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Methods for the discovery of cap-like structures

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

Cap analysis



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

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

MS & X-ray crystallography 

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

Poly A

Ramanathan A, Robb GB, Chan SH. mRNA capping: biological functions and applications. Nucleic Acids Res. 2016 Sep 19;44(16):7511-26.  [PMC]

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

Links to 5'-capped oligonucleotides!!


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Bio-Synthesis also provides biotinylated and capped mRNA as well as long circular oligonucleotides".