Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

Demidov, Vadim V.; Yavnilovich, Michael V.; Belotserkovskii, Boris P.; Frank‐Kamenetskii, Maxim D.; Nielsen, Peter E.

doi:10.1073/pnas.92.7.2637

Cited by 195 publications

(154 citation statements)

References 20 publications

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“…As it was expected for the double-duplex mode of pcPNA invasion, k ps is not affected by the change of pH in the range from 6.4 to 7.6 (data not shown). This is in a sharp contrast to the case of pyrimidine (T,C-containing) PNAs, which bind to dsDNA via triplex invasion (28,29).…”

Section: Resultsmentioning

confidence: 76%

“…We believe that such a complex is formed, as in the case of pyrimidine PNA triplex invasion (7,28,30,31), because of a small-scale opening of dsDNA (DNA ''breathing''; see the theoretical analysis below) and it may be schematically presented as in Fig. 1b.…”

Section: Mechanism Of the Double-duplex Invasionmentioning

confidence: 99%

“…Third, such a process may complicate the design of bis-pcPNAs, which is a next logical step in the pcPNA development to reduce the molecularity of strand-displacement reaction, as it was done in the case of pyrimidine bis-PNAs (28,29,(49)(50)(51).…”

Section: Is Approximately Valid Even Atmentioning

confidence: 99%

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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.

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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Section: Resultsmentioning

confidence: 76%

Section: Mechanism Of the Double-duplex Invasionmentioning

confidence: 99%

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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.

102

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“…2C, according to which two double-duplex complexes separated by a DNA duplex spacer do not open the spacer up, even when it consists of only 2 bp. These properties of the double-duplex invasion complex contrast to the properties of the triplexinvasion complex formed by hpyPNAs with dsDNA, which has been extensively studied before (14,16,22). In fact, two triplexinvasion complexes form an extended open region even if separated by as many as 10 bp (43).…”

Section: Circular Permutation Assay (Cpa)mentioning

confidence: 80%

“…Certain types of peptide nucleic acids (PNAs) are known to bind complementary target sites in dsDNA sequence-specifically through invasion of the double helix (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Of those types, however, only hpyrPNAs and pseudocomplementary (pc)PNAs form stable PNA-dsDNA complexes and thus are of practical value as functional tools.…”

mentioning

confidence: 99%

Inducing and modulating anisotropic DNA bends by pseudocomplementary peptide nucleic acids

Kuhn

Cherny

Demidov

et al. 2004

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

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DNA bending is significant for various DNA functions in the cell. Here, we demonstrate that pseudocomplementary peptide nucleic acids (pcPNAs) represent a class of versatile, sequence-specific DNA-bending agents. The occurrence of anisotropic DNA bends induced by pcPNAs is shown by gel electrophoretic phasing analysis. The magnitude of DNA bending is determined by circular permutation assay and by electron microscopy, with good agreement of calculated mean values between both methods. Binding of a pair of 10-meric pcPNAs to its target DNA sequence results in moderate DNA bending with a mean value of 40 -45°, while binding of one self-pc 8-mer PNA to target DNA yields a somewhat larger average value of the induced DNA bend. Both bends are found to be in phase when the pcPNA target sites are separated by distances of half-integer numbers of helical turns of regular duplex DNA, resulting in an enhanced DNA bend with an average value in the range of 80 -90°. The occurrence of such a sharp bend within the DNA double helix is confirmed and exploited through efficient formation of 170-bp-long DNA minicircles by means of dimerization of two bent DNA fragments. The pcPNAs offer two main advantages over previously designed classes of nonnatural DNAbending agents: they have very mild sequence limitations while targeting duplex DNA and they can easily be designed for a chosen target sequence, because their binding obeys the principle of complementarity. We conclude that pcPNAs are promising tools for inducing bends in DNA at virtually any chosen site.I ntrinsic or induced DNA bending plays an important role in various biological processes such as transcription, replication, recombination, DNA packaging, and repair (1-6). It has therefore been recognized that biological functions relying on DNA bending may be influenced by nonnatural DNA-bending agents (7-11). So far, two classes of artificial DNA-bending agents have been investigated: tethered triple-helix-forming oligonucleotides (7-9) and six-zinc-finger peptides (10, 11). In both classes, two DNA-binding domains are connected by a linker and targeted to two separated sites in double-stranded DNA (dsDNA), thus inducing bending at the intervening region. Whereas tethered triplex-forming oligonucleotides can easily be designed, they suffer from severe sequence limitations because they require the presence of two Ն15-bp-long homopurine-homopyrimidine (hypr) tracts, located not far from each other in the target DNA. On the other hand, the design of artificial DNA-binding peptides such as the reported six-zinc-finger peptides is not straightforward and may require a selection process starting with a pool of peptides for any chosen DNA target sequence (12). Thus, a DNA-binding ligand capable of sequence-specific recognition and bending of DNA, which could easily be designed for a chosen DNA target sequence, would be of great interest.Certain types of peptide nucleic acids (PNAs) are known to bind complementary target sites in dsDNA sequence-specifically through invasion of the ...

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PNAs: synthetische Polyamidnucleinsäuren mit außergewöhnlichen Bindungseigenschaften

Uhlmann¹,

Peyman²,

Breipohl³

et al. 1998

Angewandte Chemie

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Äußerst überraschend war die Erkenntnis, daß Peptidnucleinsäuren (PNAs, B=Nucleobase) trotz ihrer drastisch vom natürlichen DNA‐Rückgrat abweichenden Struktur besser als die meisten Oligonucleotidderivate als Nucleinsäuremimetica genutzt werden können. Die Synthese, physikalischen Eigenschaften und biologischen Wechselwirkungen sowohl der PNAs als auch ihrer Chimären mit DNA und RNA werden hier zusammenfassend beschrieben.

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Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

Cited by 195 publications

References 20 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

Inducing and modulating anisotropic DNA bends by pseudocomplementary peptide nucleic acids

PNAs: synthetische Polyamidnucleinsäuren mit außergewöhnlichen Bindungseigenschaften

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