Triple helix formation at (AT)(n) adjacent to an oligopurine tract

Abstract

We have used DNase I footprinting to investigate the recognition of (AT)(n) tracts in duplex DNA using GT-containing oligonucleotides designed to form alternating G·TA and T·AT triplets. Previous studies have shown that the formation of these complexes is facilitated by anchoring the triplex with a block of adjacent T·AT triplets, i.e. using T11(TG)6 to recognize the target A11(AT)6.(AT)6T11. In the present study we have examined how the stability of these complexes is affected by the length of either the T·AT tract or the region of alternating G·TA and T·AT triplets, using oligonucleotides of type T(x)(TG)(y) to recognize the sequence A11(AT)11. We find that successful triplex formation at (AT)(n) (n = 3, 6 or 11) can be achieved with a stabilizing tail of 11 x T·AT triplets. The affinity of the third strand increases with the length of the (GT)(n) tract, suggesting that the alternating G·TA and T·AT triplets are making a positive contribution to stability. These complexes are stabilized by the presence of manganese or a triplex-specific binding ligand. Shorter oligonucleotides, such as T7(TG)5, bind less tightly and require the addition of a triplex-binding ligand. T4(GT)5 showed no binding under any conditions. Oligonucleotides forming a 3'-terminal T·AT are marginally more stable that those with a terminal G·TA. The stability of these complexes was further increased by replacing two of the T·AT triplets in the T(n) tail region with two C+·GC triplets.