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Nucleic Acids Research, Vol 24, Issue 16 3173-3180, Copyright © 1996 by Oxford University Press

ARTICLES

Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation

A McGregor, MV Rao, G Duckworth, PG Stockley and BA Connolly
Department of Biochemistry, The University of Newcastle, Newcastle upon Tyne, UK.

The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems.
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