Journal
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
Volume 18, Issue 17, Pages 4828-4832Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmcl.2008.07.075
Keywords
cap analog; T7 RNA polymerase; capping efficiency; in vitro transcription; mRNA stability; translation efficiency; luciferase activity; HeLa cells
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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 similar to 1.7 times more stable than the RNA with the 5'-standard cap and similar to 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 similar to 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 50-modified capped mRNA (Poly A). (C) 2008 Elsevier Ltd. All rights reserved.
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