Abstract
For site specific incorporation of 4-thiouridine into oligoribonucleotides a new phosphoramidite is proposed. It makes use of the S-pivaloyloxymethyl group for the protection of the thiol function. This group is easily introduced and removed without modification of the standard protocol for solid phase synthesis of RNA. Three 4-thiouridine-containing oligoribonucleotides (21-mers), corresponding to tRNA minisubstrates of yeast tRNA:pseudouridine-55 synthase (Pus4) were prepared. These 4-thiouridine containing substrates were characterized and used as photoaffinity probe of the enzyme:substrate complex. Irradiation resulted in the specific photocross-linking of these oligoribonucleotides with purified recombinant tRNA:pseudouridine-55 synthase.
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