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Issue 89, 2014
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The synthesis and application of a diazirine-modified uridine analogue for investigating RNA–protein interactions

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Abstract

The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate U DzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. U DzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.

Graphical abstract: The synthesis and application of a diazirine-modified uridine analogue for investigating RNA–protein interactions

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Supplementary files

Article information


Submitted
14 Aug 2014
Accepted
22 Sep 2014
First published
23 Sep 2014

This article is Open Access

RSC Adv., 2014,4, 48228-48235
Article type
Paper
Author version available

The synthesis and application of a diazirine-modified uridine analogue for investigating RNA–protein interactions

C. C. Smith, M. Hollenstein and C. J. Leumann, RSC Adv., 2014, 4, 48228
DOI: 10.1039/C4RA08682A

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