Synthesis and characterization of 5-[(4-azidophenacyl)thio]uridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analog
- 11 July 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (14), 5814-5820
- https://doi.org/10.1021/bi00440a017
Abstract
The synthesis, isolation, and characterization of a new photo-cross-linking uridine 5''-triphosphate analogue are described. This nucleotide analogue, 5-[(4-azidophenacyl)thio]uridine 5''-triphosphate (5-APAS-UTP), contains an aryl azide group apprximately 10 .ANG. from the uridine ring. The azide is photoactivated by irradiation at 300 nm, resulting in covalent attachment of the nucleotide to adjacent molecules. The nucleotide can be desulfurated with Raney nickel to cause molecular cleavage between the base and the aryl azide. Desulfuration yields uridine 5''-triphosphate and p-azidoacetophenone. If the analogue is cross-linked to another molecule, desulfuration leaves the analogue''s acetophenone group attached to that molecule. This effectively leaves behind a molecular tag on molecules that interact with the uridine analogue either as monomeric nucleotide or as part of an RNA molecule. This nucleotide analogue can be incorporated into internal positions in RNA by transcription in vitro with Escherichia coli RNA polymerase. It can therefore be used to examine interactions between RNA and other molecules (e.g., proteins or nucleic acids). Because the sulfur atom can be selectivity removed, the covalent bonds formed between analogue-containing RNA and other molecules can be cleaved, when desired, to facilitate identification of the cross-linked molecules and RNA nucleotides in the cross-linked complex.This publication has 13 references indexed in Scilit:
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