Sequence‐specific DNA‐triplex formation at imperfect homopurine‐homopyrimidine sequences within a DNA plasmid

The ability of pyrimidine‐rich and purine‐rich oligodeoxynucleotides to form stable triple‐helical structures with imperfect R · Y target sites, containing C interruptions in the purine strand (CG inversions) and located within Bluescript KS+, a plasmid of 2959 bp, has been investigated by electrophoresis, ultraviolet absorbance and cleavage‐protection experiments. First, we synthesized double‐stranded oligonucleotides corresponding to the plasmid sites and studied their interaction with oligopyrimidines which oppose either G or T to CG inversions. The resulting imperfect DNA triplexes were detected by gel‐mobility shift. Their melting profiles were found to be biphasic, and the triplex‐to‐duplex plus single‐strand transition was affected by hysteresis. The 21‐nucleotide triplex containing three GC · G mismatched triads had t m = 45°C, while the same triplex, but with three GC · T triads, had t m = 31°C. Moreover, replacing C with 5‐methylcytosine in the third strand resulted in a significant stabilization of the defective triplex, t m = 49°C. The potential of the 21‐nucleotide oligopyrimidines to recognize and bind in a sequence‐specific manner to imperfect R · Y sequences in Bluescript KS+ has been investigated by means of a restriction‐endonuclease‐protection assay, taking advantage of the fact that one R · Y sequence of Bluescript KS+ was partially overlapped with a Hae II site. Effective endonuclease inhibition was observed with oligopyrimidines opposing G‐to‐GC inversions, at 10–50 μM. By contrast, the oligopyrimidine opposing T‐to‐CG inversions did not exhibit any interference with endonuclease activity in our standard conditions. Finally, we have tested the ability of purine‐rich strands to bind the R · Y sites of Bluescript KS+. A very weak cleavage protection was observed by using an oligomer (130 μM) with a polarity antiparallel to the purine strand of the target site. The resulting Y · R · R triplex was stabilized by CG · G (GC · G) and TA · A base triplets. This triplex denatures with a low cooperative melting profile suggesting the absence of strong interactions between the third strand and the target site.