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Analysis and preparation of capped RNAs

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

Wang et al. in 2019 with the help of CapQuant discovered 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. A different approach combined mass spectrometric characterization with crystallography for the determination of equilibrium dissociation constants values for different N7-alkylated caps interacting with eukaryotic translation initiation factor eIF4E (Brown et al., Wang et al.).  

Figure 1: 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. Multiple quantities of heterogeneous nuclear RNA (hnRNA) transcripts are turned over in the nucleus. After apparent quality control in the nucleus in eukaryotic cells, mRNAs emerge from precursor hnRNAs through a series of modification reactions. 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 (Nakazato et al. 1975).

Since transcripts are produced at different rates from different loci, the position of a gene or allele in a chromosome, the classification of transcripts is 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. 

Preparation of capped RNA

In 2003, Huang F. reported a method for the preparation of RNAs modified with small caps. The following small caps added to RNA molecules were coenzyme A (CoA), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD). All three coenzymes contained an adenosine group. This approach allows the preparation of coenzyme-linked RNA libraries for in vitro selection, coenzyme-coupled specific RNA sequences for other uses such as fluorescent labeling to detect specific RNA or DNA sequences, the investigation of RNA structures, and the study of RNA-RNA and RNA-protein interactions.

Adenosine derivatives such as ATP, 3’-Dephospho-coenzyme A (De-P-CoA), NAD, and FAD with the help of a transcription promoter sequence derived from T7 class II promoters initiate transcription. Using adenosine makes the preparation of adenosine-initiated RNA with free 5’-hydroxyl groups possible.

T7 RNA polymerase only requires the adenosine group for recognition and to initiate transcription. Therefore, other adenosine derivatives also allow the preparation of adenosine derivative-linked RNAs.

The conjugation of other biologically active molecules such as coenzymes S-adenosylcysteine, S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (SAM), the sugar-containing molecule adenosine 5’-diphosphoglucose (ADPG) and the signaling molecules diadenosine polyphosphates Ap(3)A and Ap(4)A to the 5'-end of RNAs is also possible.

T7 Class II promoter Sequence

Figure 2: Adenosine derivative-initiated transcription under the T7 class II promoter (φ2.5).

In this approach, in addition to nucleoside 5′-triphosphates (NTPs), an adenosine derivative (R-A) is added to the transcription solution. R-A serves as the transcription initiator to produce R-A-RNA. Adenosine triphosphate (ATP) competes with R-A for transcription initiation, resulting in normal RNA with 5′-triphosphate, pppRNA.

Figure 3: Model for initiation of T7 DNA at the primary origin as proposed by Saito et al., 1980.



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.

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

Huang F. Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription. Nucleic Acids Res. 2003 Feb 1;31(3):e8. doi: 10.1093/nar/gng008.

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

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

Saito H, Tabor S, Tamanoi F, Richardson CC. Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3917-21.

Saito H, Richardson CC; Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants. Journal of Virology Jan 1981, 37 (1) 343-351.

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.