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Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG

Published online by Cambridge University Press:  11 January 2002

JANUSZ STEPINSKI
Affiliation:
Department of Biophysics, University of Warsaw, 02-089 Warsaw, Poland
CLINT WADDELL
Affiliation:
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, 71130-3932, USA
RYSZARD STOLARSKI
Affiliation:
Department of Biophysics, University of Warsaw, 02-089 Warsaw, Poland
EDWARD DARZYNKIEWICZ
Affiliation:
Department of Biophysics, University of Warsaw, 02-089 Warsaw, Poland
ROBERT E. RHOADS
Affiliation:
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, 71130-3932, USA
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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.

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RNA , Volume 7 , Issue 10 , October 2001 , pp. 1486 - 1495
Copyright
2001 RNA Society

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Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG
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Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG
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Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG
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