Chemically cleavable biotin-labeled nucleotide analogs.

Publisher Summary This chapter describes a procedure to synthesize the chemically cleavable biotinylated nucleotide Bio-19-SS-dUTP. The ability to incorporate biotin-labeled nucleotide analogs into DNA and RNA has stimulated interest in the use of the avidin–biotin affinity system to isolate protein–DNA and protein–RNA complexes. The development of the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP 6 appears to be a general solution to this problem. Bio-12-SS-dUTP contains a disulfide bond in the 12-atom linker arm joining biotin to the 5-carbon of uridine. Following the binding of biotinylated DNA to an avidin affinity column, a reducing agent, such as dithiothreitol is used to cleave the linker arm and release the DNA. The synthesis of Bio-19-SS-dUTP consists of three distinct steps. First, dUTP is reacted with mercury acetate to modify the 5-carbon of uracil. Second, allylamine is added to this position on the pyrimidine ring in the presence of a palladium catalyst. Third, allylamine-dUTP is reacted with an N -hydroxysuccinimide ester of biotin to produce the final product, Bio-19-SS-dUTP.