39 research outputs found
Bimolecular quadruplexes and their transitions to higher-order molecular structures detected by ESI-FTICR-MS
Computing DNA duplex instability profiles efficiently with a two-state model: trends of promoters and binding sites
Pseudocomplementary PNAs as selective modifiers of protein activity on duplex DNA: the case of type IIs restriction enzymes
Circular dichroism of DNA frayed wires.
Ultraviolet circular dichroism spectra are reported for the oligonucleotide d(A15G15) in aqueous solutions containing 5 mM MgCl2 at several temperatures and in the presence of partially complementary oligonucleotides. Oligonucleotides with several consecutive terminal guanine residues self-associate to form aggregates, called frayed wires, that consist of integer numbers of strands. A "stem" is formed through interactions between the guanine residues of the associated oligonucleotides, whereas the adenine "arms" remain single stranded. Upon subtracting the circular dichroism spectrum of d(A15) from that of d(A15G15), one obtains a spectrum that closely resembles previously published spectra of poly(G). Subtracting spectra measured at temperatures between 10 degrees C and 60 degrees C reveals the resultant spectra to be independent of temperature, consistent with the extreme thermal stability observed for the aggregated structures. Upon the addition of d(T15) to the solution, complexes with the adenine portion of the d(A15G15) frayed wires are formed. Subtraction of d(A15):d(T15) spectra measured at several temperatures from those of the d(A15G15):d(T15) does not significantly alter the spectrum of the guanines. The helix-coil transition temperature of d(A15):d(T15) duplex is identical to that of the unbinding of d(T15) from d(A15G15):d(T15) complexes. Experiments using oligonucleotides in which the adenines were replaced with sequences of bases yielded similar results. By varying the length of the nonguanine tract, it is shown that the solubility of the complexes increases with the length of the nonguanine region of the oligonucleotide
Pseudocomplementary PNAs as selective modifiers of protein activity on duplex DNA: the case of type IIs restriction enzymes
This study evaluates the potential of pseudocomplementary peptide nucleic acids (pcPNAs) for sequence-specific modification of enzyme activity towards double-stranded DNA (dsDNA). To this end, we analyze the ability of pcPNA–dsDNA complexes to site-selectively interfere with the action of four type IIs restriction enzymes. We have found that pcPNA–dsDNA complexes exhibit a different degree of DNA protection against cleaving/nicking activity of various isoschizomeric endonucleases under investigation (PleI, MlyI and N.BstNBI) depending on their type and mutual arrangement of PNA-binding and enzyme recognition/cleavage sites. We have also found that the pcPNA targeting to closely located PleI or BbsI recognition sites on dsDNA generates in some cases the nicking activity of these DNA cutters. At the same time, MlyI endonuclease, a PleI isoschizomer, does not exhibit any DNA nicking/cleavage activity, being completely blocked by the nearby pcPNA binding. Our results have general implications for effective pcPNA interference with the performance of DNA-processing proteins, thus being important for prospective applications of pcPNAs
of protein activity on duplex DNA: the case of type IIs restriction enzymes
Pseudocomplementary PNAs as selective modi®er
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 (≈37°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 kJ⋅mol(−1), contributing to an anomalously high activation energy of ≈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