• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of rnaThe RNA SocietyeTOC AlertsSubscriptionsJournal HomeCSHL PressRNA
RNA. Oct 2001; 7(10): 1486–1495.
PMCID: PMC1370192

Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG.

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, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-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 K(I) 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.

Full Text

The Full Text of this article is available as a PDF (500K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Pasquinelli AE, Dahlberg JE, Lund E. Reverse 5' caps in RNAs made in vitro by phage RNA polymerases. RNA. 1995 Nov;1(9):957–967. [PMC free article] [PubMed]
  • Cai A, Jankowska-Anyszka M, Centers A, Chlebicka L, Stepinski J, Stolarski R, Darzynkiewicz E, Rhoads RE. Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation. Biochemistry. 1999 Jun 29;38(26):8538–8547. [PubMed]
  • Canaani D, Revel M, Groner Y. Translational discrimination of 'capped' and 'non-capped' mRNAS: inhibition of a series of chemical analogs of m7GpppX. FEBS Lett. 1976 May 1;64(2):326–331. [PubMed]
  • Carberry SE, Darzynkiewicz E, Stepinski J, Tahara SM, Rhoads RE, Goss DJ. A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E. Biochemistry. 1990 Apr 3;29(13):3337–3341. [PubMed]
  • Contreras R, Cheroutre H, Degrave W, Fiers W. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic Acids Res. 1982 Oct 25;10(20):6353–6362. [PMC free article] [PubMed]
  • Hendler S, Fürer E, Srinivasan PR. Synthesis and chemical properties of monomers and polymers containing 7-methylguanine and an investigation of their substrate or template properties for bacterial deoxyribonucleic acid or ribonucleic acid polymerases. Biochemistry. 1970 Oct 13;9(21):4141–4153. [PubMed]
  • Hickey ED, Weber LA, Baglioni C. Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. Proc Natl Acad Sci U S A. 1976 Jan;73(1):19–23. [PMC free article] [PubMed]
  • Konarska MM, Padgett RA, Sharp PA. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. [PubMed]
  • Lankhorst PP, Haasnoot CA, Erkelens C, Altona C. Carbon-13 NMR in conformational analysis of nucleic acid fragments. 2. A reparametrization of the Karplus equation for vicinal NMR coupling constants in CCOP and HCOP fragments. J Biomol Struct Dyn. 1984 Jun;1(6):1387–1405. [PubMed]
  • Lassota P, Stolarski R, Shugar D. Conformation about the glycosidic bond and susceptibility to 5'-nucleotidase of 8-substituted analogues of 5'-GMP. Z Naturforsch C. 1984 Jan-Feb;39(1-2):55–63. [PubMed]
  • Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK. Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell. 1997 Jun 13;89(6):951–961. [PubMed]
  • Matsuo H, Li H, McGuire AM, Fletcher CM, Gingras AC, Sonenberg N, Wagner G. Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nat Struct Biol. 1997 Sep;4(9):717–724. [PubMed]
  • Mattaj IW. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986 Sep 12;46(6):905–911. [PubMed]
  • Pasquinelli AE, Dahlberg JE, Lund E. Reverse 5' caps in RNAs made in vitro by phage RNA polymerases. RNA. 1995 Nov;1(9):957–967. [PMC free article] [PubMed]
  • Shatkin AJ. mRNA cap binding proteins: essential factors for initiating translation. Cell. 1985 Feb;40(2):223–224. [PubMed]
  • Shatkin AJ. mRNA caps--old and newer hats. Bioessays. 1987 Dec;7(6):275–277. [PubMed]
  • Wieczorek Z, Zdanowski K, Chlebicka L, Stepiński J, Jankowska M, Kierdaszuk B, Temeriusz A, Darzynkiewicz E, Stolarski R. Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues. Biochim Biophys Acta. 1997 Nov 1;1354(2):145–152. [PubMed]
  • Yisraeli JK, Melton DA. Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol. 1989;180:42–50. [PubMed]

Articles from RNA are provided here courtesy of The RNA Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...