Poster abstracts

Poster number 61 submitted by Eduardo Paredes

A universal initiator for co- and post-transcriptional RNA labeling and conjugation

Eduardo Paredes (Department of Chemistry, Carnegie Mellon University), Debasish Grahacharya (Department of Chemistry, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University)

Abstract:
Derivatization of RNA has greatly facilitated investigations and applications of RNA structure and function. For 5'-terminal derivatives of RNA, current methodologies involve either complex syntheses of transcriptional initiator molecules or post-transcriptional conjugations with amine or thiol reactive groups. We describe here the simple synthesis and enzymatic incorporation of 5'-propargyl adenosine into RNA. Unlike other analogs of guanosine or adenosine with a monophosphate that cause premature transcriptional arrests, 5'-propargyl adenosine as the transcriptional initiator can produce RNA that bears a 5'-alkyne. The alkyne can be used in post-transcriptional conjugations through the highly efficient Cu(I)-catalyzed azide alkyne cycloaddition reaction (click-chemistry). As click-chemistry is bioorthogonal, the conjugation reaction can be performed directly in the transcription reaction mixture. Additionally, we show that not only is 5'-propargyl adenosine a transcriptional initiator but that it can be used as an universal intermediate in near quantitative click-reactions to rapidly generate triazolyl linked adenosine analogs, each of which can then be used directly as a transcriptional initiator. These transcriptional initiators produce quantitatively 5'-functionalized RNA. This significantly expands the conditions for and simplifies access to RNA functionalized with useful molecules.

Keywords: Transcriptional priming, Conjugation, RNA modification