Terminal Modifications of PNA and Their Use in Diagnostic and Antisense Technologies

Abstract

Peptide nucleic acids (PNA) are analogues of DNA that bind to DNA and RNA via Watson-Crick base-pairing rules. Due to the lack of a negatively-charged backbone, hybridisation of PNA to DNA or RNA occurs without electrostatic repulsion thus binding is typically stronger and more rapid than when traditional DNA probes are used. This is reflected in the increased melting temperature (Tm) of the conjugates. These properties, as well as the chemical and biological stability of PNA, make these molecules attractive for use in diagnostic and therapeutic applications. Amino acids are routinely conjugated to PNA probes to enhance the synthesis and solubility of the probes or assist with their cellular delivery, however little thought is given to the impact these modifications have on their hybridisation properties. In this work, a series of PNA-peptide chimeric assemblies based around a single PNA sequence were used to investigate the effect different amino acids have on the stability and specificity of PNA/DNA hybridisation. These experiments demonstrated that the positively charged amino acid lysine, which is routinely conjugated to PNA probes, increases the stability of the resultant PNA/DNA duplexes such that at many experimental temperatures, single base mismatched target DNA will also stably hybridise with PNA. In contrast, the negatively charged amino acid glutamic acid decreases the thermal stability of the mismatch duplexes sufficiently so that they are not stable at most experimental temperatures, whilst the fully complementary duplex is. This indicates that glutamic acid should replace lysine as the routine solubility enhancing group used for PNA probes.