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Titolo:
Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3 '-O-methyl)GpppG and 7-methyl(3 '-deoxy)GpppG
Autore:
Stepinski, J; Waddell, C; Stolarski, R; Darzynkiewicz, E; Rhoads, RE;
Indirizzi:
Louisiana State Univ, Hlth Sci Ctr, Dept Biochem & Mol Biol, Shreveport, LA 71130 USA Louisiana State Univ Shreveport LA USA 71130 ol, Shreveport, LA 71130 USA Univ Warsaw, Dept Biophys, PL-02089 Warsaw, Poland Univ Warsaw Warsaw Poland PL-02089 Dept Biophys, PL-02089 Warsaw, Poland
Titolo Testata:
RNA-A PUBLICATION OF THE RNA SOCIETY
fascicolo: 10, volume: 7, anno: 2001,
pagine: 1486 - 1495
SICI:
1355-8382(200110)7:10<1486:SAPOMC>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
MESSENGER-RNA 5'-CAP; NMR COUPLING-CONSTANTS; PROTEIN-SYNTHESIS; KARPLUS EQUATION; AQUEOUS-SOLUTION; IN-VITRO; TRANSLATION; POLYMERASES; BINDING; INVITRO;
Keywords:
bacteriophage polymerases; cap analog inhibition; cap-dependent translation; in vitro transcription; ribonuclease T2; tobacco acid pyrophosphatase; translational efficiency;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
28
Recensione:
Indirizzi per estratti:
Indirizzo: Rhoads, RE Louisiana State Univ, Hlth Sci Ctr, Dept Biochem & Mol Biol, Shreveport, LA 71130 USA Louisiana State Univ Shreveport LA USA 71130 ort, LA 71130 USA
Citazione:
J. Stepinski et al., "Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3 '-O-methyl)GpppG and 7-methyl(3 '-deoxy)GpppG", RNA, 7(10), 2001, pp. 1486-1495

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 m(7)G Moiety of m(7)GpppG 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, P-1-3'-deoxy-7-methyguanosine-5' P-3- guanosine-5' triphosphate and P-1-3'-O,7-dimethylguanosine-5' P-3-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 solutionsemploying the catalytic properties of zinc salts. Structures were proven by H-1 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 m(7)GpppG or GpppG. Analysis ofthe 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 K-1 similar to that of m(7)GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m(7)GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/09/20 alle ore 20:43:06