Fidelity of Binding of the Guanidinium Nucleic Acid (DNG) d(Tg)₄-T-azido with Short Strand DNA Oligomers (A₅G₃A₅, GA₄G₃A₄G, G₂A₃G₃A₃G₂, G₂A₂G₅A₂G₂). A Kinetic and Thermodynamic Study^†

Short strand DNA oligomers (A₅G₃A₅, GA₄G₃A₄G, G₂A₃G₃A₃G₂, and G₂A₂G₅A₂G₂) and the guanidinium (g) linked thymidyl nucleoside d(Tg)₄-T-azido associate as triplexes. The melting temperatures, T_m, the association and dissociation kinetic and thermodynamic parameters and activation energies for the triplexes were determined by UV thermal analysis. The hypochromic shift and T_m for triplex formation increases with increase in concentration and decreases with the number of mismatches. The melting temperatures are between 35 and 55 °C in the range of ionic strength of 0.06−0.24 and decrease with increase in ionic strength at 100 deg/(ionic strength unit). The melting and cooling curves exhibit hysteresis behavior in the temperature range 5−95 °C at 0.2 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (k_on, E_on) and dissociation (k_off, E_off) processes were obtained. The second-order rate constants, k_on, for the triplex formation at 288 K are between 10 and 500 M^-1 s^-1. Values of k_on increase with the decrease in the ionic strength. The first order rate constants for the dissociation, k_off, at 288 K are between 10^-6 and 40 × 10^-6 s^-1 and increase with increase in ionic strength. The energies of activation for the association and dissociation processes are in the range −22 to −9 kcal/mol and 8 to 29 kcal/mol, respectively. At 6.3 × 10^-5 M/base and at the physiological ionic strength (0.15−0.30) and below, the triplex structures formed with d(Tg)₄-T-azido and A₅G₃A₅ and GA₄G₃A₄G have well-defined T_m values. The melting curves with G₂A₃G₃A₃G₂ and G₂A₂G₅A₂G₂ are very shallow with small hypochromic shifts denoting negligible binding at physiological ionic strength. Therefore, with the increase in the G content (mismatched base pairs) at a certain concentration (e.g., 6.3 × 10^-5 M/base), discrimination (change in fidelity) occurs in the formation and strength of binding of d(Tg)₄-T-azido to d(pA_npG_m) oligomers. The standard molar enthalpies for triplex formation have in general larger negative values at low ionic strength than at high ionic strength, indicating that at lower μ values the formation of triplexes of d(Tg)₄-T-azido with d(pA_npG_m) are more favorable. The values of ΔH°(288) calculated from the activation parameters are between −17 and −49 kcal/mol, and the values of ΔG°(288) are between −7.5 and −11.8 kcal/mol for A₅G₃A₅, GA₄G₃A₄G, G₂A₃G₃A₃G₂, and G₂A₂G₅A₂G₂, respectively. There is a linear relationship in the enthalpy−entropy compensation for the triplex melting thermodynamics.