Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids.
If adenines and thymines in two mutually complementary mixed-base 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 self-hybridization of pcPNA oligomers have been assayed by mixing curve (Job plot) and thermal melting experiments. The data indicate that, at physiological temperatures ( approximately 37 degrees 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 degrees C. At elevated temperatures (>50 degrees 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 kJ.mol(-1), contributing to an anomalously high activation energy of approximately 150 kJ.mol(-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.
Demidov
Vadim V
VV
Center for Advanced Biotechnology, Boston University, 36 Cummington Street, Boston, MA 02215, USA. vvd@bu.edu
Protozanova
Ekaterina
E
Izvolsky
Konstantin I
KI
Price
Christopher
C
Nielsen
Peter E
PE
Frank-Kamenetskii
Maxim D
MD
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D018076:DNA, Complementary; D008024:Ligands; D020135:Peptide Nucleic Acids; D010455:Peptides; D004247:DNA