End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes

Abstract

Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine–homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA–DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

Highly selective single nucleotide polymorphism recogniton by a chiral (5S) PNA beacon

“…16 Dabcyl was used as quencher, linked to the e-amino group of lysine at the C-terminal part and carboxyfluorescein was used as fluorophore, linked to the N-terminus of the PNA molecule; two additional charges (a positive arginine side chain and a negative glutamate unit) were inserted just before and after the PNA segment, according to the design described by Frank-Kamenetskii and coworkers. 16,17 Arginine was used instead of lysine as originally proposed, in order to avoid any possible competition with the side chain amino group of the chiral monomer during attachment to a solid support or micro-and nanoparticles. Recent studies carried out in our laboratory have shown that substitution of the achiral N-(2-aminoethyl) amino acid backbone with chiral units derived from amino acid can affect the DNA binding ability depending on the position and the stereochemistry of the stereogenic centers introduced.…”

“…Achiral PNA beacons have been successfully used in many applications and have shown a strong discrimination ability between full matched and single mismatched DNA sequences; 16,17 however, it was interesting to verify if the selectivity in recognizing single-point mutations could be further improved by the presence of a stereogenic center in the backbone. The hybridization properties of the chiral PNA beacon were examined using UV melting temperatures and compared with those observed with the achiral unmodified beacon (Table 1).…”

“…[9][10][11][12] An important class of probes is represented by molecular beacon (MB) oligonucleotides, which are modified with a fluorophore and a quencher (or a quenching surface) at each end, held together by a dsDNA stem made of complementary sequences; this structure allows to produce a fluorescence ''switch-on'' upon interaction with the target DNA. [13][14][15] PNA beacons [16][17][18] and the related ''light up probes'' [19][20][21] have also been recently described, displaying the advantages of higher selectivity and simpler design. Peptide nucleic acids (PNAs) are oligonucleotide mimics based on a polyamide backbone, superior to oligonucleotides in the recognition of single base mutations.…”

Sequence‐Unrestricted, Watson–Crick Recognition of Double Helical B‐DNA by (R)‐MiniPEG‐γPNAs

“…Though promising as antigene reagents, because of the specificity of recognition and generality in sequence design, PNA binding is presently limited to mostly homopurine [22] and homopyrimidine targets. [23] Mixed-sequence PNAs have been shown to be capable of invading topologically constrained supercoiled plasmid DNA, [24][25][26][27] conformationally perturbed regions of genomic DNA [28] and duplex termini; [29] however, they are unable to invade the interior regions of double helical B-form DNA (B-DNA)-the most stable form of DNA double helix. "Tail-clamp" [30,31] and "doubleduplex invasion" [32] strategies have subsequently been developed and have enabled mixed-sequence PNAs to invade B-DNA, but they are not without limitations.…”

Strand Invasion of Mixed-Sequence, Double-Helical B-DNA by γ-Peptide Nucleic Acids Containing G-Clamp Nucleobases under Physiological Conditions

“…More recently, we showed that sequence restriction can be relaxed by utilizing conformationally preorganized γPNAs as molecular reagents (Chart 1a, bottom) (19). However, like all the other studies, with the exception of triplex-binding by homopyrimidine PNAs (20), strand invasion of linear double helical B-DNA was performed in buffers containing relatively low salt concentrations (11, 18, 19, 21, 22). When subjected to physiological ionic strengths, little to no binding was observed (23).…”