KINETIC SEQUENCE DISCRIMINATION OF CATIONIC BIS-PNAS UPON TARGETING OF DOUBLE-STRANDED DNA

Strand displacement binding kinetics of cationic pseudoisocytosine-con taining linked homopyrimidine peptide nucleic acids (bis-PNAs) to full y matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding w as obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to mat ched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formatio n decreases exponentially Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three po sitive charges differ in their specificity of binding by one order of magnitude, Under appropriate experimental conditions the kinetic speci ficity for bis-PNA targeting of dsDNA is as high as 300, Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhi bit very high sequence specificity, thereby making such reagents extre mely efficient for sequence-specific targeting of duplex DNA.