Surface plasmon resonance study of PNA interactions with double-stranded DNA

Peptide nucleic acid (PNA) is a well known DNA analogue bearing a N-(2-aminoethyl)glycine backbone (aegPNA). This molecule is able to not only form a duplex with single stranded (ss) nucleic acids but also higher-order (i.e., three- and four-stranded) complexes with double-stranded (ds) DNA in a sequence specific manner. Here, the application of surface plasmon resonance (SPR) to study the binding of PNA to dsDNA is reported for the first time. SPR protocols were developed to verify the sequence rules and conditions for binding (pH and ionic strength) of homopyrimidine and homopurine aegPNAs to dsDNA, for which the solution phase behaviors are known, allowing a direct comparison. Then, using real-time SPR measurements, the hybridization efficiency, binding direction (antiparallel and parallel direction), sequence-dependent binding modes of the PNA to dsDNA (triplex formation and duplex invasion) and the binding kinetics associated with the binding mode were all ascertained. These SPR protocols were then further applied to study the dsDNA binding properties of a new conformationally rigid PNA bearing a D-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone (acpcPNA), which revealed that acpcPNA cannot form higher-order complexes with dsDNA through either triplex formation or duplex invasion. The SPR technique is thus shown to be a powerful technique for studying higher-order nucleic acid complexes. The binding behaviors of aegPNA obtained from the SPR analysis in the solid–liquid phase measurement correlate well with those in the literature derived from solution phase measurements.