To study the helical structure in a P-loop formed by an invasion of oligopyrimidine peptide nucleic acid (PNA) into DNA duplex, bent DNA fragments containing a homopurine-homopyrimidine sequence between two bent DNA loci were prepared. As the spacer DNA length between the two bent loci varied by 1 bp over one helical turn, the electrophoretic mobility, reflecting the overall extent of DNA bending, was modulated sinusoidally in non-denaturing 5% polyacrylamide gel. When the bent DNA fragments differing in the spacer DNA length were preincubated with an oligopyrimidine PNA, the gel mobilities were changed due to a P-loop formation. By analyzing the gel mobility data with variations of the P-loop size, average helical parameters at the P-loop structure were determined. (PNA)2·(DNA) triplex within a P-loop had the helical periodicities of 15.6 (±0.2) bp per turn at 20°C and 17.4 (±0.7) bp per turn at 10°C. In addition, the results indicate that a helical unwinding by 57 (±7) degrees at 20°C and 37 (±13) degrees at 10°C is present at the two junctions between a P-loop and its adjacent DNA duplex.
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