Specificity of Antiparallel DNA Triple Helix Formation

Abstract

We have used DNase I footprinting to examine the formation of antiparallel DNA triple helices on DNA fragments containing the homopurine target sites (GGA)2GGX(GGA)2GG.(CCT)2CCZ(CCT)2CC (where X.Z is each base pair in turn), with the GA- and GT-rich oligonucleotides, (GGA)2GGN(GGA)2GG and (GGT)2GGN(GGT)2GG (N = each base in turn). These were designed to form G.GC and A.AT or T.AT triplets with a central N.XZ mismatch, which should bind in an antiparallel orientation. We find that almost all combinations generate DNase I footprints at low micromolar concentrations. At each target site, the relative binding of the GA- and GT-containing oligonucleotides was not the same, suggesting that these two triplexes adopt different conformations. For a central GC base pair, the most stable complex is observed with a third strand generating a G.GC triplet as expected. A.GC is also stable, especially in the GT oligonucleotides. For a central AT base pair, all four bases form stable complexes though T.AT is favored for the GA-rich thirds strands and A.AT for the GT-rich strands. For a central CG base pair, the stable complexes are seen with third strands generating T.CG triplets, though A.CG and C.CG are stable with GT- and GA-containing oligonucleotides, respectively. C.TA is the best triplet at a central TA base pair. The third strands with central guanines avoided the formation of G.YR triplets on the fragments containing central pyrimidines, producing DNase I footprints which had slipped relative to the target site. These oligonucleotides bound at a different location, generating complexes containing 11 contiguous stable triplets at the 3'-end of the third strand. The results suggest rules for designing the best third strand oligonucleotides for targeting sequences in which homopurine tracts are interrupted by pyrimidines.

Targeting DNA with Triplexes

“…Similarly in a REPSA selection assay for oligonucleotides which generate stable triplexes at a 19-mer target site, perfect matches were observed for the 13 bases at the 3'-end, while mismatches were selected at the 5'-end, and shorter oligonucleotides generated complexes with much reduced stability [179]. The greater effect of the 3'-end of the third strand has also been observed in footprinting experiments with oligonucleotides containing (GGA) n repeats, for which slipped structures were observed [171]. More recently this effect has been confirmed by Arimondo et al [180] who compared the strength of triplex formation by (AGG) 4 and (GGA) 4 4 ].…”

“…In some studies antiparallel triplexes are shown to have very high affinities; short oligonucleotides exhibit nanomolar binding constants [169,170], while in other studies longer oligonucleotides, under similar conditions, fail to show binding at micromolar concentrations [137,[170][171][172][173].…”

“…It is generally agreed that GT triplexes are less stable than their GA counterparts [171,[174][175][176], possibly because the antiparallel G q GC triplet is more closely isomorphous to A q AT than Self association of these oligonucleotides can effectively compete with triple helix formation and prevent complete triplex formation since, at high concentrations, more of the oligonucleotide is trapped in self-associated structures. The self-structures adopted by GA-containing oligonucleotides explain the opposite temperature dependence of triplexes formed with GA-and GT-containing oligonucleotides.…”

Base triplet nonisomorphism strongly influences DNA triplex conformation: Effect of nonisomorphic G∗︁ GC and A∗︁ AT triplets and bending of DNA triplexes

“…60 This is in addition to the GA and GT TFOs themselves exhibiting different binding affinities to the target DNA duplex. 61 Most interestingly, the possibility of inducing a bend in a purine-rich tract of DNA duplex using a TFO is perceived when a G * GC triplet mediates between mini A * AT triplexes and vice versa (Figures 9-12). Twist angle and conformational variations in the RH strand together with an increase in WH groove width and variations in X-displacement of the WC duplex pairs at the nonisomorphic junctions are found to be the cause for bending.…”

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn2+ ions

“…Triple helical structures can also be obtained with a third strand containing pyrimidines and purines (GT-rich oligonucleotides) through formation of G•G.C and T•A.T base triples. Most reports concerning such GT third strand triple helices also considered an antiparallel third strand orientation (11)(12)(13)(14)(15)(16). It has been proposed that the number of GpT/TpG steps in the third strand will determine the orientation of the third strand with respect to the purine duplex strand.…”