title: Synthesis and application of a new 2',3'-isopropylidene guanosine substituted cap analog.
abstract: The synthesis and biological evaluation of a new cap analog, which is modified at the C2' and C3' positions of N(7)-methylguanosine is reported. The new cap analog, P(1)-2',3'-isopropylidene, 7-methylguanosine-5'P(3)-guanosine-5'triphosphate was assayed with respect to its effects on efficiency of incorporation into RNAs during in vitro transcription, and intracellular stability and translational activity of its 5'-capped mRNAs, upon transfection into HeLa cells. The intracellular stability of 5'-capped and uncapped full length test mRNAs was measured by using a real-time RT-PCR assay. The RNA with the 5'-modified cap was found to be approximately 1.7 times more stable than the RNA with the 5'-standard cap and approximately 2.5 times more stable than the uncapped control RNA. The translational efficiency was monitored by measuring the luciferase activity of a variety of in vitro synthesized and capped RNAs coding for a luciferase fusion protein after transfection into HeLa cells. The RNA capped with the 2',3'-isopropylidene substituted analog, (m(7,2',3'-isopropylidene)G[5']ppp[5']G), was translated the most efficiently, with approximately 2.9-fold more activity than the standard cap (m(7)G[5']ppp[5']G). The observed increase in the level of protein synthesis is likely resulted as a consequence of exclusively forward capped transcripts and increased cellular stability of the 5'-modified capped mRNA (Poly A).
authors.affiliation: Ambion, Inc., An Applied Biosystems Business, Bioorganic Chemistry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA.
authors.forename: Anilkumar R
authors.initials: AR
authors.lastname: Kore
authors.forename: Muthian
authors.initials: M
authors.lastname: Shanmugasundaram
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Vlassov
mesh_terms: D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D006151:Guanosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis / Q000502:physiology; D012314:RNA Cap Analogs / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C427043:2',3'-isopropylideneguanosine; D012314:RNA Cap Analogs; D014764:Viral Proteins; D006151:Guanosine; D008156:Luciferases; D049409:Luciferases, Firefly; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases