Decapping Enzyme NUDT12 Partners with BLMH for Cytoplasmic Surveillance of NAD-Capped RNAs. Cell Rep. 2019 Dec 24;29(13):4422-4434.e13. [PubMed]

---...---

Humans need cobalamin (vitamin B12) for producing red blood cells and a healthy nervous system. Vitamin B12 is required in humans to produce healthy red blood cells in the bone marrow. Since vitamin B12 is only readily available in a human diet that consumes animal meats and dairy products or yeast extracts, modern humans may not get enough vitamin B12 from their diet. The human body metabolizes the water-soluble B vitamines very fast. Therefore, humans need to consume B vitamins daily. A diet supplemented with vitamin B12 will avoid a vitamin B12 deficiency; this is especially important for aging people. Therefore, older people and vegans may need vitamin B12 fortified foods or supplements to maintain a healthy vitamin B12 level in their blood.

Vitamin B12 is essential for folate metabolism and the synthesis of a citric acid cycle intermediate called succinyl-CoA. Vitamin B12 and folate are also crucial for homocysteine metabolism. To maintain the integrity of DNA, folate and vitamin B12 are needed.

In humans, vitamin B12 is a cofactor-precursor for two biochemical reactions. After ingestion, a multistep transport system transports cobalamins into the bloodstream. The soluble protein transcobalamin (TC) binds cobalamin. Next, receptor-mediated endocytosis transports the TC-cobalamin complex into the cell. Bloch et al., in 2017, studied the transportation complex assembly by solving four structures of the beta domain of human transcobalamin bound to different cyanocobalamin. Figure 1 shows structural models for transcobalamin-2 in complex with cobalt cyanocobalamin.  

Figure 1: Beta domain of human transcobalamin in complex with cyanocobalamin [PDB ID 5NP4].

Gut microbes in humans synthesize vitamin B12. Vitamin B12 functions as a modulator of the gut microbial ecology. Limitation of B12 can result from uptake disorders or dietary deficiencies. Deficiencies of the vitamin can cause anemia and permanent nerve and brain damage. Vitamin B12 is a precious resource in the gut and may not be available to the human host in significant quantities. Also, the vitamin may help to shape the structure and function of human gut microbial communities.

Johnson et al., in 2012, reported the structures of cobalamins and cobalamin riboswitches. A riboswitch is a regulatory segment of a messenger RNA that binds to small molecules. The riboswitch changes its conformation when it attaches to the target molecule - the switched RNA conformation results in a change in the production of the protein encoded by the mRNA.

Bacterial mRNAs often contain riboswitches in the 5’-untranslated regions (UTRs) of their mRNAs. The riboswitch's aptamer domain adopts a compact three-dimensional fold that acts as a scaffold for the ligand-binding pocket. Oligomers that fold into conformationally ordered structures in solution are also known as foldamers.

Cobalamin riboswitches occur widely in bacteria. The solved vitamin B12 riboswitch structures revealed insight into how the receptor and regulatory domains communicate in a ligand-dependent fashion to regulate mRNA expression.

Reference

Aptamers [ Custom Aptamer Synthesis]

Bloch JS, Ruetz M, Kräutler B, Locher KP. Structure of the human transcobalamin beta domain in four distinct states. PLoS One. 2017 Sep 14;12(9):e0184932. [ PMC]

Garst AD, Edwards AL, Batey RT. Riboswitches: structures and mechanisms. Cold Spring Harb Perspect Biol. 2011 Jun 1;3(6):a003533. [PMC]

Foldamers [What-is-a-Foldamer ?]

Johnson JE Jr, Reyes FE, Polaski JT, Batey RT. B12 cofactors directly stabilize an mRNA regulatory switch. Nature. 2012 Dec 6;492(7427):133-7.[PMC]

---...---

Unlike the conventional vaccines that require preparing a large titer of infectious viruses, nucleic acid vaccines could be prepared in a fraction of the time.  This has prompted the development and clinical testing of the mRNA vaccines targeting the mutated spike protein of COVID-19 in a record time.  At the time of writing of this article, FDA has issued EUA (emergency use authorization) for mRNA-based anti-COVID-19 vaccines developed by Moderna Therapeutics (USA) and Pfizer/BioNTech (USA/Germany).  The key difference between the two vaccine preparations is the storage temperature (-20^oC for Moderna's; -60 to -80^oC for BioNTech's) required for preservation.

An ideal vaccine is expected to meet several objectives.  The objectives may include blocking the spread of the virus, preventing symptoms associated with the infection, hospitalization, deaths, etc.  Following the completion of preclinical testing on animals, clinical trials may begin after submitting an Investigational New Drug (IND) application and obtaining clearance from FDA.  Normally, after completing Phase I, II and III studies, a new drug application (NDA) may be filed to FDA for market approval.  During the unprecedented times like the COVID-19 pandemic, the above process may be bypassed in lieu of issuing EUA as a temporary measure.

In the case of Moderna Therapeutics, the process of developing mRNA vaccine began soon after learning the genomic sequence of COVID-19 in January, 2020.  Phase I study (120 individuals; began March 16, 2020) assessed dose range, IgG antibody binding response by ELISA, and T cell response by intracellular cytokine stimulation assay, and safety of the mRNA-1273 vaccine.  Upon finding that two consecutive injections of 100 ug or 250 ug dose (given 28 days apart) yielded similar clinical responses and that a lower reactogenicity (adverse reaction) was observed with 100 ug dose, the latter was selected to proceed to Phase II and III studies.  Phase 2 study (600  individuals) examined the efficacy of 50 ug dose versus 100 ug dose and introduced saline solution as a placebo (negative control) using the previous dosing schedule.  Additional laboratory studies assessed kidney function, liver function, blood count, coagulation, etc.  


 

Phase III study included ~30,000 participants (47% female, 52% male) with the mean age of 51.4 (24.8% >65 y).  As of 14 day post 2^nd injection, the study found a higher percentage of COVID-19 infected cases in placebo (negative control) group (0.7%, 0.4%, 0.4% for age group 18-65y, 65-75y, >75y, respectively) than vaccine treated group (<0.1%, 0%, 0% for age group 18-65y, 65-75y, >75y, respectively).  Though saline water was used as the placebo, an alternate vaccine targeting an irrelevant virus may have been more informative.  Intriguingly, the study also showed similar efficacy for those with risk factors such as chronic lung disease, cardiac disease, obesity, liver disease, HIV infection (COVID-19 infection rate: 0.8% in placebo group, <0.1% for vaccine treated group). [https://www.fda.gov/advisory-committees/advisory-committee-calendar/vaccines-and-related-biological-products-advisory-committee-december-17-2020-meeting-announcement#event-materials ].    Phase I, II and III studies are still ongoing and may yield valuable information regarding side effects or long-term benefit.  Some preliminary information for the vaccination of cancer patients is provided by the European Society for Medical Oncology (https://www.esmo.org/covid-19-and-cancer/covid-19-vaccination).

 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.  For medicinal chemistry, it specializes in peptide synthesis, characterization, modification, purification to generate various peptide-based building blocks as well as pharmaceutical intermediates—in addition to peptide libraries, peptide arrays, peptidomimetics.   Antibody purification, characterization/quantification, modification and labeling are also offered.  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 conjugate to peptides.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology 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/Messenger-RNA-(mRNA)-for-Vaccine-Development-Against-Coronavirus.aspx

https://www.biosyn.com/tew/Ribose-2’-O-methylation,-“self-and-non-self,”-and-Coronaviruses.aspx

https://www.biosyn.com/tew/RNA-Capping-and-De-Capping.aspx

 
References

Dai L, Gao GF. Viral targets for vaccines against COVID-19.   Nat Rev Immunol. 18:1-10 (2020).  PMID: 33340022

Lindsay KE, Bhosle SM, et al. Visualization of early events in mRNA vaccine delivery in non-human primates via PET-CT and near-infrared imaging.  Nat Biomed Eng. 3:371-380 (2019).  PMID: 30936432

Rinaldi A. RNA to the rescue: RNA is one of the most promising targets for drug development given its wide variety of uses.  EMBO Rep. 21:e51013 (2020).  PMID: 32588530

In cells, RNA transcripts are bound to proteins to form RNA binding protein complexes (RBPs). RBPs play essential roles in regulating cellular processes, such as RNA metabolism, trafficking, mRNA splicing, localization, translation, and degradation. The interaction of coding and non-coding RNAs with RNA-binding proteins regulate critical cellular processes post-transcriptionally. The 3’-untranslated regions (3'-UTRs) contain regulatory motifs for RNA-binding protein involved in the control of mRNA expression levels and stability.

In recent years, scientists developed many strategies to analyze complex and dynamic RNA-protein networks in cells. However, identifying proteins and other molecules that specifically bind to RNAs for studying their biological function is challenging.

Specific RNA affinity probes allow the study of RNA interactions with molecules such as DNA, RNA, and proteins. For example, synthetic probes allow localization of a gene or a DNA and RNA sequence within a biological context. Historically, for this approach, a small piece of DNA or RNA labeled with a radioisotope (³H, ³²P, or ¹²⁵I), a fluorescent dye, or a ‘tag’ that can be recognized by a specific antibody, has been used for localization of the particular target, a gene sequence, via hybridization. These probes are known as hybridization probes. Biotin is now a common tag used for many applications.

" Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as mRNAs or siRNAs, containing natural or modified backbone, base, sugar and internucleotide linkages. Bio-Syntheis also provides biotinylated mRNA".

Synthetic biotinylated miRNA probes 

Biotin tagged synthetic microRNA (miRNA) duplexes allow the identification of messenger RNAs (mRNAs) associated with miRNAs. 

Ørom and Lund, in 2007, showed that the production of synthetic miRNA duplexes enables the identification of mRNAs associated with miRNAs. The approach involved producing synthetic miRNA duplexes carrying a biotin group at the 3’-end of the miRNA sense strand. The tagged miRNA sense strand incorporates into the miRISC complex and associates with endogenous target mRNAs after cell transfection. Mild cell lysis allows the capture of the miRNA-mRNA complexes on streptavidin beads now ready for purification and analysis. 

Biotin used in combination with the proteins avidin or streptavidin also enables labeling gene segments. These “pull-down probes” allow the purification of molecular complexes associated with the targeted DNA or RNA sequence. Reactive derivatives of biotins such as para-nitro-phenyl or N-hydroxy-succinimide esters enable the chemical covalent attachment or conjugation of biotin to side-chain groups of proteins, polysaccharides, oligonucleotides, as well as others. A variety of biotinylation reagents are now commonly available from various companies. 

Enzymatic biotinylated RNA tags 

RNA transcripts containing a short, encoded hairpin recognition motif can be enzymatically labeled. 

Recently, Busby et al. showed that RNA transcripts containing a short, encoded hairpin recognition motif could be enzymatically labeled. The labeling reaction utilizes a preQ1-biotin tag and bacterial tRNA guanine transglycosylase (TGTase). The natural derivative preQ1 allows marking the RNA sequence of interest containing a TGT recognition hairpin. PreQ1 is a metabolic intermediate in the synthetic pathway that produces the hypermodified guanine nucleotide, queuosine (Q). In eubacteria, TGTase inserts preQ1 into the wobble position of specific tRNAs, modified further to give queuosine.

Biotinylation RNA can be efficiently purified using avidin or streptavidin beads. Bacterial TGT covalently labels RNAs containing a short, encoded hairpin recognition motif using nucleobase derivatives.

Figure 1: Site-specific incorporation of nucleobase derivatives. Derivatives can be affinity labels or fluorophores (L), including biotin, BODIPY, thiazole orange, and Cy7 linked through a PEG linker attached to PreQ1 (Adapted from Alexander et al.).

tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a base-exchange reaction resulting in an anticodon modification of certain tRNAs. Prokaryotic TGT replaces guanine (G) with 7-aminomethyl-7-deazaguanine (PreQ1) at the wobble position of four specific tRNAs.

Figure 2: Crystal structure of the Zymomonas mobilis tRNA-guanine transglycosylase in complex with PreQ1 (Brenk et al.). The substrate PreQ1 is shown in the binding pocket as a doted structure (left) and as a yellow dot structure (middle) in the binding pocket of the enzyme.

Queuosine is a nucleoside found in tRNA with an additional cyclopentenyl ring added via an amino group to the methyl group of 7-methyl-7-deazaguanosine. This nucleoside is present in the first position of the anticodon of tRNA-tyrosine, tRNA-histidine, tRNA-asparagine, and tRNA-aspartic acid found in many organisms. Queuosine may play a role in the regulatory function of tRNA.

The RNA methyltransferase Dnmt2‐dependent tRNA methylation is dynamically modulating queuosine‐tRNA levels. Queuosine‐tRNA levels control the translation speed of queuosinylated‐tRNA decoded codons. An altered translation in the absence of queuosine results in misfolded aggregates. These aggregates trigger endoplasmic reticulum stress and the unfolded protein response.

Figure 3: Chemical structures of PreQ1, queuosine (Q), and guanosine (G).

Figure 4: Crystal structure of a class I PreQ1 riboswitch in complex with PreQ1 (left) and the structural model or PreQ1 (right). Riboswitches are natural RNA aptamers regulating gene expression by binding to specific small molecules. Connelly et al., in 2019, reported the discovery of synthetic small molecules that specifically bind to the PreQ1 riboswitch aptamer. Alteration of the chemical structure of the ligand was found to cause changes in the mode of RNA binding affecting regulatory functions.

FIGURE 5: PreQ1-biotin. RNA molecules containing a TGT recognition hairpin can be labeled with the preQ1-biotin deivative using bacterial tRNA Guanine Transglycosylae (TGT). 

Xie et al., in 2003, achieved trapping of a covalent intermediate of Zymomonas mobilis TGT and an RNA substrate by adding 9-deazaguanine (9dzG) to the reaction mixture. The purification and crystallization of the complex allow the research group to solve the intermediate X-ray structure (figure 6).

FIGURE 7: Crystal structure of the chemically trapped catalytic tRNA guanine transglycosylase covalent intermediate (Xie et al., 2003; PDB ID 1Q2R). Different models of the complex are shown. The structure shows the deglycosylated state of the guanine. 9-Deazaguanine is shown in blue.

Cap-biotinylated RNAs

Cap-biotinylated RNAs allow quantifying cap-dependent translation of mRNAs, mRNA processing, and turnover. 

Co-transcriptional incorporation of cap analogs allows studying translation efficiency, capping efficiency, and the susceptibility to decapping by cap-specific mRNA-decapping pyrophosphatase, Dcp1–Dcp2. Furthermore, biotinylated caps can serve as capture probes. Bednarek et al., in 2018, reviewed a general synthesis strategy for the synthesis of biotinylated 5’-cap RNA analogs.

FIGURE 7: Structure of a biotinylated cap (Adopted from Bednarek et al.). Chemical synthesis allows the production of cap analogs with a modified triphosphate bridge as well. The biotin affinity tag is attached to the 2’-position of m⁷G via an amide bond.  

According to Nierves & Lange, biotin labels or tags allow detection sites of biotinylation combined with mass spectrometry. This approach is beneficial for the proteomic study of biotinylated peptides. Enzymatic site-specific labeling of RNA targets is possible with RNA guanine transglycosylase from E. coli using unnatural nucleobase substrates. Busby et al. recently demonstrated the identification of RNA–protein interactions and the selective enrichment of cellular RNA in mammalian systems. The research group showed that an engineered enzyme variant achieved high labeling selectivity levels against the human transcriptome. The research group observed a 145-fold enrichment of cellular RNA directly isolated from mammalian cell lysates.

Reference

Alexander, S. C.; Busby, K. N.; Cole, C. M.; Zhou, C. Y.; Devaraj, N. K. Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation. J. Am. Chem. Soc. 2015, 137 (40), 12756–12759. [PubMed]

Sylwia Bednarek, Vanesa Madan, Pawel J. Sikorski, Ralf Bartenschlager, Joanna Kowalska and Jacek Jemielity; mRNAs biotinylated within the 5′ cap and protected against decapping: new tools to capture RNA–protein complexes.  Philosophical Transactions of the Royal Society B: Biological SciencesVolume 373, Issue 1762. [The Royal Socieity]

Bioconjugation for drug discovery

Ruth Brenk, Milton T Stubbs, Andreas Heine, Klaus Reuter, Gerhard Klebe; Flexible adaptations in the structure of the tRNA-modifying enzyme tRNA-guanine transglycosylase and their implications for substrate selectivity, reaction mechanism and structure-based drug design. Chembiochem. 2003 Oct 6;4(10):1066-77. [PubMed]

Kayla N. Busby, Amitkumar Fulzele, Dongyang Zhang, Eric J. Bennett, and Neal K. Devaraj; Enzymatic RNA biotinylation for affinity purification and identification of RNA-protein interactions. ACS Chemical Biology 2020 15 (8), 2247-2258 . [bioRxiv]

Connelly CM, Numata T, Boer RE, Moon MH, Sinniah RS, Barchi JJ, Ferré-D'Amaré AR, Schneekloth JS Jr. Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure. Nat Commun. 2019 Apr 2;10(1):1501. [PMC]

Jenkins JL, Krucinska J, McCarty RM, Bandarian V, Wedekind JE. Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation. J Biol Chem. 2011 Jul 15;286(28):24626-37. [PMC]

D. Metzler. Biochemistry. The chemical reactions of living cells. 2nd Edition). [Book]

Lorenz Nierves and Philipp F. Lange; DETECTABILITY OF BIOTIN TAGS BY LC-MS/MS. [bioRxiv]

Ulf Andersson Ørom and Anders H. Lund; Isolation of microRNA targets using biotinylated synthetic microRNAs. Methods 43, 2, 162-165. [PubMed] [Europe PMC]

Queuosine

Francesca Tuorto, Carine Legrand, Cansu Cirzi, Giuseppina Federico, Reinhard Liebers, Martin Müller, Ann E Ehrenhofer‐Murray, Gunnar Dittmar, Hermann‐Josef Gröne, Frank Lyko; Queuosine‐modified tRNAs confer nutritional control of protein translation. EMBO J (2018)37:e99777 [EMBO] 

Winkler W. C., Breaker R. R.; Regulation of bacterial gene expression by riboswitches. (2005) Annu. Rev. Microbiol. 59, 487–517. [PubMed]

Xie W, Liu X, Huang RH. Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat Struct Biol. 2003 Oct;10(10):781-8. doi: 10.1038/nsb976. Epub 2003 Aug 31. Erratum in: Nat Struct Biol. 2004 Jul;11(7):678. [PubMed]

---...---

Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as mRNAs or siRNAs, containing natural or modified backbone, base, sugar and internucleotide linkages.

Bio-Synthesis specializes in complex oligonucleotide modifications using phosphodiester backbone, purine and pyrimidine heterocyclic bases, and sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

Furthermore, Bio-Synthesis specializes in biomolecular conjugation for drug discovery.

---...---

Messenger RNA (mRNA) regulates cell proliferation. 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.

4E-binding proteins (4E-BPs) block the interaction of eIF4E with eIF4G to negatively regulate the formation of the eIF4F complex.

RNA-based pharmaceutical therapeutics and vaccines are a new approach to treating chronic and rare diseases, including COVID-19. Fore example, the deregulation of translation control appears the cause of many cancer types. The expression of eIF4E influences cell growth and phenotype. The overexpression of eIF4E leads to accelerated cell division and malignant transformation.

Structural complexes between proteins and their substrates can be studied in the gas-phase to determine equilibrium dissociation constants. Brown et al., in 2007, showed that mass spectrometry allows the measurement of the apparent gas-phase equilibrium dissociation constants (Kd) values for the specific molecular binding events. The Kds of guanosine triphosphate (GTP), GMP, and cap derivatives interactions with eIF4E were determined.

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]

Jin Wang, Bing Liang Alvin Chew, Yong Lai, Hongping Dong, Luang Xu, Seetharamsingh Balamkundu, Weiling Maggie Cai, Liang Cui, Chuan Fa Liu, Xin-Yuan Fu, Zhenguo Lin, Pei-Yong Shi, Timothy K. Lu, Dahai Luo, Samie R. Jaffrey, Peter C. Dedon; Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA. bioRxiv 683045. [Nucleic Acids Research]
 

---...---

Therapeutic synthetic messenger RNAs (mRNAs) are now famous for developing vaccines against viruses and the treatment of cancers. The use of mRNA as a therapeutic has virtually no risk of genomic integration and mutagenesis of the host genome's critical regions. Synthetic mRNA used as a vaccine does not change the human (the host's) genome. Also, the gene expression platform based on mRNA does not need to enter the nucleus to function. Hence, mRNA can express a protein inside a cell, even in a cell that is not dividing. Because the expression of a protein from mRNA is transient, the mRNA platform is safer than plasmid RNA (pRNA) vector-based platforms. The idea of using mRNA as a protein replacement therapy is already over 20 years old. Since mRNAs are relatively labile and immunogenic, this approach did not become popular until more recently.

A study by Kariko's research group in 2005 demonstrated that the incorporation of base modifications found in natural RNAs into mRNA could reduce TLR mediated immunogenicity of mRNAs. The natural occurring nucleobases 5-methylcytidine (m5C), N6 -methyladenosine (m6A), pseudouridine (Ψ), 5-methyluridine (m5U), and 2-thiouridine (s2U) can be incorporated into mRNA either separately or in combination.

More recently, Andries et al. in 2015 tested if nucleobase modifications found in natural RNAs are superior to pseudouridine (Ψ) at enhancing the translational capacity of mRNA. The research group found that mRNAs containing the N¹-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the pseudouridine (Ψ) and/or m5C/Ψ-modified mRNAs.

The study observed an up to ~44-fold higher gene expression for double modified mRNAs or ~13-fold higher gene expression for single modified mRNAs upon cell line transfection. Modification of mRNA with (m5C/m1Ψ) resulted in reduced intracellular innate immunogenicity and improved cellular viability. The researchers suggest that this may be due to the mRNA's increased ability to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling.

 Figure 1: Chemical structures of modified nucleosides investigated in the studies.

Reference

Oliwia Andries, Séan Mc Cafferty, Stefaan C. De Smedt, Ron Weiss, Niek N. Sanders, Tasuku Kitada; N1-methyl-pseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. Journal of Controlled Release 217 (2015) 337–344. [PubMed]

K. Kariko, M. Buckstein, H. Ni, D. Weissman, Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23 (2005) 165–175. [PubMed]

Bio-Synthesis provides a full spectrum of high-quality custom oligo modification services.
Bio-Synthesis uses direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, modified nucleic acids, sugar, and internucleotide linkages.
Bio-Synthesis also specializes in complex oligonucleotide modifications. Examples are phosphodiester backbone, purine, pyrimidine heterocyclic bases.
Bio-Synthesis can also incorporate sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

Of interest is the non-immunological application of mRNA technology to counter the COVID-19 pandemic.  Human angiotensin-converting enzyme 2 (ACE2) was identified as the receptor for COVID-19 in keeping with the prior finding that it represents the receptor for HCoV-NL63 and SARS-causing coronaviruses.  Docking of the spike protein (of COVID-19) to ACE2 allows the endocytosis of the bound complex.  This has prompted the development of various peptides and peptide mimetics targeting either the spike protein or ACE2 to disrupt the COVID-19 entry (VanPatten, et al 2020).  The therapeutic efficacy of multiple peptide drugs against COVID-19 is being assessed through clinical trials.

An alternative to the above approach involves expressing a surplus of decoy receptors to divert COVID-19 away from respiratory cells.  Of relevance, in 2013, soluble form of recombinant human ACE2 (hrsACE2) has been tested for treating patients afflicted with pathologically high level of angiotensin II (as ACE2 converts angiotensin II to angiotensin I) (Haschke et al., 2013).   In 2017, it was further tested to treat patients with acute respiratory distress syndrome (Khan et al., 2017).  After the emergence of COVID-19 coronavirus in 2020,  the investigators at the Karolinska Institute and Karolinska University Hospital (Sweden) reported that clinical grade of hrsACE2 could suppress the infection of human kidney organoids (organ-like structures formed in vitro from progenitor cells) by COVID-19 (Montell et al., 2020).

          

 

A drawback to the above methodology is that hrsACE2 exhibits relatively short half-life in circulation, requiring multiple administrations for extended periods to treat the COVID-19 infected.  To improve, an mRNA was designed to ectopically express human ACE2 after transfection, which could then be secreted to the extracellular milieu (Kim et al., 2020).  The ACE2-encoding mRNAs produced by in vitro translation were packaged in lipid-based nanoparticles for the cellular uptake.  Its systemic injection resulted in the hepatic delivery, followed by the secreted ACE2 which was detectable in the circulation.   Nevertheless, it remains to be seen whether the mRNA transfected cells expressing secreted ACE2 in vivo constitute a novel target for COVID-19 coronavirus. 

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 conjugate to peptides.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology 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/Messenger-RNA-(mRNA)-for-Vaccine-Development-Against-Coronavirus.aspx

https://www.biosyn.com/tew/Ribose-2’-O-methylation,-“self-and-non-self,”-and-Coronaviruses.aspx

https://www.biosyn.com/tew/RNA-Capping-and-De-Capping.aspx

References

Haschke M, Schuster M, et al. Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects.   Clin Pharmacokinet. 52:783-92 (2013). PMID: 23681967

Khan A, Benthin C, et al. A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome.   Crit Care. 21:234 (2017).  PMID: 28877748

Kim J, Mukherjee A, et al. Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2.  bioRxiv. 2020 Jul 25.   PMID: 32743574

Monteil V, Kwon H, et al. Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2.  Cell  181:905-913.e7 (2020).  PMID: 32333836

VanPatten S, He M, Altiti A, et al. Evidence supporting the use of peptides and peptidomimetics as potential SARS-CoV-2 (COVID-19) therapeutics.   Future Med Chem. 12:1647-1656 (2020).  PMID: 32672061

Therapeutic synthetic messenger RNAs (mRNAs) are now famous for developing vaccines against viruses and the treatment of cancers. The use of mRNA as a therapeutic has virtually no risk of genomic integration and mutagenesis of the host genome's critical regions. Synthetic mRNA used as a vaccine does not change the human (the host's) genome. Also, the gene expression platform based on mRNA does not need to enter the nucleus to function. Hence, mRNA can express a protein inside a cell, even in a cell that is not dividing. Because the expression of a protein from mRNA is transient, the mRNA platform is safer than plasmid RNA (pRNA) vector-based platforms. The idea of using mRNA as a protein replacement therapy is already over 20 years old. Since mRNAs are relatively labile and immunogenic, this approach did not become popular until more recently.

A study by Kariko's research group in 2005 demonstrated that the incorporation of base modifications found in natural RNAs into mRNA could reduce TLR mediated immunogenicity of mRNAs. The natural occurring nucleobases 5-methylcytidine (m5C), N6 -methyladenosine (m6A), pseudouridine (Ψ), 5-methyluridine (m5U), and 2-thiouridine (s2U) can be incorporated into mRNA either separately or in combination.

More recently, Andries et al. in 2015 tested if nucleobase modifications found in natural RNAs are superior to pseudouridine (Ψ) at enhancing the translational capacity of mRNA. The research group found that mRNAs containing the N¹-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the pseudouridine (Ψ) and/or m5C/Ψ-modified mRNAs.

The study observed an up to ~44-fold higher gene expression for double modified mRNAs or ~13-fold higher gene expression for single modified mRNAs upon cell line transfection. Modification of mRNA with (m5C/m1Ψ) resulted in reduced intracellular innate immunogenicity and improved cellular viability. The researchers suggest that this may be due to the mRNA's increased ability to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling.

 Figure 1: Chemical structures of modified nucleosides investigated in the studies.

Reference

Oliwia Andries, Séan Mc Cafferty, Stefaan C. De Smedt, Ron Weiss, Niek N. Sanders, Tasuku Kitada; N1-methyl-pseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. Journal of Controlled Release 217 (2015) 337–344. [PubMed]

K. Kariko, M. Buckstein, H. Ni, D. Weissman, Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23 (2005) 165–175. [PubMed]

Bio-Synthesis provides a full spectrum of high-quality custom oligo modification services.
Bio-Synthesis uses direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides containing backbone, modified nucleic acids, sugar, and internucleotide linkages.
Bio-Synthesis also specializes in complex oligonucleotide modifications. Examples are phosphodiester backbone, purine, pyrimidine heterocyclic bases.
Bio-Synthesis can also incorporate sugar modified nucleotides such as our patented 3rd generation Bridged Nucleic Acids.

Incorporating chemical modifications will stabilize small interfering RNAs (siRNAs) metabolically and improve tissue distribution.

RNA interference (RNAi) is an endogenous regulatory pathway silencing a gene in a sequence-specific manner. However, a significant barrier for efficient gene silencing using RNAi is the difficulty of delivering small interfering RNAs (siRNAs) in vivo. Design strategies addressing low stability and non-targeted biodistribution are needed. Also, therapeutic siRNAs must avoid the stimulation of undesirable innate immune responses.

Gavrilov & Saltzman in 2012 reviewed the mechanistic principles of RNA interference and its use in biomedical applications. This review also described the use of chemical modifications for siRNA sugars and backbone, including bridged nucleic acids (BNAs) and others. 

In 2016, Schirle et al. solved the crystal structure of human Argonaute-2 (Ago2) bound to a modified, metabolically stable siRNA guide sequence with extensive backbone modifications to show that Argonaute-2 can bind pharmacologically stable siRNAs.

The comparison of a complex with an un-modified siRNA and modified siRNA guide revealed that the Ago2 structure is relatively unaffected by chemical modifications in the bound siRNA. According to Schirle et al., the modified siRNA appeared to be more plastic and shifts, relative to the unmodified siRNA, to optimize contacts with Ago2. The structural study revealed that even significant conformational perturbations in the 3′-half of the siRNA seed region have a relatively modest effect on knock-down potency. These findings explained why various modification patterns are tolerated in siRNAs and revealed the structural basis for new therapeutic siRNA designs.

RNA modifications used by Schile et al. are shown in figure 1.

Figure 1: Structures with RNA modifications. Blue: 2′-O-Me, Green: 2′-F, purple: 2′-O-MOE, red: VP-T = 2′-O-MOE-thymidine-(E)-5′- vinylphosphonate; s: phosphorothioate. All backbone linkages are phosphodiesters except those indicated with s (Adapted from Schirle et al.).

Example of a modified siRNA sequence used in the study:

VP-TsUsAUsCUsAUsAAsUGsAUsCsAsGsGsUsAsA

   ------------

Figure 2; Various views of a Human Argonaute2-siRNA complex.

Reference

Gavrilov K, Saltzman WM. Therapeutic siRNA: principles, challenges, and strategies. Yale J Biol Med. 2012 Jun;85(2):187-200. Epub 2012 Jun 25. PMID: 22737048; PMCID: PMC3375670. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375670/

Schirle NT, Kinberger GA, Murray HF, Lima WF, Prakash TP, MacRae IJ. Structural Analysis of Human Argonaute-2 Bound to a Modified siRNA Guide. J Am Chem Soc. 2016 Jul 20;138(28):8694-7. doi: 10.1021/jacs.6b04454. Epub 2016 Jul 12. PMID: 27380263; PMCID: PMC4993527.

https://www.rcsb.org/structure/5JS2

---...---

Molecular probes containing bridged nucleic acids (BNAs) at selected positions in combination with real-time PCR allow the detection of pathogen mutants that can cause various disorders in humans.

Nontuberculous mycobacteria are such pathogens. Nontuberculous mycobacteria (NTM) are mycobacteria other than M. tuberculosis and M. leprae. NTM are known as atypical mycobacteria, mycobacteria other than tuberculosis (MOTT), or environmental mycobacteria.

Hirama et al. designed a BNA probe detection system based on real-time PCR (called BNA-PCR) to identify point mutations at position 2058 or 2059 in domain V of the 23S rRNA gene responsible for clarithromycin resistance. 

BNA are artificial nucleic acids with a high binding affinity to specific sequences allowing the design of probes with improved hybridization capability and enhanced biochemical stability. Specific designed BNA probes enable discrimination of single mismatches within a target sequence of amplified products. 

The BNA-PCR assay allows the identification of Mycobacterium avium complex (MAC) isolates from clinical samples.

Clarithromycin resistance gene BNA-PCR primer and probe set

Forward primer:     5′-GTAACGACTTCCCAACTGTCTC-3′

Reverse Primer:     5′-ACCTATCCTACACAAACCGTACC-3′

BNA Probe:           Alex532-CGCGGCAGGACGAAAAGAC-BHQ1  ;  AA = Placing of BNAs

The BNA FRET probe contains two BNA bases for detecting MAC isolates' clarithromycin resistance gene (BNAs are colored). If a point mutation is present, the resulting sequence mismatch leads to a decrease in the melting temperature (Tm) value, preventing the BNA probe from annealing to the PCR product, resulting in a reduced emitted fluorescence (Hirama et al.).

Recently Bio-Synthesis also developed an “Ultra-Sensitive BRAF Codon 600 (V600E) Mutation Analysis Kit” based on BNAs for rapid and convenient real-time PCR detection of the BRAF-V600E mutation with high sensitivity. The kit enables efficient discrimination of the mutant from the wild-type gene. It allows laboratories to detect levels down to 0.1% or lower of the BRAF V600E mutant in a wild-type background.

Figure 1: Clarithromycin, structures. Clarithromycin is a ribosome-targeting macrolide antibiotic that interacts with a bacterial 23S rRNA hairpin-like structure in domain II and the peptidyl transferase loop in domain V. 

Reference

23S rRNA

Alexa 532 labeling 

BHQ1 modification 

BNA Clamp PCR

Bridged Nucleic Acids (BNAs)

CDC nontuberculous-mycobacteria  

Clarithromycin

FRET molecular beacon

Dye and Quencher Pairs for FRET

Sung-Kun Kim, Klaus D. Linse, Parker Retes, Patrick Castro, Miguel Castro; Bridged Nucleic Acids (BNAs) as Molecular Tools.J Biochem Mol Biol Res 2015 September 1(3): 67-71. [PDF]

Hirama T, Shiono A, Egashira H, Kishi E, Hagiwara K, Nakamura H, Kanazawa M, Nagata M. PCR-Based Rapid Identification System Using Bridged Nucleic Acids for Detection of Clarithromycin-Resistant Mycobacterium avium-M. intracellulare Complex Isolates. J Clin Microbiol. 2016 Mar;54(3):699-704.  [PMC]

Imanishi T, Obika S. 2002. BNAs: novel nucleic acid analogs with a bridged sugar moiety. Chem Commun (Camb.) 16:1653–1659. [ Article ]

Rahman SA, Seki S. 2008. Design, synthesis, and properties of 2′,4′-BNANC: a bridged nucleic acid analogue. J Am Chem Soc 130:4886–4896. [PDF]

“Ultra-Sensitive BRAF Codon 600 (V600E) Mutation Analysis Kit” based on BNAs.

---...---