Synthetic oligonucleotide-based therapeutics, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and modified oligonucleotides, require high-quality products at high quantities and free of impurities.
Phosphorylated oligonucleotides such as 5′-triphosphates are necessary biochemical and therapeutic tools. The 5’-triphosphate modification is an essential nucleic acid modification found in all living organisms taking part in metabolic processes providing energy to drive many processes in living cells.
5'-triphosphate oligonucleotides can act as antiviral and anticancer inhibitors, as substrates for polymerase chain reactions, and nucleic acids ligation reactions, allowing structural and mechanistic studies. Also, 5'-triphosphate oligonucleotides can stimulate an immune response stimulation and are intermediates in the enzymatic synthesis of m7G-5′-ppp capped RNAs.
Oligonucleotides containing a 5'-triphosphate group are short chains of nucleotides modified with a triphosphate group at their 5' end. Various applications in molecular biology and biotechnology utilize 5'-triphosphate oligonucleotides in molecular biology and biotechnology, particularly in nucleic acid-based therapeutics.
Some key features and applications of 5'-TP oligonucleotides are:
1. Immunostimulatory Properties: 5'-triphosphate (5'-TP) oligonucleotides can activate the immune system by interacting with pattern recognition receptors, for example, Toll-like receptors (TLRs), specifically TLR3, TLR7, TLR8, and TLR9. This activation leads to the induction of cytokines and other immune response mediators, making them useful in immunotherapy and vaccine development.
2. Antiviral Activity: Certain 5'-TP oligos containing specific sequences of nucleotides can exert antiviral effects by targeting viral nucleic acids or proteins. They can interfere with viral replication and trigger immune responses against viral infections.
3. RNA Interference (RNAi): 5'-TP oligonucleotides as part of small interfering RNA (siRNA) molecules trigger RNA interference. RNAi is a process that silences or knocks down specific genes. The presence of the triphosphate group at the 5' end enhances the efficiency of siRNA uptake and subsequent gene silencing.
4. Gene Editing and Genome Engineering: In the gene-editing technique CRISPR-Cas9, 5'-TP oligonucleotides can guide the Cas9 nuclease to specific genomic targets. 5'-TP oligonucleotides serving as the template for repairing or modifying DNA sequences enable precise gene editing and genome engineering.
5. Diagnostic and Therapeutic Applications: 5'-TP oligonucleotides are used in diagnostics and therapeutics, for example, as probes in molecular diagnostic techniques such as polymerase chain reaction (PCR) or situ hybridization (ISH).
5'-TP oligonucleotides can be modified with various functional groups or conjugated to other molecules (for example, targeting ligands or therapeutic agents) for enhanced specificity and efficacy in therapeutic applications.
The cytosolic pattern recognition receptor RIG-I detects negative-stranded RNA viruses that do not have double-stranded RNA but contain panhandle blunt short double-stranded 5′-triphosphate RNA in their single-stranded genome. In 2009, Schlee et al. reported that synthetic single-stranded 5′-triphosphate oligoribonucleotides could not bind and activate RIG-I. However, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I.
Reference
ATP [ NIH , wiki ]
Bare, G. A. L., Horning, D. P. Chemical Triphosphorylation of Oligonucleotides. J. Vis. Exp. (184), e63877, doi:10.3791/63877 (2022). [ jove ]
Cytosolic pattern recognition receptor Rig-I [wiki/RIG-I ]
Schlee M, Roth A, Hornung V, Hagmann CA, Wimmenauer V, Barchet W, Coch C, Janke M, Mihailovic A, Wardle G, Juranek S, Kato H, Kawai T, Poeck H, Fitzgerald KA, Takeuchi O, Akira S, Tuschl T, Latz E, Ludwig J, Hartmann G. Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity. 2009 Jul 17;31(1):25-34.[ PMC ]
Zlatev I, Lackey JG, Zhang L, Dell A, McRae K, Shaikh S, Duncan RG, Rajeev KG, Manoharan M. Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA. Bioorg Med Chem. 2013 Feb 1;21(3):722-32. [ sciencedirect ]
---...---
Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.
---...---