Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na(+). From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na(+). From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence

Physicochemical characterization of multistranded DNA assemblies, DNA frayed wires

grantor: University of TorontoPhysicochemical methods have been employed to characterise multistranded DNA assemblies--DNA frayed wires. Frayed wires arise from self-association of oligodeoxyribonucleotides with long terminal runs of consecutive guanines, e.g. d(A15G15). Intermolecular guanine-guanine interactions lead to the formation of high molecular weight complexes comprised of several strands of parent oligonucleotide. There are two distinct conformational domains in the frayed wire. Self-complexed guanines form a guanine stem located in the core of frayed wires. Non-guanine portions of the parent oligonucleotides remain single-stranded and are displaced from the stem. We refer to this part of the molecule as single stranded arms. Spectroscopic and gel electrophoretic studies showed that hybridization of short strands complementary to the arms does not interfere with the stem of frayed wires. And conversely, the stem does not affect stability of the duplex formed within the arms. The melting of this duplex proceeds at the same temperature as the melting of the short duplex in the solution. The single stranded arms of frayed wires affect their electrophoretic migration resulting in lower mobilities than those for double stranded DNA of comparable molecular weight We explored the propensity of oligonucleotides with different sequences and compositions to oligomerize into DNA frayed wires. Parent oligonucleotides with runs of 10 or more guanines at either 3' or 5 ' terminus give rise to high molecular weight complexes. The presence of the guanine base at the very terminus of the parent strand is crucial for its ability to polymerise. Neither the arm length nor the arm sequence affect self-association. However, the arms are important for the solubility of frayed wires. Aggregates arising from oligonucleotides with very short arms are prone to lateral association giving rise to very large aggregates which readily precipitate. We identified two environmental factors important for the self-assembly of DNA frayed wires. The presence of a divalent cation is essential for polymerisation of the parent strands into high molecular weight complexes. Incubations in the presence of monovalent cations result in a limited aggregation leading to formation of species consisting of 2 to 6 parent strands. The rate and the extent of polymerisation are strongly favoured by elevated temperature consistent with the positive activation energy for the reaction of DNA frayed wire formation. This behaviour is atypical for the nucleic acid folding and may indicate the presence of a secondary conformation within the stem or the arm region precluding the polymerisation at low temperatures. Upon heat denaturation of this secondary structure self-association of the parent strands proceeds.Ph.D

Specific versus Nonspecific Binding of Cationic PNAs to Duplex DNA

Although peptide nucleic acids (PNAs) are neutral by themselves, they are usually appended with positively charged lysine residues to increase their solubility and binding affinity for nucleic acid targets. Thus obtained cationic PNAs very effectively interact with the designated duplex DNA targets in a sequence-specific manner forming strand-invasion complexes. We report on the study of the nonspecific effects in the kinetics of formation of sequence-specific PNA-DNA complexes. We find that in a typical range of salt concentrations used when working with strand-invading PNAs (10–20 mM NaCl) the PNA binding rates essentially do not depend on the presence of nontarget DNA in the reaction mixture. However, at lower salt concentrations (<10 mM NaCl), the rates of PNA binding to DNA targets are significantly slowed down by the excess of unrelated DNA. This effect of nontarget DNA arises from depleting the concentration of free PNA capable of interacting with DNA target due to adhesion of positively charged PNA molecules on the negatively charged DNA duplex. As expected, the nonspecific electrostatic effects are more pronounced for more charged PNAs. We propose a simple model quantitatively describing all major features of the observed phenomenon. This understanding is important for design of and manipulation with the DNA-binding polycationic ligands in general and PNA-based drugs in particular

Effect of phosphorylation state of 5′ nt at the nick site on stacked-unstacked equilibrium

<p><b>Copyright information:</b></p><p>Taken from "Base-stacking and base-pairing contributions into thermal stability of the DNA double helix"</p><p>Nucleic Acids Research 2006;34(2):564-574.</p><p>Published online 31 Jan 2006</p><p>PMCID:PMC1360284.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Stacking parameters measured in 1× TBE at 37°C of A•T-containing (top panel) and G•C-containing (bottom panel) nicked dinucleotide stacks before (circles) and after (triangles) dephosphorylation are compared. White and gray fills are used for fragments with the nick in the forward and reverse strand, respectively. Nicked dinucleotide stacks with a purine at a 5′-side of the nick are underscored (see text). () Salt dependence of Δ values of nicked contacts indicated to the right of each panel before (circles) and after (triangles) dephosphorylation. Total concentration of sodium assuming 1× TBE to be equivalent to 15 mM Na is indicated, see

Effect of ambient conditions—temperature () and ionic strength ()—on DNA stacking parameters for A•T- and G•C-containing contacts

<p><b>Copyright information:</b></p><p>Taken from "Base-stacking and base-pairing contributions into thermal stability of the DNA double helix"</p><p>Nucleic Acids Research 2006;34(2):564-574.</p><p>Published online 31 Jan 2006</p><p>PMCID:PMC1360284.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Dinucleotide stacks are shown at the top of each panel. Error bars represent the scatter range of the experimentally determined values of nicked stacks (see text). This data is tabulated in Supplementary Tables 2 and 3

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

Overall stability, stacking and base pairing contributions for DNA polymers at different temperatures () Temperature dependence of the stacking contributions to the stability of A•T- (red circles) and G•C- (blue circles) containing polymers

<p><b>Copyright information:</b></p><p>Taken from "Base-stacking and base-pairing contributions into thermal stability of the DNA double helix"</p><p>Nucleic Acids Research 2006;34(2):564-574.</p><p>Published online 31 Jan 2006</p><p>PMCID:PMC1360284.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Straight solid line of the same color gives the temperature dependence of the stability parameter of corresponding polymer calculated using and at [Na] = 15 mM. () Temperature dependence of A•T (red) and G•C (blue) base pairing parameters calculated as a difference between stability and stacking terms using data in (a). Horizontal broken lines correspond to mean values of kcal/mol and kcal/mol