Studies of DNA dumbbells VIII. Melting analysis of DNA dumbbells with dinucleotide repeat stem sequences

Abstract

Melting curves and circular dichroism spectra were measured for a number of DNA dumbbell and linear molecules containing dinucleotide repeat sequences of different lengths. To study effects of different sequences on the melting and spectroscopic properties, six DNA dumbbells whose stems contain the central sequences (AA)(10), (AC)(10), (AG)(10), (AT)(10), (GC)(10), and (GG)(10) were prepared. These represent the minimal set of 10 possible dinucleotide repeats. To study effects of dinucleotide repeat length, dumbbells with the central sequences (AG)(n), n = 5 and 20, were prepared. Control molecules, dumbbells with a random central sequence, (RN)(n), n = 5, 10, and 20, were also prepared. The central sequence of each dumbbell was flanked on both sides by the same 12 base pairs and T(4) end-loops. Melting curves were measured by optical absorbance and differential scanning calorimetry in solvents containing 25, 55, 85, and 115 mM Na(+). CD spectra were collected from 20 to 45 degrees C and [Na(+)] from 25 to 115 mM. The spectral database did not reveal any apparent temperature dependence in the pretransition region. Analysis of the melting thermodynamics evaluated as a function of Na(+) provided a means for quantitatively estimating the counterion release with melting for the different sequences. Results show a very definite sequence dependence, indicating the salt-dependent properties of duplex DNA are also sequence dependent. Linear DNA molecules containing the (AG)(n) and (RN)(n), sequences, n = 5, 10, 20, and 30, were also prepared and studied. The linear DNA molecules had the exact sequences of the dumbbell stems. That is, the central repeat sequence in each linear duplex was flanked on both sides by the same 12-bp sequence. Melting and CD studies were also performed on the linear DNA molecules. Comparison of results obtained for the same sequences in dumbbell and linear molecular environments reveals several interesting features of the interplay between sequence-dependent structural variability, sequence length, and the unconstrained (linear) or constrained (dumbbell) molecular environments.

DNA Thermal Stability Depends on Solvent Viscosity

“…The solid line was calculated from the structure-prediction program DINAMELT; , the predicted melting temperatures agree with the measured values. The slope of the line can be used to estimate the number of cations released per phosphate upon denaturation of a duplex or hairpin using eq where [M + ] is the cation concentration, R is the gas constant, α is a factor that accounts for changes in the activity coefficient of the cation with concentration and is often set equal to 0.9, Δ H ° is the enthalpy of melting per nucleotide, and Δ n is the number of cations released per nucleotide. ,− Cation release occurs because the hairpin has a higher charge density than the random coil so that a greater number of counterions are condensed in the ion atmosphere and released upon melting. ,, The factor in parentheses on the right-hand side of eq is not significantly salt- and temperature-dependent and is often given the value of 50. ,, Using this value, the slope of the line suggests that 1.4 Na + ions are released per hairpin or 0.093 Na + ions per phosphate. This value is within the range of values observed for small DNA and RNA oligomers in solutions containing less than 1 M Na + ions. ,− ,− …”

“…where [M + ] is the cation concentration, R is the gas constant, α is a factor that accounts for changes in the activity coefficient of the cation with concentration and is often set equal to 0.9, 39 ΔH°is the enthalpy of melting per nucleotide, and Δn is the number of cations released per nucleotide. 8,39−44 Cation release occurs because the hairpin has a higher charge density than the random coil so that a greater number of counterions are condensed in the ion atmosphere and released upon melting.…”

Torsional Rigidities of Weakly Strained DNAs

“…These attempts all invoke the independent dinucleotide step model, wherein the secondary structure and torsional and bending rigidities associated with a given base-pair step are assumed to be completely independent of the sequence of its flanking DNA, or of any altered state of its flanking DNA that is induced by protein or other ligand binding, or by a B-to-Z transition. However, this assumption has been unequivocally contradicted by numerous published NMR structures of small duplexes, which yield different structural parameters for particular steps, e.g., A-A steps, embedded in different flanking sequences (46), as well as by numerous other published experiments (6,36,(47)(48)(49)(50)(51)(52) and unpublished studies of S. A. Winkle (Florida International University, personal communication, 1997) that were reviewed previously (36). Other evidence indicates that dinucleotide step models for directional permanent bends, or for A d , cannot account satisfactorily for the behavior of all sequences (43,53,54).…”

A Structural Transition in Duplex DNA Induced by Ethylene Glycol

“…Numerous conformational substates within the right-handed families have also been detected by various methods, although specific structural details are lacking in most cases . Average properties that reflect changes in secondary structure respond to practically every conceivable perturbation, including the presence of small neutral osmolytes. − Such changes appear to arise from shifts of population (base pairs) among different conformational substates. Conformation-sensitive properties include the elastic constants, α and κ β , for torsion and bending, respectively, the twist energy parameter, E T , that governs the supercoiling free energy, the intrinsic twist, l 0 (turns), the intrinsic binding constant for ethidium, and the circular dichroism spectrum .…”