Editing Capped mRNA (section)

== Introduction ==

Most naturally occurring eukaryotic mRNAs and many viral RNAs have a modified nucleotide structure known as a "cap" on their 5' terminus. In vivo, the cap structure is involved in the initiation of protein synthesis and in mRNA processing and stability. Many viral RNAs are infectious only when capped, and uncapped RNAs introduced into cells via transfection or microinjection are rapidly degraded by cellular RNases. Therefore, RNA prepared for microinjection into oocytes or for transfection into eukaryotic cells should be capped. In vitro, capped mRNAs are translated more efficiently than uncapped transcripts in rabbit reticulocyte lysate or wheat germ translation systems. The trimethylated cap is involved in&nbsp; RNA transport and RNA translation in nematodes.&nbsp; Transcripts synthesized for use in in vitro RNA splicing experiments may also need to be capped.



The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 μg RNA per 20 μl reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed. Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability, efficient translation, nuclear transport and splicing. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or&nbsp; co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications such as generating viral genomic RNA for reverse-genetic systems and crystallographic studies of cap binding proteins such as eIF4E.

The 5' cap structure improves stability of the RNA and translation efficiency, and hence is important for microinjection and transfection experiments. The co-transcriptional capping method generates 40-50 μg of ~80% capped RNA. It is important to note that using 3'-0-Me-m7G(5')ppp(5')G RNA cap analog (ARCA), which is blocked at the 3'-hydroxyl of the m7G ensures incorporation of the cap in the correct orientation. To cap larger quantities of RNA it is possible to scale up the standard reactions for both capping methods. However, using the Vaccinia Capping System would be a more feasible option cost-wise since co-transcriptional capping utilizes cap analog, which is a relatively expensive component. The Vaccinia Capping System is also a better method since it has capping efficiency of almost 100%