ALBERT

Notes: [Auszug] Fig. 1 The 95-, 144-, 203- and 301-base pair restriction fragments containing part of the lactose operon transcription initiation region. Each fragment is fully embodied within the next largest. A partial restriction map indicates location between restriction sites, lac z is the region coding for b ...

Notes: Theoretical melting curves were calculated for four DNA restriction fragments, 157-257 base pairs (bp), and a series of hypothetical block DNAs with sequences d(C2xAxC2x). d(C2xTxG2x), 5 ≤ x ≤ 40. These DNAs provided a mixture of A·T/G·C sequence distributions with which to investigate the effects of parameters and base-pair changes on the melting of short DNAs. The sensitivity of DNA melting curves to changes in internal loop melting parameters σ and κ was examined. As Expected, theoretical melting curves of short DNAs with a quasirandom base-pair sequence vary little with changes in internal loop parameters. End melting dominates the transition behaviour of these moleucles. This was also observed for the block DNAs up to x = 22. Beyond this length, melting curves are highly sensitive to the internal loop parameters. Sensitivity is also predicted for a 157-bp fragment with a block distribution of A·T and G·C pairs. These results indicate that accurate evaluation of internal loop parameters is possible with short DNAs (100-200 bp) containing a G·C/A·T/G·C block distribution with at least 22 bp in each block. Duplex-to-single-strands dissociation parameters were reevaluated form experimental melting curve data of eight DNA fragments using a least squares fit approach. This analysis confirmed parameter values previously found with a simplified dissociation model. A Priori predictions are made on the effects of base-pair changes on the melting curves of three characterized DNA restriction fragments. Single base-pair changes are predicted to induce small but measurable changes in the melting curves. The characteristics of the altered melting curves depend on the location of the base-pair change.

Notes: Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3°C for the T4 loop hairpin to 55.3°C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop for mation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions.Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop. In contrast, the evaluated free-energy of forming an A4 loop is approximately 400 cal/mole higher for each nearest-neighbor set indicating unfavorable interactions between A bases in a loop-affect loop formation and overall hairpin stability, (2) The absolute value for the free-energy of loop formation at the Tm of each hairpin varies by about 1 kcal/mole depending on the set of nearest-neighbor interactions employed and the relative hierarchy of stability for each loop is conserved for different nearest neighbor sets, (3) The melting process of each hairpin deviates from strict two-state behavior in the order according to loop sequence of T 〉 A 〉 G 〉 C, (4) Results of our analysis are compared with the early work of Scheffler et al., 1970 on the hairpins formed from the copolymer sequences d(T - A)q where q = 9-21. Comparisons with the more recent works a DNA dumbbell (Benight et al., 1988) and the very similar DNA hairpins studied by Senior et al., 1988 are presented.

Notes: CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T4 single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ∼20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying.On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (∼ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of fT(T), the fraction of triplex as a function of temperature, was obtained. Analysis of fT vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔHT = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔST = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from fT = 0 to 0.55 and a higher, sharper transition above fT = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′T = 44.8 ± 9.6 kcal/mol and ΔS′T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′T = 128.0 ± 2.3 kcal/mol and ΔS′T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.

Notes: The probability of DNA base-pair opening was calculated at temperatures below the denaturation region. The helix-coil transition theory, modified to include different nearest-neighbor interactions, was employed. Predictions of base-pair opening employing DNA melting-curve parameters differed considerably from predictions based on parameters evaluated from synthetic RNA oligomer data of Gralla and Crothers (G-C) and formaldehyde-DNA binding experiments of McGhee and von Hippel (M-vH). Calculations based on the latter parameters indicate a base-pair-opening probability of 10-2-10-3 at 35°C in 0.1M NaCl. DNA melting-curve parameters predict values about 103 smaller. At a temperature 10°C below the transition midpoint of a specific DNA sequence, DNA melting parameters predict base-pair opening of about 10-3, whereas the G-C and M-vH parameters predict that ≳0.16 of the DNA is melted. Experiments and theoretical assumptions relevant to the calculations are analyzed. Evidence suggests that DNA melting parameters are valid when the average loop size exceeds some minimum value, whereas the G-C and M-vH parameters appear more valid for single base-pair loops. A reconciliation of the two sets of predictions can be made if interactions extending beyond neighboring base pairs are considered. Such interactions will make the parameters of the nearest-neighbor model appear to change with the average loop size. Experiments that may provide further measurements of base-pair opening are discussed.

Notes: The conformation and internal dynamics of supercoiled pUC 8 DNA (2717 bp) are examined by dynamic light scattering, and the magnitude and uniformity of its torsional rigidity are determined using time-resolved fluorescence polarization anisotropy of intercalated ethidium dye. Neither measurement gives any indication of an appreciably reduced bending or twisting rigidity, or anomalously rapid internal motions. For 31P, in supercoiled pUC 8, we measure T2 = (2.0 ± 0.5) × 10-3 s. This lies within the range of present theoretical estimates obtained using normal rigidities. The proton linewidths observed for pUC 8 and pBR322 (4363 bp) DNAs are within a factor of 2-3 of those similarly estimated assuming ordinary rigidities.According to Bendel, Laub and James [(1982) J. Am. Chem. Soc. 104, 6748-6754], supercoiled pIns36 DNA (7200 bp) exhibits an astonishingly long T2 = 1.17 s for 31P, a slowest rotational relaxation time, τ = 5 × 10-9 s, and an enormously reduced bending rigidity. Serious questions raised by these findings are examined here. The 5 × 10-9 s slowest rotational relaxation time is shown to be physically inadmissible.The nmr relaxation theory developed previously by Allison, Shibata, Wilcoxon, and Schurr [(1982) Biopolymers 21, 729-762], is modified to incorporate new results for deformable filaments, which directly introduce the highly nonexponential tumbling correlation function for reorientation of the local helix axis. Essential requirements for a complete calculation of R2, including estimation of the tumbling correlation function and evaluation of the still unknown DIP/CSA cross-term, are described in detail. Slow coil-deformation modes analogous to the Rouse-Zimm modes of linear DNAs are shown to make an important, if not dominant, contribution to the R2 relaxation rate. Geometrical parameters in the theory are chosen to provide good agreement with literature data for 600-bp linear DNA. Using this theory and an informed guess for the tumbling correlation function, we find that the 31P-nmr relaxation data of Bendel et al., if correct, necessarily impose on their DNA one or more extreme properties, such as enormously reduced bending or twisting rigidities. In contrast, the same theory yields reasonable agreement with the T2 reported here for 31P in supercoiled pUC 8 DNA when its rigidities are assumed to be quite ordinary.

Notes: Dynamic and static light scattering, CD, and optical melting experiments have been conducted on M13mp19 viral circular single-strand DNA as a function of NaCl concentration. Over the 10,000-fold range in concentration from 100 μM to 1.0M NaCl, the melting curves and CD spectra indicate an increase in base stacking and stability of stacked regions with increased salt concentration. Analysis of dynamic light scattering measurements of the single-strand DNA solutions as a function of K2 from 1.56 to 20 × 1010 cm-2 indicates the collected autocorrelation functions are biexponential, thus revealing the presence of two decaying dynamic components. These components are taken to correspond to (1) global translational motions of the molecular center of mass and (2) motions of the internal molecular subunits. From the evaluated relaxation rates of these components, diffusion coefficients D0 and Dplat are determined. The center of mass translational diffusion coefficient D0, varies in a nonmonotonic manner, by 10%, from 3.75 × 10-8 to 3.39 × 10-8 cm2/s over the NaCl concentration range from 100 μM to 1.0M. Likewise, the radius of gyration RG, obtained from static light scattering experiments, varies by 15% from 699 to 830 Å over the same NaCl range. Dplat, the diffusion coefficient of the internal subunits, displays a different dependence on the NaCl concentration and decreases, by nearly 22% in a titratable fashion, from 12.46 × 10-8 to 10.26 × 10-8 cm2/s, when the salt is increased from 100 μM to 1.0M. A semiquantitative interpretation of these results is provided by analysis of the light scattering data in terms of the circular Rouse-Zimm chain. Rouse-Zimm model parameters are estimated from the experimental results, assuming the circular chains are composed of a fixed number of Gaussian segments, N + 1 = 15. The rms displacement of the internal segments, b, is estimated to be the smallest (442 Å) in 100 mM NaCl. Increases of b to 467 Å in 100 μM and 524 Å in 1.0M NaCl are observed. Meanwhile, the hypothetical friction factor of the internal subunits, f, progressively increases as the NaCl concentration is raised. It is inferred from the evaluated Rouse-Zimm model parameters that both the static flexibility of the circular chain and diffusive displacements of the internal subunits decrease with increases in NaCl concentration from 100 mM to 1.0M. These decreases directly contract the salt-dependent behavior of double-stranded DNA, where greater flexibility is observed when the Na+ concentration is increased. The melting and CD measurements indicate the decrease in flexibility and internal motions is due to the formation of nucleotide stacking in the higher NaCl environments. In 100 μM NaCl, where stacking is highly unfavored, a significant electrostatic contribution to the persistence length likely acts to stiffen the molecule. It appears the observable changes in the internal dynamics of M13mp19 single-strand DNA are associated with increases in base stacking that occur from 100 μM to 1.0M NaCl, which apparently induce relatively small perturbations in the overall global tertiary conformation of the DNA.