Melting of Oligodeoxynucleotides with Various Structures

Abstract

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62- bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end- structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na⁺] < 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30–40 bp-long conforms to the two state model.

From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na⁺.     

Melting of short DNA fragments. Relation between the nucleation constant of a spiral region and the ionic strength

Melting of two DNA duplexes of known nucleotide sequences containing 14 and 36 base pairs has been investigated within the range of ionic strength from 0.2 to 0.02 M [Na+]. The values of melting enthalpy of base pair delta H were measured for the duplex of 14 base pairs in the solutions of varying ionic strength. The values of delta H were obtained from slopes of linear plots of reciprocal melting temperature versus logarithm of oligonucleotide chains concentration. In the aforementioned range the decrease of the ionic strength causes a 5% decrease of delta H. By fitting the theoretical profiles to the experimental ones the ionic strength dependence of the nucleation constant beta was measured for DNA fragments of various lengths. With the decrease of the ionic strength the value of beta drops 2 times for the short duplex and 8 times for the long one.     

The kinetics of DNA helix-coil subtransitions

The kinetic analysis of individual helix-coil subtransitions were performed by comparing melting and renaturation profiles obtained at different temperature change rates. The duration of the three transition stages and its dependence on temperature and ionic strength were determined for a T7 phage DNA fragment. The obtained temperature dependence of the melting time for a stretch flanked by melted regions is in quantitative agreement with that predicted by the theory of slow processes (V.V. Anshelevich, A.V. Vologodskii, A.V. Lukashin, M.D. Frank-Kamenetskii, Biopolymers 23, 39 (1984)). The reasons are discussed for the increasing relaxation time of this stretch in the middle of its transition with decreasing ionic strength. The zipping kinetics of a melted region under essentially nonequilibrium conditions was examined for T7 fragment and pAO3 DNAs. The obtained temperature dependence of the zipping time is in quantitative agreement with calculations based on the theory of slow processes. The renaturation times of stretches flanked by helical regions proved fairly small even at a low ionic strength. These times are several orders of magnitude smaller than the renaturation times of the same stretches with one helical boundary. A formal application of the theory of slow processes failed to account for the small renaturation times of stretches that are zipped from both ends. This is probably due to the non-allowance for the changing entropy of the loop linking two helix-coil boundaries migrating towards each other. Slow processes have been revealed in the intramolecular melting of Col E1 DNA at a high ionic strength. The reason for the long relaxation time of one subtransition is the large size of the loop that separates the melting stretch from the helical part of the molecule. This result can be accounted for by the theory of slow processes.     

Relationship between helix-coil transition and gene organization of ColE1 plasmid DNA. Differential scanning calorimetric and theoretical studies

Differential scanning calorimetry (DSC) was carried out to analyze the transition of helix to coil state of DNA, using ColE1 DNA molecules digested with EcoRI. The DSC curves showed multimodal transition, consisting of nine to 11 peaks over a temperature range, depending on the ionic strength of the DNA solution. These DSC curves were essentially in good agreement with the optical melting curves of ColE1 DNA. The theoretical melting profiles of ColE1 DNA were predicted from calculations based on the helix-coil transition theory and the nucleotide sequence of the DNA. These profiles resembled the DSC curves and made it possible to assign the peaks seen in the DSC curves to the helix-coil transition of particular regions of the nucleotide sequence of ColE1. The helix-coil transition of each of the small genes gave rise to a single peak in the DSC curve, while the helix-coil transition of large genes contributed to two or more peaks in the DSC curve. This multimodal transition within a single coding region might correspond to the melting of individual segments encoding the different domains of the proteins. The helix-coil transition at the specific sites including ori, the origin of replication of ColE1, was also found to occur in a particular temperature range. DSC, a simple method, is thus useful for analyzing the multimodal helix-coil transition of DNA, and for providing information on the genetic organization of DNA.     

The Kinetics of DNA Helix-Coil Subtransitions

Abstract

The kinetic analysis of individual helix-coil subtransitions was performed by comparing melting and renaturation profiles obtained at different temperature change rates. The duration of the three transition stages and its dependence on temperature and ionic strength were determined for a T7 phage DNA fragment. The obtained temperature dependence of the melting time for a stretch flanked by melted regions is in quantitative agreement with that predicted by the theory of slow processes (V.V. Anshelevich, A.V. Vologodskii, A.V. Lukashin, M.D. Frank-Kamenetskii, Biopolymers 23, 39 (1984)). The reasons are discussed for the increasing relaxation time of this stretch in the middle of its transition with decreasing ionic strength.

The zipping kinetics of a melted region under essentially nonequilibrium conditions was examined for T7 fragment and pAO3 DNAs. The obtained temperature dependence of the zipping time is in quantitative agreement with calculations based on the theory of slow processes.

The renaturation times of stretches flanked by helical regions proved fairly small even at a low ionic strength. These times are several orders of magnitude smaller than the renaturation times of the same stretches with one helical boundary. A formal application of the theory of slow processes failed to account for the small renaturation times of stretches that are zipped from both ends. This is probably due to the non-allowance for the changing entropy of the loop linking two helix-coil boundaries migrating towards each other.

Slow processes have been revealed in the intramolecular melting of Col E1 DNA at a high ionic strength. The reason for the long relaxation time of one subtransition is the large size of the loop that separates the melting stretch from the helical part of the molecule. This result can be accounted for by the theory of slow processes.     

Studies of DNA dumbbells VII: evaluation of the next-nearest-neighbor sequence-dependent interactions in duplex DNA

Melting experiments were conducted on 22 DNA dumbbells as a function of solvent ionic strength from 25-115 mM Na(+). The dumbbell molecules have short duplex regions comprised of 16-20 base pairs linked on both ends by T(4) single-strand loops. Only the 4-8 central base pairs of the dumbbell stems differ for different molecules, and the six base pairs on both sides of the central sequence and adjoining loops on both ends are the same in every molecule. Results of melting analysis on the 22 new DNA dumbbells are combined with our previous results on 17 other DNA dumbbells, with stem lengths containing from 14-18 base pairs, reported in the first article of this series (Doktycz, Goldstein, Paner, Gallo, and Benight, Biopoly 32, 1992, 849-864). The combination of results comprises a database of optical melting parameters for 39 DNA dumbbells in ionic strengths from 25-115 mM Na(+). This database is employed to evaluate the thermodynamics of singlet, doublet, and triplet sequence-dependent interactions in duplex DNA. Analysis of the 25 mM Na(+) data reveals the existence of significant sequence-dependent triplet or next-nearest-neighbor interactions. The enthalpy of these interactions is evaluated for all possible triplets. Some of the triplet enthalpy values are less than the uncertainty in their evaluation, indicating no measurable interaction for that particular sequence. This finding suggests that the thermodynamic stability of duplex DNA depends on solvent ionic strength in a sequence-dependent manner. As a part of the analysis, the nearest-neighbor (base pair doublet) interactions in 55, 85, and 115 mM Na(+) are also reevaluated from the larger database.     

Differential scanning calorimetric and theoretical studies of ColE1 DNA

The differential scanning calorimetry (DSC) of plasmid ColE1 DNA was carried out. The DSC curve under the solvent condition of 1.0 X SSC buffer gave eleven clear peaks over the temperature range of 83 to 98 degrees C. The DSC curves obtained here were essentially in good agreement with the optical melting curves of ColE1 DNA reported previously. The theoretical melting profiles of ColE1 DNA calculated from its entire nucleotide sequence showed a good agreement with the DSC curves. The theoretical analysis made by constructing the thermal stability map showed that there was the positional correlation between the boundaries of the cooperatively melting regions and the ends of the protein coding regions of genes of ColE1. It was shown that the helix-coil transition of many of the small genes had a single cooperatively melting region. However, the large genes such as cea and mob3 had two or more cooperatively melting regions. It was suggested that this is closely related to the domain structures of the proteins encoded by such genes.     

Determination of DNA cooperativity factor.

The paper presents measurements of the difference in the melting temperature of a colE1 DNA region when it is located inside the DNA helix and at its end. A direct comparison of calculations based on the rigorous theory of helix-coil transition with experimental data for .2 M Na+ (the conditions for fully reversible melting) yielded the value of 2.5-5x10(-5) for the cooperatively factor sigma. We discuss the reversibility of DNA melting and the possibility of applying the "all-or-nothing" concept to the melting of DNA regions.     

Thermodynamic stability of the 5' dangling-ended DNA hairpins formed from sequences 5'-(XY)2GGATAC(T)4GTATCC-3', where X, Y = A, T, G, C

Expressions for the partition function Q (T) of DNA hairpins are presented. Calculations of Q (T), in conjunction with our previously reported numerically exact algorithm [T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight (1990) Biopolymers, 29, 1715-1734], yield a numerical method to evaluate the temperature dependence of the transition enthalpy, entropy, and free energy of a DNA hairpin directly from its optical melting curve. No prior assumptions that the short hairpins melt in a two-state manner are required. This method is then applied in a systematic manner to investigate the stability of the six basepair duplex stem 5'-GGATAC-3' having four-base dangling single-strand ends with the sequences (XY)2, where X, Y = A, T, G, C, on the 5' end and a T4 loop on the 3' end. Results show that all dangling ends of the sample set stabilize the hairpin against melting. Increases in transition temperatures as great as 4.0 degrees C above the blunt-ended control hairpin were observed. The hierarchy of the hairpin transition temperatures is dictated by the identity of the first base of the dangling end adjoining the duplex in the order: purine greater than T greater than C. Calculated melting curves of every hairpin were fit to experimental curves by adjustment of a single parameter in the numerically exact theoretical algorithm. Exact fits were obtained in all cases. Experimental melting curves were also calculated assuming a two-state melting process. Equally accurate fits of all dangling-ended hairpin melting curves were obtained with the two-state model calculation. This was not the case for the melting curve of the blunt-ended hairpin, indicating the presence of a four-base dangling-end drives hairpin melting to a two-state process. Q (T) was calculated as a function of temperature for each hairpin using the theoretical parameters that provided calculated curves in exact agreement with the experimentally obtained optical melting curves. From Q (T), the temperature dependence of the transition enthalpy delta H, entropy delta S, and free energy delta G were calculated for every hairpin providing a quantitative assessment of the effects of dangling ends on hairpin thermodynamics. Comparisons of our results are made with those of the Breslauer group [M. Senior, R. A. Jones, and K. J. Breslauer (1988) Biochemistry 27, 3879-3885] on the T2 5' dangling-ended d(GC)3 duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physical properties of moloney murine leukemia virus high-molecular-weight RNA: a two subunit structure.

The high-molecular-weight RNA of Moloney murine leukemia virus (MuLV) was analyzed by sedimentation equilibrium ultracentrifugation. Molecular weights of 7.2 x 10(6) and 3.4 x 10(6) were found for the native and subunit forms, respectively, indicating that the native structure is a dimer. S20,w and frictional coefficients were determined for MuLV RNA by analytical velocity centrifugation as a function of ionic strength. The apparent S20,w of native MuLV RNA was 47.3, 57.4, and 66.5 in 0.01, 0.1, and 0.20 M Na+, respectively; the corresponding frictional coefficients were 5.44, 4.48, and 3.87. Native RNA was estimated by circular dichroism to be 85% helical, whereas denatured RNA was 54% helical. Thermal denaturation profiles were obtained from uv absorbance scans. Melting temperatures of 57 and 68 C were obtained for high-molecular-weight RNA in 0.01 M Na+ and 0.122 M Na+, 1mM Mg2+, respectively. van't Hoff plots of the thermal denaturation data gave enthalpies for the helix-coil transition of 21,600 cal (ca. 90,500 J) per mol of cooperatively melting unit in high salt and 19,600 cal (ca. 82,100 J) per mol in low salt, consistent with both base stacking and pairing. The melting of Mu LV RNA occurred over a broad temprange and van't Hoff plots were linear over most of the melting range, indicating a noncooperative process of helix stabilization.     

Thermodynamics of the various forms of the dodecamer d(ATTACCGGTAAT) and of its constituent hexamers from proton nmr chemical shifts and UV melting curves: three-state and four-state thermodynamic models

Chemical shifts of base and H1' protons of the single-stranded hexamers d(ATTACC) and d(GGTAAT), of the 1:1 mixtures of these complementary hexamers, and of the self-complementary dodecamer d(ATTACCGGTAAT) were measured at various temperatures in aqueous solution. Four different sample concentrations were used in the case of the dodecamer and of the mixture of the complementary hexamers; the individual hexamers were measured at two different DNA concentrations. Absorbance temperature profiles at five different NaCl concentrations were measured for the dodecamer in order to quantify the effect of the ionic strength on the duplex formation. Under suitable conditions of nucleotide concentration, temperature, and ionic strength, the dodecamer adopts either a B-DNA duplex or a hairpin-loop structure. Chemical shift vs temperature profiles, constructed for all samples, were used to obtain thermodynamic parameters either for the various stacking interactions in the single strands or for the duplex or the hairpin-loop formation. In the analysis of the duplex formation of the hexamers, a two-state approach appeared too simple, because systematic deviations were revealed. Therefore, a new three-state model (DUPSTAK) was developed. In order to investigate the magnitude of error arising from the use of the two-state approach in cases where the DUPSTAK model appears more appropriate, a series of test calculations was made. The magnitude of error in the enthalpy and in the entropy of duplex melting is found to depend linearly upon the actual melting temperature and not upon the individual delta Hd degrees and delta Sd degrees values. Thermodynamic analysis of the chemical shift vs temperature profiles in D2O solution (no added salt) yields an average Tmd value of 341 K (1M DNA) and delta Hd degrees of - 121 kJ.mol-1 for the dimer/random-coil transition of the hexamer duplex d(ATTACC).d(GGTAAT). For the duplex in equilibrium random-coil transition of the 12-mer d(ATTACCGGTAAT) an average Tmd value of 336 K (1M DNA) and delta Hd degrees of -372 kJ.mol-1 are found. The hairpin/random-coil transition of d(ATTACCGGTAAT) is characterized by a rather large delta Hh degrees value, -130 kJ.mol-1, and an average Tmh value of 304 K.     

Influence of base sequence on the stability of the double helix of DNA.

On the basis of published measurements of the melting transitions of synthetic polydeoxyribonucleotides with known sequences we have determined the parameters of the interplane (stacking) interactions of base pairs in DNA over the range of ionic strengths from 0.01 to 0.1 M Na+. We found that deviations of the stacking-interaction energy from the mean value of 7-8 kcal/mole were extremely small and did not exceed 0.2 kcal/mole. We report an analysis of the influence of the heterogeneity of the stacking interactions on the melting parameters of polynucleotides with random sequences (models of natural DNA's). Inclusion of this effect does not significantly distort the linear dependence of the melting temperature on the relative content of G-C pairs and insignificantly affects the width of the helix-coil transition in DNA under normal conditions. However it is the heterogeneity of the stacking interactions that plays the crucial role in the melting of DNA under conditions where the difference between the relative stabilities of the A-T and G-C pairs tends to zero, as in concentrated solutions of tetraethylammonium and tetramethylammonium salts.     

Effect of divalent copper ions on heat denaturation of DNA

Using the thermal denaturation method the effect of bivalent copper of (4-10(-6)-10(-3)) M concentrations on the helix-coil transition of DNA was studied in the solution of Na+ concentrations 10(-3)-10(-1) M. Unlike the previous studies, this paper makes allowance for the effect of impurity ions present in DNA and deionized water. It has been shown that in the region of low Cu2+ and Na+ concentrations, thermal stability increases, the melting range extends and the denaturation curves become asymmetric. At concentrations more than approximately 3-10(-5) M Cu2+, melting temperature starts to fall, and the range reduces to 1-1.5 degrees at [Cu2+] greater than or equal to 2-10(-4) M. As [Cu2+] reaches these values, the denaturation curve asymmetry and melting range increase again, which is due to the inversion of the relative stability of AT- and GC-pairs. Employing experimental and phase-transition-theory data for homopolymers, the constants of Cu2+ binding with phosphates and DNA bases were calculated. The concentration dependence of the DNA denaturation parameters was shown to be governed by the superposition of binding Cu2+ with phosphates and nucleic acid bases.     

Fine structures in denaturation curves of bacteriophage lambda DNA. Their relation to the intramolecular heterogeneity in base compositon.

Precise recording of polyphasic optical melting curves was carried out for three kinds of bacteriophage lambda DNA differing in length (lambdac1857s7, lambdacIb2 and lambdacIb2b5). Each of denaturation steps in melting profiles was characterized by two parameters, the melting temperature and the relative size. Any difference in fine structures in melting profiles was not recognized between the intact lambdacI857s7DNA and the DNA fragmented into halves. The change in fine structures in melting profiles caused by the deletions of the b2 and b5 region agreed qualitatively well with the prediction based on the physical and the genetical maps of phage lambda chromosome. The combined results indicate that, first, the well-known linear relationship between melting temperature and G+C content may apply also to each of denaturation steps in polyphasic melting curves due to heterogeneity of nucleotide distribution in a single DNA species, and, second, the effect of molecular ends on melting fine structures can be neglected at moderate salt concentration (0.01 M less than or equal to Na+ less than or equal to 0.2 M) for such a high molecular weight DNA. The heterogeneous distribution of nucleotides was derived for lambdaDNA and for its b2 and b5 regions.     

The nonequilibrium character of DNA melting: effects of the heating rate on the fine structure of melting curves.

To investigate the effects of heating rate on the DNA melting profile and to test the predictions of the theory of slow relaxation processes in DNA melting (1) concerning these effects, we obtained differential melting curves for the Bsp I C1 fragment of T7 DNA (1461 bp) and the Sma I-Eco RI fragment of Col E1 DNA (1291 bp) at heating rates of 0.05 and 0.5 deg/min. At low ionic strength (0.02 M Na+) the heating rate has been shown to affect the position of the third peak in melting curve for C1 fragment. According to the melting maps (2), this peak corresponds to the unwinding of the section between the end of the molecule and the region already melted. At high ionic strength (0.2 M Na+), when the melting of this DNA is reversible (3), the position of the peaks does not depend on the heating rate. In the case of the Col E1 DNA fragment the heating rate affects, as might be expected from the melting maps (4), only the last peak, as the melting of the last section is always nonequilibrium. The results of the study are in good qualitative agreement and in satisfactory quantitative agreement with the theoretical predictions (1).     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Volumetric properties of the formation of double stranded DNA: a nearest-neighbor analysis

The kinetics of the helix-coil transition have been studied by performing UV-monitored melting and reannealing curves of DNA and analyzing the resultant hysteresis between these curves. The analysis assumes a single-step bimolecular transition with duplex formation defined as the forward reaction. Volume parameters of the helix-coil transition were obtained by measuring the pressure dependence of the rate constants from 5-200 MPa. The data were interpreted in terms of several possible nearest-neighbor models, ranging from one to eleven parameters. Twenty-four oligonucleotide duplexes 22 base pairs in length were used to solve for individual nearest-neighbor activation volumes and transition volumes. Statistically, the most valid fit of the volumetric data was obtained with a six-parameter model in which the directionality of the dinucleotide steps is not considered, for example, 5'AG/CT is the same as 5'GA/TC. The resultant transition volumes at 48 degrees C ranged from -7.1 +/- 0.8 mL/mol (GC/CG) to +2.9 +/- 0.3 mL/mol (AA/TT). The success of the six-parameter model suggests that the relative size of the nearest-neighbor dinucleotides is the most important factor determining the magnitude of the volumetric parameters. The finding that the magnitude of the volumetric parameters correlates with the change in the solvent accessible surface area of the bases during the helix-coil transition corroborates this hypothesis.     

Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation delta Gloop (n) and single-strand circles delta Gcircle (N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy delta H degrees and entropy delta S degrees for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, pp. 377-399).(ABSTRACT TRUNCATED AT 400 WORDS)     

Counterion association with native and denatured nucleic acids: an experimental approach

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.     

Studies of DNA dumbbells. VI. Analysis of optical melting curves of dumbbells with a sixteen-base pair duplex stem and end-loops of variable size and sequence

Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5′-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3′ linked on both ends by single-strand loops of A_t or C_t sequences (˛ = 2, 3, 4, 6, 8, 10, 14), T_t sequences (˛ = 2, 3, 4, 6, 8, 10), and G_t sequences (t = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM Na⁺. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation ΔG_loop(n > t) and single-strand circles, ΔG_cir(N) as a function of end-loop size, t = 2-14, circle size, N = 32 + 2_t, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy ΔH, entropy ΔS, and free energy ΔG, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na⁺]. Since the multistate analysis is based on the numerically exact model, and considers a statistically significant number of theoretically possible partially melted states, it does not require prior assumptions regarding the nature of the melting transition, i.e., whether or not it occurs in a two-state manner. For comparison with the multistate analysis, thermodynamic transition parameters were also evaluated directly from experimental melting curves assuming a two-state transition and using the graphical van't Hoff analysis. Comparisons between results of the multistate and two-state analyses suggested dumbbells with loops comprised of six or fewer residues melted in a two-state manner, while the melting processes for dumbbells with larger end-loops deviate from two-state behavior.Dependence of thermodynamic parameters on[Na⁺] as a function of loop size suggests single-strand end-loops have different counterion binding properties than the melted circle. Results are compared with those obtained in an earlier study of dumbbells with the slightly different stem sequence 5'-G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C-3' linked on the ends by T loops (˛ = 2,3,4,6,8,10,14).© 1996 John Wiley &Sons, Inc.