Labeling and quantitative analysis of i6A-incorporated RNA via In-situ azidation of prenyl functionality and click reaction

Abstract

We report here the efficient synthesis of prenyl-containing i6A-RNA bearing the representative ACGCUAAAAA sequence through phosphoramidite chemistry. To determine proof-of-concept, we have developed two approaches for selectively fluorescent labeling of this i6A-RNA, one relied on direct use of probe PTAD-DBCO-Cy5 via Ene-ligation in 37% yield, another one constituted two sequential processes including azidation by use of TMSN3/ Selectfluor and subsequent click reaction with DBCO-Cy5 in total 89% yield.