ALBERT

Notizen: The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400-500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.

Notizen: The Tm of internal loop-forming (dA · dT)N domains in pBR322 DNA has been measured over a tenfold range of [Na+]. The slopes SN = dTm/d log[Na+] are linear and decrease in magnitude with decreasing loop size N, signaling a reduction in Na+ released during the transition of these domains to the coil state. Values of SN decrese linearly with increasing N-1 in accordance with the expectation of a simple model for the occurrence of a gradient of long-range electrostatic forces at helix-coil boundaries, and extrapolate almost precisely to the value of S∞ observed for (dA · dT)∞. These results indicate (1) less counterion is released per phosphate residue from the finite loop than from the infinitesized loop, and (2) the difference in binding is constant for each boundary formed and independent of the size of the loop within the range examined: ∼ 350 base pair (bp) 〉 N 〉 71 bp. The slope of the dependence of SN on N-1 indicates the region of higher charge density at the boundary extends at least 18 Å into the coil and probably 40-50 Å before dropping to a value characteristic of the unperturbed coil. The free energy for excess counterion binding at boundaries can be expressed by \documentclass{article}\pagestyle{empty}\begin{document}$$ {{ - \Delta G} \mathord{\left/ {\vphantom {{ - \Delta G} {RT}}} \right. \kern-\nulldelimiterspace} {RT}} = 10.47{\rm log}\left[{{\rm Na}^ + } \right] + 5.234 $$\end{document} When the loop entropy function in a statistical mechanical algorithm for the dissociation of DNA is weighted by this quantity, calculated Tm are seen to vary by only ±0.09°C from observed.

Notizen: No Abstract

Notizen: We describe the capabilities of a method for obtaining high-resolution melting profiles of short, homogeneous DNAs using a thermo-differential absorbance technique. The absorbance difference of two identical DNA solutions, raised linearly in temperature and maintained at a constant temperature difference, is monitored using a double-beam spectrophotometer. A specially constructed temperature controller and cell holder enable the temperature of the DNA samples to be controlled and monitored directly. A heating rate of 6.75°C/hr has been found to give reproducible results at ionic strengths 〉 0.01M. A method of reconstructing the true derivative from experimental data using a Taylor series expansion is described and shown to work well when the difference in temperature between samples is in the range of 0.2°C. Reconstructed derivative profiles are further analyzed by deconvolution into distinct Gaussian components. The melting profile of PM-2 DNA is shown to consist of 14 components, while the much longer lambda DNA yields 55. Related techniques such as data management and analysis for the fractional G·C content of specific domains are also described.

Notizen: On the basis of elementary two-state, ideal solution thermocynamics, a modified expression for the melting of oligo. polynucleotide helices is derived which is applicable to variations in TmN and/or oligomer concentration, Cm with oligomer length, N: (I) \documentclass{article}\pagestyle{empty}\begin{document}$$ \{ 1/T_m{}^N - [1 - 0.67/(n + 1)N]/T_m{} ^\infty \} = (R/N \cdot \Delta H_{\rm r} )\,{\rm ln}\ (c_m \cdot N \cdot V_{\rm r} ^{\rm f} ) $$ \end{document} ΔHr is the enthalpy per helix residue, i.e., per base-pair or base-triplet, Vrf is the thermodynamic “available” or “reaction” volume, in liters/mole of helical residues; and n is the number of polynucleotide strands, e.g., n = 2 for oligo (A)N·2 poly(U)∞. Some earlier treatments have engendered confusion in the interpretation of the “reaction volume,” but with the derivation herein, the entropic origin and physical significance of Vrf is unequivocal.The following approximation was arrived at for the reduction expected in the configurational entropy, ΔSrconf, ∞, for (A)∞·2(U)∞, when the poly(A), strand is substituted for by an equivalent strand of contiguous oligo(A)N,′s: (II) \documentclass{article}\pagestyle{empty}\begin{document}$$ \Delta S_{\rm r}{}^{{\rm conf,}N} =(1 - 0.67/3N)\Delta S_{\rm r}{}^{{\rm conf,}\infty} $$\end{document} This adjustment of ΔSrconf, ∞ represents the source of the coefficient to 1/Tm∞ in expression (I). The expectation that ΔSrconf, N 〈 ΔSrconf, ∞ is due to the effect of releasing normal internucleotide configurational restrictions every Nth residue in one-third of the strands of the (A)N·2(U)∞ helix.Although the reduction in ΔSrconf, ∞ (II) may seem small (i.e., only 5.5% for the tetramer), its effect on the magnitude of Vrf in expression (I) is exponential. Thus, without these considerations the quantitative applicability of earlier expressions is questionable.By examining the variation in TmN with cm for a single N, all assumptions, required for evaluating Vrf or the entropic effects of discontinuities in the (A)N strand are avoided in the determination of a reliable enthalpy. We have therefore examined the system (III) \documentclass{article}\pagestyle{empty}\begin{document}$$ \left( {\rm A} \right)_4 \cdot 2\left( {\rm U} \right)_\infty \rightleftharpoons {\rm tetra}\left({\rm A} \right)_4 + 2{\rm poly}\left( {\rm U} \right)_\infty $$\end{document} and obtained a ΔHr = 12.58 ± 0.08 kcal per mole (A)·2(U) base-triplets between 5 and 2.5°C. That this value for ΔHr is in such excellent agreement with all calorimetric values reported for (A)∞·2(U)∞ suggests that the enthalpy for reaction(III) is not significantly affected by disconnections in the backbone of (A)4·2(U)∞. From (I), Vrf = 6.0 × 10-4 1/mole or 1 Å 3per helical residue. ΔHr°, corrected for residual single-strand stacking in (A)4, is in excellent agreement with that found earlier for (A)1·2(U)∞. A residual heat capacity of 90 kcal(±20) per mole (A)·2(U) base-triplets per °C is deduced from the decrease of ΔHr° with temperature.

Notizen: The variation in the helix-coil transition temperature, TmN, with oligomer length, N, for the system (I) \documentclass{article}\pagestyle{empty}\begin{document}$$ \left( {\rm A} \right)_N \cdot 2\left( {\rm U} \right)_\infty \mathop \to \limits_\Delta \left( {\rm A} \right)_N + 2\left( {\rm U} \right)_\infty $$\end{document} has been examined. The results for N = 4-13, measured in 0.2M Na+, have been analyzed in terms of the expression of Blake (1972): (II) \documentclass{article}\pagestyle{empty}\begin{document}$$ 1/T_{m^N } \, - \,\,(1\, - \,0.67/3N)/T_{m^\infty } \, = \,(R/N\,\Delta H_r )\,\ln \,(c_m NV_{r^{\rm f} } )\,({\rm II}) $$\end{document} where cm is the free oligomer concentration at TmN, and Vrf is the thermodynamic free volume available to a helical base-triplet residue. The correlation coefficient for the fit to expression (II) of data obtained over a 50° temperature range is 0.997 when ΔHr = -12.6 kcal/mole of base-triplets (independent of oligomer length (N ⋝ 4) or temperature), the value previously obtained from both calorimetry of (A)∞·2(U)∞ and (A)4 concentration dependence of Tm. It is found that Vrf = 8.0 × 10-4 1/mole (± 30%) or 1.33 Å3 per helical base-triplet, and is constant with temperature. A maximum value for Vrf of 21.0 × 10-4 1/M (± 1.3%), equivalent to 3.54 Å3 per helical basetriplet is obtained by the same treatment of the helix-coil transition data for the three-stranded helix formed by adenosine (N = 1) and 2(U)∞ obtained by Davies and Davidson (1971).

Notizen: The thermal dissociation of DNA by five distinct local processes as outlined by Azbel [(1980) Biopolymers 19, 61-80] is treated as a collection of two-state substransitions. With the traditional expression for loop entropy and the assumption of a small, positive loop-initiation enthalpy, explicit relationships between thermodynamic parameters are derived for all five processes. The effect of each process on subtransitional width, unit enthalpy, and melting temperature is estimated; the latter being in excellent agreement with that predicted by Azbel. Criteria proposed for the deconvolution of the oscillatory high-resolution melting profiles exhibited by short, homogeneous DNAs [Yen & Blake (1980) Biopolymers 19, 681-700] have been applied to profiles of SV40 fragment [Gabbarro-Arpa et al. (1979) Nature 280, 515-517] and φx174-RF DNA at two ionic strengths [Vizard et al. (1978) 275, 250-251] and indicate that dissociation of the strands proceeds as a series of local two-state subtransitions of domains 200-300 base pairs in length. Although the obvious overlap of some subtransitions raises questions about resolution, most sections are very well resolved with the minimum number of subtransitions and yield values for the apparent van't Hoff enthalpy in excellent agreement with those expected by calorimetric measurements. The flexibility of such an approach, where the enthalpy is recognized explicitly as an adjustable parameter, is especially suited for the analysis of profiles from unkown DNA sequences, and for the evaluation of extraordinary variations in free energy, such as that associated with loop formation or that resulting from variations in the linear charge density at low ionic strengths.

Notizen: A series of high-resolution melting curves were obtained by the continuous direct-derivative method [Blake, R. D. & Lefoley, S. G. (1978) Biochim. Biophys. Acta 518, 233-246] on lambda DNA (cI857S7 strain) under varying conditions of [Na+]. Examination of the denaturation patterns at close intervals of [Na+] indicates that frequent changes in mechanism occur below 0.04M Na+, while almost none occurs above 0.1M Na+. Changes at low [Na+] generally occur in an abrupt fashion, in most cases within a 3 mM change in [Na+], and in at least one case within 0.6 mM, indicating the balance between alternative mechanisms is frequently quite delicate. These changes involve segments of between 900 and 1500 or more base pairs in length and are therefore not insignificant. Changes at low [Na+] reflect a perturbation of the energetic balance between competing mechanisms by weakly screened long-range electrostatic forces. Some perturbation probably also arises from variations in the linear charge density of the double helix induced by the proximity of premelted loop segments; however, this contribution cannot be evaluated without a detailed denaturation map.At high [Na+] the mechanism of melting is more conserved, permitting the dependence of subtrasitional melting temperature tm(i) on [Na+] to be examined for almost all 34 ± 2 subtransitions. The G + C composition of segments responsible for each subtransition was determined by a quantitative spectral method. Analysis according to the Manning-Record expression [Manning, G. (1972) Biopolymers 11, 937-949; Record, M. T., Jr., Anderson, C. F. & Lohman, T. M. (1978) Q. Rev. Biophysics 11, 103-178] relating ΔHm and dtm(i)/d log[Na+] to the fraction of Na+ released during melting, appears to indicate almost 40% more Na+ is bound to the single-stranded G and/or C residues than to A and T residues. This is consistent with a much shorter mean axial spacing and higher charge density in the former, particularly single-stranded G residues, which have an extraordinary tendency to stack.

Notizen: The mean cooperative length of domains of DNA, determined from the variance in (G + C) content in derivative melting curves of large bacterial DNAs, varies from 230 base pairs (bp) for (A - T)-rich domains to 580 bp for (G - C) domains. These values correspond to values for the cooperativity parameter of 2(±2) × 10-5 and 3(±2) × 10-6, respectively, and to +7.2 and +9.6 kcal for the free energy of helix interruption in those regions.

Notizen: The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3-4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.