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, participating in metabolic processes and providing energy to drive many processes in living cells.
The 5-triphosphate group in nucleotides plays a fundamental role in energy transfer, nucleic acid synthesis, transcription, signaling, and enzymatic reactions. Its presence provides chemical properties and molecular interactions required for these crucial biological processes.
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 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 molecular biology and biotechnology applications utilize 5'-triphosphate oligonucleotides, particularly in nucleic acid-based therapeutics.
The 5'-triphosphate (5'-TP) group plays a crucial role in the function of nucleotides and nucleic acids. 5’-TP refers to three phosphate groups attached to the 5'-carbon of a nucleotide.
The triphosphate moiety is responsible for several essential functions associated with nucleic acids:
High Energy Activated Molecules: The presence of the triphosphate group in nucleotides, such as adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), provides a high-energy bond. Hydrolysis of the terminal phosphate group releases energy, which cells can harness for various cellular processes, including muscle contraction, active transport of ions, DNA and RNA synthesis, and enzymatic reactions.
Nucleic Acid Synthesis: During DNA and RNA synthesis, the triphosphate group is critical for adding nucleotides to the growing nucleic acid chain. In DNA replication, for example, the 5'-TP group of a deoxyribonucleoside triphosphate (dNTP) is a substrate for DNA polymerase enzymes. The formation of a phosphodiester bond between the 5'-phosphate of the incoming dNTP and the 3'-hydroxyl group of the growing DNA chain is catalyzed by a polymerase, resulting in the elongation of the DNA strand.
RNA Transcription: Transcription requires the triphosphate group to initiate and elongate RNA chains. The RNA polymerase enzyme recognizes the triphosphate group at the 5'-end of the incoming ribonucleoside triphosphate (rNTP) and incorporates it into the growing RNA molecule during transcription. The 5'-TP group stabilizes the RNA polymerase binding and facilitates proper transcription initiation and elongation.
Signaling Molecules: Certain nucleotides with a triphosphate group, such as GTP and ATP, function as important signaling molecules within cells. GTP is a molecular switch in G-protein signaling pathways and several cellular processes, including cell growth, differentiation, and intracellular signaling cascades. In addition to its energy-carrying role, ATP acts as a signaling molecule in cell signaling, muscle contraction, and enzymatic reactions.
Enzymatic Reactions: The triphosphate group can participate in enzymatic reactions as a phosphate donor or acceptor. Kinases, for example, transfer a phosphate group from ATP to target molecules, regulating their activity. Similarly, phosphatases remove phosphate groups, modulating the function of their target molecules.
Some key features and applications of 5'-TP oligonucleotides are:
Immunostimulatory Properties: 5'-triphosphate (5'-TP) oligonucleotides can interact with pattern recognition receptors, Toll-like receptors 3 (TLR3), TLR7, TLR8, and TLR9 to activate the immune system. This activation leads to the induction of cytokines and other immune response mediators, making them useful in immunotherapy and vaccine development.
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.
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.
RNA Interference (RNAi): 5'-TP oligonucleotides as part of small interfering RNA (siRNA) molecules trigger RNA interference. RNAi 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.
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 serve as the template for repairing or modifying DNA sequences and enable precise gene editing and genome engineering. Here, 5΄-phosphate mimics stabilize the 5΄-end of the guide strand. The modification protects the guide RNA from phosphatase degradation and 5΄- to 3΄-exonucleases. This modification significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. 5΄-(E)-vinylphosphonate stabilizes the 5΄-phosphate group, enabling systemic delivery and silencing in the kidney and heart.
Diagnostic and Therapeutic Applications: Diagnostic and therapeutic approaches utilize 5'-TP oligonucleotides as probes in the polymerase chain reaction (PCR) or in-situ hybridization (ISH).
Modification: 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.
Synthetic 5'-TP oligonucleotides are typically chemically synthesized and may require modifications to enhance stability, cell penetration, or target specificity, depending on the desired application.
Reference
ATP [Adenosine_triphosphate]
Bare, G. A. L., Horning, D. P. Chemical Triphosphorylation of Oligonucleotides. J. Vis. Exp. (184), e63877, doi:10.3791/63877 (2022). [jove]
Rig-I [RIG-I]
Thillier Y, Decroly E, Morvan F, Canard B, Vasseur JJ, Debart F. Synthesis of 5' cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase. RNA. 2012 Apr;18(4):856-68. [PMC]
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. [article]
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