EFFECTS OF SITE-SPECIFIC SUBSTITUTION OF 5-FLUOROURIDINE ON THE STABILITIES OF DUPLEX DNA AND RNA

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studi ed to determine how FUrd substitution in nucleic acids may alter the e fficiency of biochemical processes that require complementary base pai ring for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, co ntains two non-equivalent uridines. Eight oligonucleotides (four RNA a nd four DNA) were prepared with either zero, one or two Urd substitute d by FUrd. The stability of each self-complementary duplex was determi ned by measuring the absorbance at 260 nm as a function of temperature . T-m values were calculated from the first derivative of the absorban ce versus temperature profiles and values for Delta H-0 and Delta S-0 were calculated from the concentration dependence of the T-m. Individu al absorbance versus temperature curves were also analyzed by a parame tric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters f or each oligonucleotide revealed that FUrd substitution had sequence-d ependent effects in both A-form RNA and B-form DNA duplexes, Conservat ion of helix geometry in FUrd-substituted duplexes was determined by C D spectroscopy, FUrd substitution at a single site in RNA stabilized t he duplex (Delta Delta G(37) = 0.8 kcal/mol), largely due to more favo rable stacking interactions, FdUrd substitution at a single site in DN A destabilized the duplex (Delta Delta G(37) = 0.3 kcal/mol) as a cons equence of less favorable stacking interactions. Ail duplexes melt via single cooperative transitions.