The 5 Triphosphate Cap

A) 5’ Triphosphate cap                          B) 5’ Triphosphate cap depicted with two adenosines

The 5’ Triphosphate cap adds a mass of ~237 dalton to the capped oligonucleotide.

RNA oligonucleotides containing 5′-triphosphate termini have been used to study the mechanism of initiation of DNA replication with DNA primase. DNA primases are enzymes whose continual activity is required at the DNA replication fork. DNA primase catalyzes the synthesis of short RNA molecules used as primers for DNA polymerases. The enzyme uses strands of nucleic acids called primers, which serve as a starting point for DNA synthesis. DNA polymerases can only add new nucleotides to an existing strand of DNA. The primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. For example, in E. coli, a typical primer is 10 to 12 nucleotides long and the consensus sequence is pppAG(N) 8-10. In human herpes simplex virus (HSV) primers are also 10 to 12 nucleotides long and the consensus sequence is pppGG(N) 8-10, whereas human (homo sapiens) primers are reported to be 11 to 14 nucleotides long with the following consensus sequence, ppp(A/G)(N)8-13. Note that the primers have a triphosphate at the 5’ terminus.

Primases catalyze the synthesis of oligonucleotides in at least five

  1.  Template binding
  2.  Nucleotide triphosphate binding
  3.  Initiation
  4.  Extension to a functional primer
  5.  Primer transfer to DNA polymerase

The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA. The presence of the DNA primase is required at the DNA replication fork. They catalyze “de novo” synthesis of short RNA molecules carrying 5′-triphosphate groups, which are used as primers by DNA polymerases.

Two research groups, Clemens and Williams and Kerr & Brown, in 1978, reported that the oligonucleotide pppA2’p5’A2’p5’ A is synthesized by extracts from interferon-treated mouse L cells in the presence of double-stranded RNA. They also reported that this compound is a potent inhibitor of protein synthesis in cell-free systems prepared from L cells or rabbit reticulocytes. The scientists reported that this inhibitor activates a nuclease which prevents mRNA from being utilized for protein synthesis. These type of oligonucleotides are a unique class of 5′-triphosphorylated oligonucleotides with 2′-5′internucleotide linkages [ppp(A2′p)nA, n = 2 to ≥ 4] involved in immune response and activation of an endogenous ppp(A2′p)nA-dependent RNase, which degrades foreign mRNA and rRNA.

Bagloni et al., in 1978, also reported that this unique oligonucleotide mediates the action of interferon by mediating the activity of a nuclease that degrades mRNA, thereby inhibiting protein synthesis. Furthermore, Wreschner et al. in the same year reported that interferon treatment of cells induces the synthesis of several proteins,  which is an enzyme, 2-5A synthetase and that  in the presence of double-stranded RNA (dsRNA) and ATP, this enzyme synthesizes 2-5A (ppp(A2'p)nA where n = 2 to 4. The 2-5A in turn activates an RNase which can inhibit protein synthesis by RNA degradation.

RNA molecules containing a 5′-triphosphate cap are also implicated in the innate immune system. The innate immune system of eukaryotes represents the first line of defense made up of the cells and mechanisms that defend the host from infection by other organisms and viruses, and it is able to detect viral nucleic acids via the family of RIG-I-like receptors. RIG-I is a cytosolic sensor of viral RNA.

In 1996, Ekland and Bartel described an RNA molecule called a ribozyme, which synthesizes RNA using the same reaction as that employed by protein enzymes that catalyze RNA polymerization. In the presence of the appropriate template RNA and nucleoside triphosphates, the ribozyme extends an RNA primer by successive addition of up to six mononucleotides. The added nucleotides are joined to the growing RNA chain by 3',5'-phosphodiester linkages. In addition, in the same year Olsen et al. used synthetic triphosphorylated RNAs to elucidate the basic mechanistic and kinetic properties of influenza endonuclease.

In 2006, Horning et al. demonstrated that the 5′-triphosphate end of RNA generated by viral polymerases is responsible for retinoic acid–inducible protein I (RIG-I)–mediated detection of RNA molecules. The researchers showed that the RNA is not detected if the 5′-triphosphate end is capped or modified. The researchers reported that RIG-I specifically detects the 5′-triphosphate end of viral RNAs. Also in 2006, Pichlmair et al. showed that influenza A virus infection does not generate dsRNA. Viral genomic single-stranded RNA (ssRNA) that contains 5′-phosphates activates RIG-I. This is blocked by the influenza protein nonstructural protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as ssRNA sensor and a potential target of viral immune evasion. These results also suggest that its ability to sense 5’-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself. The notion is that the cytoplasmic presence of RNA containing accessible 5-phosphates allows discrimination between self and viral RNA, indicating that, similar to dsRNA, 5′ phosphate containing ssRNA constitutes a viral “pathogen-associated molecular pattern.” Furthermore, RIG-I recognition is specific for viral RNA. Influenza viral RNA (vRNA) is uncapped. Phosphorylated 5′ termini present in siRNA and ssRNAs generated by in vitro transcription have been reported to induce IFN-α/β when transfected into cells.

It was found that genomic RNA prepared from a negative-strand RNA virus and RNA prepared from virus-infected cells but not from noninfected cells, triggered a potent interferon-α response in a phosphatase-sensitive manner. 5′-triphosphate RNA directly binds to RIG-I. As a result, uncapped 5′-triphosphate RNA or 3pRNA present in some viruses serve as the molecular signature for the detection of viral infection by RIG-I. Apparently, influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription.

Host defense against infection by invading viral and bacterial pathogens is dependent on the initiation and maintenance of the finely tuned primary innate immune response. This rapid protective response is coupled to subsequent adaptive immunity that provides long-term protection based on immunological memory. Pattern recognition receptors, as part of the innate immune system, detect characteristic microbial components, including foreign nucleic acids and their characteristic structures. The recognized structures range from long double-stranded RNA (dsRNA) and 5’-triphosphorylated (ppp) RNA, to unmethylated CpG DNA. RNA oligonucleotides that contain a 5′-triphosphate group can be used as mimics instead of viral RNAs to study induction of antiviral immune response. The terminal 5′-triphosphate group on an RNA oligonucleotide can be converted to the 7mGpppN or 5' N7-Methylguanosine-triphosphate Cap structure commonly found on mature mRN

Next, 5′-ppp RNA can mimic ribozymes that catalyze the ligation of oligonucleotides with the same chemistry used by polymerases. Some of these ribozymes may be suitable starting points for the in vitro evolution of ribozyme polymerases that use oligonucleotide triphosphates as substrates.

As an antisense agent, Poeck et al. in 2008 designed a small RNA that acted as an antitumor agent to not only silence the antiapoptotic gene Bcl2, but also to bind and activate retinoic acid-inducible gene-I (RIG-I). The silencing of Bcl2 promoted tumor cell apoptosis, and activating RIG-I stimulated the immune system to destroy the tumor. It was shown that the antitumor effect of the dually active molecule in a mouse model of metastatic melanoma to the lung was more potent than small RNAs that just activate RIG-I or just knock down Bcl2.

In vitro transcribed RNA generated during in vitro RNA synthesis also carry 5’-triphosphate termini. These 5’-triphosphate capped RNAs can be directly used for PCR reactions but must be removed to yield a 5’-hydroxyl group prior to end-labeling or RNA ligation.

Synthesis and purification of 5’ triphosphate caps and capped structures

Capped RNA oligonucleotides can be synthesized enzymatically by in vitro transcription and chemically using phosphoramidite chemistries. The use of chemical synthesis allows the reproducibly of obtaining capped RNAs at almost any scale independent from the RNA sequences and also allows the addition of modifications at specific positions. The resulting modified oligonucleotides can be purified to a high purity and analyzed by a number of established methods, including PAGE, reverse phase HPLC, anion-exchange HPLC and mass-spectrometry. The capped RNA oligonucleotides can be modified with fluorescent dyes as well. The following example of a dye labeled 5’ triphosphate capped oligonucleotide illustrates the synthesis capabilities at Biosynthesis Incorporated.

The next figure shows experimental data for synthetic 5’-triphsophate capped oligonucleotide. The QC/QA data for the fluorescently labeled 5’ triphosphate capped oligonucleotide is illustrated.

A synthetic 5’ triphosphate capped

A synthetic 5’ triphosphate capped oligonucleotide.

This experimental data show that Biosynthesis has the capability to routinely synthesize this type of modified oligonucleotide using natural nucleotide monomers or artificial nucleotide monomers, such as bridged nucleic acids (BNAs). Furthermore, after each successful synthesis data data report is provided as a QC/QA report.


Baglioni C, Minks MA, Maroney PA. Interferon action may be mediated by activation of a nuclease by pppA2’p5’A2’p5’A. Nature. 1978 Jun 22;273(5664):684-7.

Clemens MJ, Williams BR. Inhibition of cell-free protein synthesis by pppA2’p5’A2’p5’ A: a novel oligonucleotide synthesized by interferon-treated L cell extracts. Cell. 1978 Mar;13(3):565-72.

Ekland EH, Bartel DP. RNA-catalysed RNA polymerization using nucleoside triphosphates. Nature. 1996 Jul 25;382(6589):373-6.

Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, Endres S, Hartmann G. 5'-Triphosphate RNA is the ligand for RIG-I. Science. 2006 Nov 10;314(5801):994-7.

Kerr IM, Brown RE.;pppA2’p5’A2’p5’A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):256-60.

Olsen DB, Benseler F, Cole JL, Stahlhut MW, Dempski RE, Darke PL, Kuo LC. Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs. J Biol Chem. 1996 Mar 29;271(13):7435-9.

Andreas Pichlmair et al.; RIG-I-Mediated Antiviral Responses to Single-Stranded RNA Bearing 5'-Phosphates. Science 314, 997 (2006); DOI: 10.1126/science.1132998.

Poeck H, et. al. 5'-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat Med. 2008 Nov;14(11):1256-63.

Wreschner DH, James TC, Silverman RH, Kerr IM. Ribosomal RNA cleavage, nuclease activation and 2-5A(ppp(A2'p)nA) in interferon-treated cells. Nucleic Acids Res. 1981 Apr 10;9(7):1571-81.