Single and Bis Peptide Nucleic Acids as Triplexing Agents: Binding and Stoichiometry

Griffith, Michael; Risen, Lisa M.; Greig, Michael J.; Lesnik, Elena A.; Sprankle, Kelly G.; Griffey, Rich; Kiely, John S.; Freier, Susan M.

doi:10.1021/ja00107a033

Cited by 148 publications

(129 citation statements)

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“…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%

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: Is Approximately Valid Even Atmentioning

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.

102

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“…sequence will on average occur by chance every 16.4 kb in nucleic acids (assuming a 50% GC content), small triplex forming PNAs ought to function as generic probes for the capture of large nucleic acids. This contention was verified using biotinylated bisPNA-T 7 (16,17) in combination with streptavidin-coated magnetic particles to efficiently purify human genomic DNA from whole blood. When lysed blood was added to the PCR reaction without prior purification as little as 5µl totally inhibited the amplification process.…”

Section: Introductionmentioning

confidence: 86%

Purification of Nucleic Acids by Hybridization to Affinity Tagged PNA Probes

1999

Current Issues in Molecular Biology

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The use of affinity tagged PNA capture probes offers an efficient means for the purification of nucleic acids by hybridization. Two different approaches are described. A sequence specific method and a generic method. The sequence specific method requires sequence information on the target and synthesis of a dedicated PNA. It can be used to selectively purify the nucleic acid containing the target from non-related nucleic acids and other cellular components. The generic method uses a "universal" triplex forming PNA and requires no sequence information on the target. It can be used in the bulk purification of large nucleic acids.

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“…in which the Watson-Crick PNA strand is connected by continuous synthesis via ethylene glycol type linkers to the Hoogsteen PNA strand (28,32). The optimal constructs employ bis-PNAs where the cytosines in the Hoogsteen strand are replaced by pseudoisocytosine (J) (Figure 6), which has a hydrogen binding pattern that is equivalent to protonated cytosine, as this eliminates the requirement for a low pH (28).…”

Section: Triplex Formation and Bis-pnasmentioning

confidence: 99%

“…The optimal constructs employ bis-PNAs where the cytosines in the Hoogsteen strand are replaced by pseudoisocytosine (J) (Figure 6), which has a hydrogen binding pattern that is equivalent to protonated cytosine, as this eliminates the requirement for a low pH (28). An important consequence of linking the two PNA strands together is that strong hysteresis (>20ºC) observed in the melting curves of the 2PNA/DNA triplexes is reduced to a few degrees centigrade (32). Typical T m 's for bis-PNAs are about 100ºC for a 10-mer and about 65ºC for a 7-mer.…”

Section: Triplex Formation and Bis-pnasmentioning

confidence: 99%

An Introduction to Peptide Nucleic Acid

1999

Current Issues in Molecular Biology

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Single and Bis Peptide Nucleic Acids as Triplexing Agents: Binding and Stoichiometry

Cited by 148 publications

References 0 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

Purification of Nucleic Acids by Hybridization to Affinity Tagged PNA Probes

An Introduction to Peptide Nucleic Acid

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