Peptide nucleic acid-assisted topological labeling of duplex dna

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Hydrogen-bonding effects and 13C-NMR of the DNA double helix

13C-nmr chemical shifts of the nucleotides in DNA are sensitive to hydrogen bonding, especially for three of the carbons immediately bonded to exocyclic oxygen or nitrogen atoms acting as H-bond acceptors or donors. GuoC2, GuoC6 and ThdC4 are strongly deshielded (about 1 ppm) upon Watson-Crick pairing in oligodeoxynucleotide duplexes, regardless of the base sequence. Deshielding at these sites may be useful to distinguish bases involved in Watson-Crick pairs from unpaired bases.     

Tau could protect DNA double helix structure

The hyperchromic effect has been used to detect the effect of tau on the transition of double-stranded DNA to single-stranded DNA. It was shown that tau increased the melting temperature of calf thymus DNA from 67 to 81 degrees C and that of plasmid from 75 to 85 degrees C. Kinetically, rates of increase in absorbance at 260 nm of DNA incubated with tau were markedly slower than those of DNA and DNA/bovine serum albumin used as controls during thermal denaturation. In contrast, rates of decrease in the DNA absorbance with tau were faster than those of controls when samples were immediately transferred from thermal conditions to room temperature. It revealed that tau prevented DNA from thermal denaturation, and improved renaturation of DNA. Circular dichroic spectra results indicated that there were little detectable conformational changes in DNA double helix when tau was added. Furthermore, tau showed its ability to protect DNA from hydroxyl radical (.OH) attacking in vitro, implying that tau functions as a DNA-protecting molecule to the radical.     

Human telomeric DNA: G-quadruplex,i-motif and Watson-Crick double helix

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson-Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C*C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding.     

Intercalation of cationic dyes in the DNA double helix: introductory theory

The effect of salt on the intercalation of acridine dyes and DNA is rather well explained by the Gouy-Chapman double-layer theory as applied to a cylinder model of the DNA–dye complex. The free energy of transfer of a dye ion from the bulk solution to the complex is divided into several parts, one of which, ΔF₀, accounts for the short-range, nonelectrostatic interactions. The assumption that ΔF₀ should not depend on the amount of dye in the complex leads to an internal dielectric constant of the cylinder of about D_i = 7. The scatter in ΔF₀ values, as calculated from individual experimental points, is of order 0.5 kT per dye ion. This scatter is large enough to mask possible effects of heterogeneity in DNA sequences. The calculations are made for a long cylinder with radius 10 Å, with the DNA phosphate charges smeared uniformly at the surface, a uniform spacing of dye charges at the cylinder axis, and a length of b = 3.37 Å per base pair. Each intercalated dye ion also adds a length b to the total length of the cylinder. The salt-dependent part of the electric free energy of intercalation, ΔF₁, is tabulated for complexes with r = 0–0.24 dye ions per DNA phosphate in 0.002–0.2M monovalent salt and dye solutions.     

Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

Sequence-dependent base pair opening in DNA double helix

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.     

The energetic basis of the DNA double helix: a combined microcalorimetric approach

Microcalorimetric studies of DNA duplexes and their component single strands showed that association enthalpies of unfolded complementary strands into completely folded duplexes increase linearly with temperature and do not depend on salt concentration, i.e. duplex formation results in a constant heat capacity decrement, identical for CG and AT pairs. Although duplex thermostability increases with CG content, the enthalpic and entropic contributions of an AT pair to duplex formation exceed that of a CG pair when compared at the same temperature. The reduced contribution of AT pairs to duplex stabilization comes not from their lower enthalpy, as previously supposed, but from their larger entropy contribution. This larger enthalpy and particularly the greater entropy results from water fixed by the AT pair in the minor groove. As the increased entropy of an AT pair exceeds that of melting ice, the water molecule fixed by this pair must affect those of its neighbors. Water in the minor groove is, thus, orchestrated by the arrangement of AT groups, i.e. is context dependent. In contrast, water hydrating exposed nonpolar surfaces of bases is responsible for the heat capacity increment on dissociation and, therefore, for the temperature dependence of all thermodynamic characteristics of the double helix.     

Upper limit for the variances of some helical parameters in DNA double helix

Assuming that the realistic DNA chains are random sequences of purines and pyrimidines and, by using the Dickerson sum functions, it is shown that there exists an upper limit for the variance of helical twist angles, the base-plane roll angles and the other helical parameters respectively in the DNA sequences. The estimates of the variances of all the above helical parameters for the DNA sequences in the Los Alamos data base have been performed and found to be in good agreement with the theoretical results obtained in this paper.     

Phased psoralen cross-links do not bend the DNA double helix

Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies base on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5 degrees [Pearlman, D. A., Holbrook, S. R., Pirkle, D. H., & Kim, S.-H. (1985) Science (Washington, D.C.) 227, 1304-1308]. On the other hand, Sinden and Hagerman [Sinden, R. R., & Hagerman, P. J. (1984) Biochemistry 23, 6299-6303] find that, in solution, psoralen cross-linked DNA is not bent. Here we use gel electrophoresis to test the validity of the current models. We have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28 degrees unwinding of the helix by HMT [Wiesehahn, G., & Hearst, J. E. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2703-2707], one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. We find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. Our observations place an upper limit of 10 degrees on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.     

Geometric features of the DNA double helix in the supercoiled state

The geometric features of the DNA molecule in the supercoiled state were considered. A model of the supercoiled structure of the DNA molecule was constructed; the model takes into account its natural helicity. The force factors arising in the molecule at various superhelix angles were calculated.     

Osmotic pressure of DNA solutions and effective diameter of the double helix

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col E1 plasmid DNA solutions in the concentration range of 1-4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A2 of the solution of DNA segments and the effective chain diameter d eff in the ionic strength range of 10(-2)-0.1 M. As the ionic strength is lowered from 0.1 to 10(-2) M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.