Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor

Abstract

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance.

Peptide nucleic acids on microarrays and other biosensors

“…In further experiments, PNAs containing a 'chiral box' at their center showed higher mismatch recognition properties than normal molecules. The chirality was introduced by PNA monomers that were based on D-lysine instead of the usual N-aminoethylglycine backbone [35][36][37].…”

Gene Modulation by Peptide Nucleic Acids (PNAs) Targeting microRNAs (miRs)

“…PNAs can be modified in order to achieve better performances in terms of cellular permeation, higher affinity, and specificity for the target DNA and RNA sequences (Corradini et al, 2004, 2007, Sforza et al, 2000, 2007a,b,c, Wojciechowski and Hudson, 2007, Dragulescu-Andrasi 2005, Rapireddy 2007. In this chapter we will describe the recent results reported in the literature by using PNAs as anti-miR agents and the perspectives of this technology for future development.…”

“…The 2D-lysine "chiral box" PNA showed also an increased sequence selectivity, both in terms of direction control and of recognition of a single base mismatch (Sforza et al, 2000). Therefore, this type of structures was found ideal for targeting point mutations in genes of diagnostic interest (Corradini et al, 2004, Tedeschi et al, 2005a. Recently chiral PNAs with L-or D-stereocenters either at the 2-or the 5-positions of the monomer or with both stereocenters simultaneously present have also been synthesized and studied .…”

A Novel Type of Nucleic Acid-based Biosensors: the Use of PNA Probes, Associated with Surface Science and Electrochemical Detection Techniques

“…In the latter case, DNA binding to PNA induces a change in the refractive index, proportional to the mass uptake on the chip surface and enabling a real-time monitoring of the hybridisation process (Jensen et al, 1997). A small review of some PNA-based biosensors has been recently published, presenting some examples of biosensors using labels or not, and showing the interest of using these DNA mimics (Brandt & Hoheisel, 2004;Corradini et al, 2004); they report in particular that PNA monomers may be modified by adding a chiral center to enhance their mismatch recognition sensitivity by SPR (Corradini et al, 2004); also, instead of using the direct grafted PNA format, PCR products may be attached to the SPR sensor surface, followed by PNA oligomer hybridisation leading to highly efficient PNA hybridisation; this has been attributed to the better accessibility of PNA probes to the immobilised PCR, compared to that of DNA oligonucleotides (Feriotto et al, 2001). However, the SPR detection sensitivity often needs to be enhanced by incorporating labels or using a second hybridisation (sandwich format).…”