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In this paper we demonstrate that peptide nucleic acids (PNAs) are excellent probes able to detect the W1282X point mutation of the cystic fibrosis (CF) gene when biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and biosensor technologies is performed. The results reported here suggest that BIA is an easy, fast, and automatable approach for detecting mutations of CF, allowing real-time monitoring of hybridization between 9-mer CF PNA probes and target biotinylated PCR products generated from healthy, heterozygous subjects and homozygous W1282X samples and immobilized on streptavidin-coated sensor chips. This method is, to our knowledge, the first application of PNAs, BIA, and SPR to a human hereditary mutation, and demonstrates the feasibility of these approaches for discriminating between normal and mutated target DNA. We like to point out that the procedure described in this paper is rapid and informative; results are obtained within a few minutes. This could be of great interest for molecular pre-implantation diagnosis to discriminate homozygous CF embryos from heterozygous and healthy embryos. Other advantages of the methodology described in the present paper are (a) that it is a nonradioactive methodology and (b) that gel electrophoresis and/or dot-spot analysis are not required. More importantly, the demonstration that SPR-based BIA could be associated with microarray technology allows us to hypothesize that the method described in the present paper could be used for the development of a protocol employing multispotting on SPR biosensors of many CF-PCR products and a real-time simultaneous analysis of hybridization to PNA probes. These results are in line with the concept that SPR could be an integral part of a fully automated diagnostic system based on the use of laboratory workstations, biosensors, and arrayed biosensors for DNA isolation, preparation of PCR reactions, and identification of point mutations.

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.

The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs). We analyzed the effect of a T10 PNA on in vitro transcription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues. At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner. The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription. Thus, an impairment of pol III recycling fully accounts for the observed inhibition. As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA adduct before reaching the terminator region. On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs. Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription.