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

Smith, Christine Catherine; Hollenstein, Marcel; Leumann, Christian (2014). The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions. RSC advances, 4(89), pp. 48228-48235. Royal Society of Chemistry 10.1039/c4ra08682a

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Smith, Christine Catherine; Hollenstein, Marcel and Leumann, Christian

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

2046-2069

Publisher:

Royal Society of Chemistry

Language:

English

Submitter:

Christian Leumann

Date Deposited:

06 Oct 2014 13:23

Last Modified:

12 Oct 2015 12:11

Publisher DOI:

10.1039/c4ra08682a

BORIS DOI:

10.7892/boris.59048

URI:

https://boris.unibe.ch/id/eprint/59048

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