RELATIVE STABILITY OF TRIPLEXES CONTAINING DIFFERENT NUMBERS OF T-CENTER-DOT-AT AND C-DOT-GC TRIPLETS(CENTER)

We have used DNase I footprinting to compare the stability of parallel triple helices containing different numbers of T.AT and C+.GC triplet s. We have targeted a fragment containing the 17mer sequence 5'-AGGAAG AGAAAAAAGAA with the 9mer oligonucleotides 5'-TCCTTCTCT, 5'-TTCTCTTTT and 5'-TTTTTTCTT, which form triplexes at the 5'-end, centre and 3'-en d of the target site respectively, Quantitative DNase I footprinting h as shown that at pH 5.0 the dissociation constants of these oligonucle otides are 0.13, 4.7 and >30 mu M respectively, revealing that increas ing the proportion of C+.GC triplets increases tripler stability, The results suggest that the positive charge on the protonated cytosine co ntributes to tripler stability, either by a favourable interaction wit h the stacked pi system or by screening the charge on the phosphate gr oups, In the presence of a naphthylquinoline tripler binding ligand al l three oligonucleotides bind with similar affinities, At pH 6.0 these triplexes only form in the presence of the tripler binding ligand, wh ile at pH 7.5 footprints are only seen with the oligonucleotide which generates the fewest number of C+.GC triplets (TTTTTTTCTT) in the pres ence of the ligand.