5-(1-propargylamino)-2′-deoxyuridine (UP):: a novel thymidine analogue for generating DNA triplexes with increased stability

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (U-P). The intramolecular triplex A(6)-L-T-6-L-(U-P)(5)T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent tripler A(6)-L-T-6-L-T-6 at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular tripler with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by U-P, in a pH dependent fashion. Tripler stability increases by similar to 12 K for each T to U-P substitution. Quantitative footprinting studies have examined the interaction of three U-P-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAG-AGAAAAAAGAA. At pH 5.0, the U-P-containing oligo; nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (U-P)(6)(CUT)-T-P binds similar to 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer U-P residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.