Quantitative investigation of the modular primer effect for DNA and peptide nucleic acid hexamers

The effect on oligonucleotide-template duplex stability upon cohybridizatio n of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexam er modules and sensor chip-immobilized template DNA strands were designed f or analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the deter mination of apparent duplex affinities from equilibrium responses. The resu lts showed that the hybridizational stability of modular hexamer pairs is s trongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in appa rent affinities could be observed for adjacent modular oligonucleotide pair s compared to affinities determined for single hexamer oligonucleotide hybr idizations. Interestingly, also for coinjections of different module combin ations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be us eful for the selection of appropriate modules and working concentrations fo r use in different modular hybridization applications. (C) 1999 Academic Pr ess.