Chemical Route to the Capped RNAs

Abstract

Eukaryotic and viral messenger RNAs contain a CAP structure that plays an important role in the initiation of translation and several other cellular processes that involve mRNAs. In this paper, we report a convenient chemical approach to the preparation of milligram quantities of short, capped RNA oligonucleotides, which overcomes some of the limitations of previous approaches. The method is based on the use of a reactive precursor, m7GppQ [P1-7-methylguanosine-5'-O-yl, P2-O-8-(5-chloroquinolyl) pyrophosphate]. The precursor reacts smoothly with 5'-phosphorylated unprotected short RNA in the presence of CuCl2 in organic media. The feasibility of this approach was demonstrated by the synthesis of the capped pentaribonucleotide m7GpppGpApCpU. The synthesized capped oligonucleotide was isolated and purified by reverse phase and ion exchange HPLC with a final yield of 37%. The structure of the m7GpppGpApCpU was confirmed by 31P NMR, mass-spectrometry and enzymatic hydrolysis.

High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions

“…Chemical approaches (the advantage being that the sequence can be varied) for the synthesis of short-capped RNAs described in the literature gave isolated yields from 2 to 37%, as summarized by Koukhareva (8). Similar or superior results have been obtained, at least for the main products, using our enzymatic approach.…”

“…They can be synthesized chemically starting from mono- or diphosphate RNA (7,8). A 7Me GpppA cap can also be added to di- or triphosphate RNA using vaccinia virus capping enzyme that contains RNA triphosphatase, guanylyltransferase and N7MTase activities (9,10).…”

Synthesis of Capped RNA using a DMT Group as a Purification Handle

“…[12] A second approach is entirely chemical, and usually involves the condensation of an activated, methylated guanosine diphosphate with a short 5 -phosphorylated oligonucleotide. [4,[13][14][15][16] These capped RNAs can be made in larger amounts. The dinucleotide cap analogues for the Synthesis of Capped RNA 41 first approach are also made using the same type of chemical condensation, although the methylated guanosine is often the nucleophile.…”

“…The dinucleotide cap analogues for the Synthesis of Capped RNA 41 first approach are also made using the same type of chemical condensation, although the methylated guanosine is often the nucleophile. In both approaches, the most common activating group is imidazole, [17] although 5-chloro-8-hydroxyquinoline [13] and morpholine [9] have also been used. The reactions often include metal salts, and are most often carried out in anhydrous organic solvents, [17] although aqueous solutions have also been used.…”

Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase

“…While the automated large-scale chemical synthesis of RNA on solid support was established more than 20 years ago, the synthesis of the 59-capped RNA is still a challenge, even at the milligram scale. Although a few attempts for their synthesis in solution were reported (Sekine et al 1985;Iwase et al 1988;Sekine et al 1989Sekine et al , 1996Sawai et al 1999;Zubereck et al 2003;Koukhareva and Lebedev 2004;Mikkola et al 2005), the associated yields were always rather low, with long reaction times (6-10 d), and associated purification difficulties to obtain material in large quantity. Regarding the synthesis of 59-capped RNAs on solid support, the instability of N 7 -methylguanosine under acidic and basic conditions has limited the efforts in this area.…”