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Synthesis of N1-(beta-D-2'-deoxyribosyl)-5-methyl-2-pyrimidinone nucleoside and its incorporation into oligodeoxyribonucleotides for the mechanistic investigation of RecA mediated homologous DNA strand exchange
Singleton, Scott F.
Master of Arts
A new method for the synthesis of N1-(beta- D-2'-deoxyribosyl)-5-methyl-2-pyrimidinone ( 1) was developed. Fluorescent nucleoside 1 was synthesized from thymidine in 5 steps and 40% overall yield. The 3-O-phosphoramidite of 1 was synthesized and oligodeoxyribonucleotides (ODNs) containing 1 were prepared in good yields using standard, automated solid-phase methodology. The ODNs were purified by RP-HPLC or PAGE. The purity of ODNs was analyzed by CE. Fluorescence spectra of the ODNs were recorded. The fluorescence of 1 was substantially quenched when 1 was incorporated into ODNs, and was quenched further after the formation of a double-stranded DNA. The binding of RecA protein to an ODN containing 1 resulted in an increase in the emission fluorescence yield in the absence or in the presence of ATPgammaS. The fluorescence emission and anisotropy changes that accompany the formation of the complexes have enabled us to establish by titration that RecA protein binds to about 3 nucleotides of ODN.