5′ Capping

A: Capping is the first step in the maturation of eukaryotic mRNA. Shortly after the start of transcription, the 5′-end of the growing RNA molecule is capped by the addition of a guanosine monophosphate residue (from GTP) via a 5′-5′ pyrophosphate linkage. After addition of the cap nucleotide, a methyl group is added to the N7 position of the guanine base producing a Cap 0 structure.

A: It has been reported that methylation at the 2′-O position of the penultimate nucleotide of capped RNA (to give a Cap 1 structure) improves translation by 20% to 50% over RNA with a Cap 0 structure. The amount of the improvement is cell-specific.

A: There are no special requirements of your template to cap with ARCA, other than the requirement for a functional T7 promoter where the first base to be transcribed (Base +1) is a “G”.

A: Co-transcriptional capping, which is how the INCOGNITO™ T7 ARCA 5mC- & Ψ-RNA Kit and MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2 work, involves the incorporation of the ARCA RNA Cap Analog dinucleotide molecule into position +1 of RNA being synthesized. Capping is performed by the T7 RNA Polymerase (RNAP) in the reaction which is also making the body of the transcript via transcription. The way the T7 RNAP is “tricked” into capping the IVT RNA, is that a dinucleotide cap analog (historically m⁷G[5′]ppp[5′]G) is included in the nucleotide substrate mix for the reaction. The cap analog molecule is recognized as GTP by T7 RNAP. Base +1 of traditional T7 RNAP-derived transcripts is a “G,” the first nucleotide incorporated into the transcript. If the nucleotide mix contains a mixture of 50% m⁷GpppG and 50% GTP, then 50% of the time, the transcript will initiate with m⁷GpppG, and 50% with GTP (ppp[5′]G). The respective RNAs would be m⁷G[5′]ppp[5′]GpNpNpNpN… and ppp[5′]GpNpNpNpN… To achieve higher capping efficiencies in this system, the ratio of m⁷GpppG to GTP is usually 4:1 or (80% + 20%). This way, based strictly on statistics, 80% of the transcripts will initiate with m⁷GpppG and thus be capped.

A: Capping is performed by the T7 RNA Polymerase (RNAP) in the reaction which is also making the body of the transcript via transcription. If the transcription template also encodes for the poly(A) tail, one can get transcription, capping and tailing all in one reaction, all done by T7 RNAP. This is one advantage to a co-transcriptional protocol.

However, there are certain disadvantages to co-transcriptional capping:

1. You can never achieve ~100% capping.
2. Overall transcription yields are limited by the fact that 80% of the normal amount of GTP in the reaction has been removed to achieve the capping. GTP is the limiting reagent substrate.
3. Also due to the limiting amount of GTP, transcripts longer than ~4 kb need to have additional GTP supplemented into the reaction for enough full-length RNA to be made. The INCOGNITO™ T7 ARCA 5mC- & Ψ-RNA Kit and MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2 contain a separate tube of GTP to supplement extra GTP into the reaction. However, when doing so, one changes the ratio of cap analog to GTP in the reaction which results in a lower capping efficiency.

A: The alternative to co-transcriptional capping is post-transcriptional capping. This method involves enzymatic treatment of the in vitro transcribed (IVT) RNA with capping enzyme as a separate reaction distinct from the transcription reaction. With this method, one can achieve virtually 100% capping efficiency and, because GTP is not limiting in the IVT reaction, IVT yields are maximized, and transcript lengths are not an issue.

A: Capping is performed by Capping Enzyme on already existing uncapped in vitro transcribed (IVT) RNA, just as is done in nature. Capping Enzyme uses GTP and S-adenosyl-methionine to build the m⁷G[5′]ppp… cap structure onto the 5′-end of the IVT RNA.

A: Since the substrate IVT RNA is made independently, and since a standard transcription reaction would not be starved for GTP, the IVT RNA yields are not limited (thus you can cap as much RNA, after the fact, as you wish), long full-length transcription products are much more easily made and capping efficiencies can approach ~100%.

The only 2 drawbacks to this method are:

1. Requirement to do the capping reaction separate from the transcription reaction.
2. Substantial secondary structure existing on the 5′-end of the IVT RNA can deny access to the 5′-most nucleotide by the capping enzyme, and thus make capping inefficient. This drawback can be addressed by pre heat denaturing the IVT RNA prior to capping. Post-transcriptional capping reaction products are thus ~100% capped correctly.