| Reference | [1]. Jemielity, J., Fowler, T., Zuberek, J., Stepinski, J., Lewdorowicz, M., Niedzwiecka, A., Stolarski, R., Darzynkiewicz, E. and Rhoads, R.E., 2003.<br />
Novel “anti-reverse” cap analogs with superior translational properties.<br />
Abstract: Synthetic analogs of the 5′-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m7Gp3G. Unfortunately, because of the presence of a 3′-OH on both the m7Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two “anti-reverse” cap analogs (ARCAs), m73′dGp3G and m27,3′-OGp3G, that cannot be incorporated in the reverse orientation because of modifications at the C3′ position of m7Guo. In the present study, we have synthesized seven new cap analogs modified in the C2′ and C3′ positions of m7Guo and in the number of phosphate residues, m27,2′-OGp3G, m72′dGp3G, m72′dGp4G, m27,2′-OGp4G, m27,3′-OGp4G, m7Gp5G, and m27,3′-OGp5G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2′, like those at C3′, prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.<br />
Rna, 9(9), pp.1108-1122.<br />
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[2]. Peng, Z.H., Sharma, V., Singleton, S.F. and Gershon, P.D., 2002.<br />
Synthesis and application of a chain-terminating dinucleotide mRNA cap analog.<br />
Abstract: We describe the synthesis of a chain-terminating mRNA cap dinucleotide and its use in the in vitro transcription of homogeneously capped RNA. Computer modeling strongly indicates that RNA capped with the new compound will be a substrate for cap-dependent translation.<br />
Organic letters, 4(2), pp.161-164.<br />
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[3]. STEPINSKI, J., WADDELL, C., STOLARSKI, R., DARZYNKIEWICZ, E. and RHOADS, R.E., 2001.<br />
Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl (3′-O-methyl) GpppG and 7-methyl (3′-deoxy) GpppG.<br />
Abstract: The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957–967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3′-5′ phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P1-3′-deoxy-7-methyguanosine-5′ P3-guanosine-5′ triphosphate and P1-3′-O,7-dimethylguanosine-5′ P3-guanosine-5′ triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures were proven by 1H NMR. Transcripts produced with SP6 polymerase using “anti-reverse” cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG. Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs. Both of the ARCAs inhibited cell-free translation with a KI similar to that of m7GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.<br />
Rna, 7(10), pp.1486-1495.
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