Reading DNA with PNA: a dynamic chemical approach to DNA sequence analysis

Abstract

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease aetiology and idiosyncratic differences in drug response. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or the hybridization of allele-specific DNA probes. Herein, a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping was developed. The approach was initially demonstrated in model systems using synthetic DNA, which was used as a template in a base-filling reductive amination reaction on a PNA backbone. The templated dynamic reaction between a free secondary amine at a ‘blank’ position on the PNA strand and four aldehyde-modified nucleobases drove selective formation of the ‘correct’ iminium intermediate according to Watson-Crick base-pairing rules. In a blind trial, the method was extended to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. Enzyme-free dynamic chemistry thus permitted successful genotyping in both singleplex and duplex formats, demonstrating the application of dynamic chemistry as a distinct method of allelediscrimination with certain advantages over those reported previously. The application of this method as a tool for the discovery of non-natural nucleobases with improved properties for antisense and genotyping applications was also investigated. Furthermore, progress was made towards the use of dynamic chemistry as a means of full nucleic acid sequence analysis, through the templated sequence-selective extension of PNA probes by reductive amination.