PNA Beacons for Duplex DNA

Kuhn, Heiko; Demidov, Vadim V.; Gildea, Brian; Fiandaca, Mark J.; Coull, James; Frank‐Kamenetskii, Maxim D.

doi:10.1089/108729001317022269

Cited by 61 publications

(45 citation statements)

References 36 publications

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“…It may also have various biotechnological applications, including the PNA-assisted DNA rare cleavage (10,(13)(14)(15)(16)(17), duplex DNA capture (8,18,19), topological DNA labeling (8,(20)(21)(22)(23), direct DNA sequencing (8,17,24), and hybridization of molecular beacons to dsDNA (8,25,26). Although the previous studies have provided convincing evidence for the double-duplex invasion mode of pcPNA binding to dsDNA (9,10), the mechanism of this process is still not well characterized, and key factors controlling the formation of pcPNA-dsDNA complexes have yet to be revealed.…”

mentioning

confidence: 99%

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

Demidov

Protozanova

Izvolsky

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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102

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If adenines and thymines in two mutually complementary mixedbase peptide nucleic acid (PNA) oligomers are substituted with diaminopurines and thiouracils, respectively, so-called pseudocomplementary PNAs (pcPNAs) are created. Pairs of pcPNAs have recently demonstrated an ability to highly selectively target essentially any designated site on double-stranded DNA (dsDNA) by forming very stable PNA-DNA strand-displacement complexes via double duplex invasion (helix invasion). These properties of pcPNAs make them unique and very promising ligands capable of denying the access of DNA-binding proteins to dsDNA. To elucidate the sequence-unrestricted mechanism of sequence-specific dsDNA recognition by pcPNAs, we have studied the kinetics of formation of corresponding PNA-DNA complexes at various temperatures by the gel-shift assay. In parallel, the conditions for possible selfhybridization of pcPNA oligomers have been assayed by mixing curve (Job plot) and thermal melting experiments. The data indicate that, at physiological temperatures (Ϸ37°C), the equilibrium is shifted toward the pairing of corresponding pcPNAs with each other. This finding explains a linear concentration dependence, within the submicromolar range, of the pcPNA invasion rate into dsDNA at 37°C. At elevated temperatures (>50°C), the rather unstable pcPNA duplexes dissociate, yielding the expected quadratic dependence for the rate of pcPNA invasion on the PNA concentration. The polycationic character of pcPNA pairs, carrying the duplicated number of protonated terminal PNA residues commonly used to increase the PNA solubility and binding affinity, also explains the self-inhibition of pcPNA invasion observed at higher PNA concentrations. Melting of pcPNA duplexes occurs with the integral transition enthalpies ranged from ؊235 to ؊280 kJ⅐mol ؊1 , contributing to an anomalously high activation energy of Ϸ150 kJ⅐mol ؊1 found for the helix invasion of pcPNAs carrying four different nucleobases. A simplified kinetic model for pcPNAs helix invasion is proposed that interprets all unusual features of pcPNAs binding to dsDNA. Our findings have important implications for rational use of pcPNAs.double-stranded DNA ͉ pseudocomplementary PNA ͉ sequence-selective recognition P eptide nucleic acids (PNAs) and their derivatives are of significant biomedical and biotechnological interest as prospective biomolecular tools for highly selective manipulation of nucleic acids (1-8). Recently, a new modification of PNAs has been introduced for sequence-unrestricted targeting of doublestranded DNA (dsDNA). Along with ordinary guanines and cytosines, these PNAs, dubbed pseudocomplementary PNAs (pcPNAs; refs. 9 and 10), carry 2,6-diaminopurines (D) and 2-thiouracils ( s U) instead of adenines and thymines, respectively.Model building revealed a steric clash between the bulky thio group of s U (or similarly modified thymine) and one of the two amino groups of D within the s U-D pair (9, 11). This clashing effect must severely destabilize the pcPNA-pcPNA duplexes whereas bindin...

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mentioning

confidence: 99%

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

Demidov

Protozanova

Izvolsky

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

102

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“…PNA beacons are convenient fluorogenic hybridization probes (21,56). We therefore decided to check whether they could sequence-specifically signal the presence of corresponding dsDNA targets via strand-invasion binding to the complementary sites with arbitrary sequences located at the end of a long linear DNA duplex.…”

Section: Resultsmentioning

confidence: 99%

“…Some of the suggested applications of strand-invading PNAs include the antigene and gene-activation strategies (13–18). PNAs have also been used as sequence-specific probes in clinical diagnostics (19,20), and as strand-specific dsDNA ‘openers’ (21–26), and as sequence-specific DNA binding anchors for biomarkers and other biologically active molecules (27–29). …”

Section: Introductionmentioning

confidence: 99%

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

Smolina

Demidov²,

Soldatenkov³

et al. 2005

Nucleic Acids Research

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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.

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“…55). The introduction of two different fluorescent labels in PNA is of interest for the preparation of molecular beacons [247,250,251], for diagnosis and biomedical studies [252].…”

Section: Labelled Pnasmentioning

confidence: 99%

Peptide Nucleic Acids (PNAs), A Chemical Overview

Porcheddu¹,

Giacomelli

2005

CMC

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Peptide nucleic acid (PNA) is a nucleic acid analogue and a fully synthetic DNA/RNA-recognising ligand with a neutral peptide-like backbone. In spite of the large change on the backbone structure, PNA molecules bind strongly to complementary DNA and RNA sequences. Originally conceived as ligand for the recognition of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a variety of research and diagnostic assays. The extraordinary properties of PNA may advance routine clinical tests and environmental analyses that will utilise the PNA technology. PNAs may also have an impact on in situ hybridisation, cytogenetics and industrial microbiology. This paper presents some recent achievements on peptide nucleic acids and discusses, from the viewpoint of literature, what the potential is and what the limitations of such compounds are. This review, which is not intended to be exhaustive, is mostly aimed at the current progress in PNA chemistry, structure, and hybridisation, highlighting some applications too.

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PNA Beacons for Duplex DNA

Cited by 61 publications

References 36 publications

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

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

Peptide Nucleic Acids (PNAs), A Chemical Overview

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