Individual basepair stability of DNA and RNA studied by NMR-detected solvent exchange

Abstract

In this study, we have optimized NMR methodology to determine the thermodynamic parameters of basepair opening in DNA and RNA duplexes by characterizing the temperature dependence of imino proton exchange rates of individual basepairs. Contributions of the nuclear Overhauser effect to exchange rates measured with inversion recovery experiments are quantified, and the influence of intrinsic and external catalysis exchange mechanisms on the imino proton exchange rates is analyzed. Basepairs in DNA and RNA have an approximately equal stability, and the enthalpy and entropy values of their basepair dissociation are correlated linearly. Furthermore, the compensation temperature, T(c), which is derived from the slope of the correlation, coincides with the melting temperature, and duplex unfolding occurs at that temperature where all basepairs are equally thermodynamically stable. The impact of protium-deuterium exchange of the imino hydrogen on the free energy of RNA basepair opening is investigated, and it is found that two A·U basepairs show distinct fractionation factors.

Figure 1

Schematic representation of the derivation of thermodynamic parameters ΔH_TR,int and ΔS_TR,int of the transition state of intrinsic catalysis of imino proton exchange in RNA and DNA duplex basepairs.(A) At low temperatures, exchange rates measured by the inversion recovery method are dominated by cross-polarization contributions. The NOE contribution, d, can be determined by the fit according to Eq. 1, included in the figure. Net exchange rates k_ex,net are then obtained by subtracting d from k_ex. (B) The ratio q of the net exchange rates, k_ex,ext and k_ex,int, is determined from exchange-rate measurement at different catalyst concentrations. Assuming that the dissociation constant K_diss is the same in the different catalysis mechanisms, q depends on the transition states of the different mechanisms exclusively. Therefore, the intrinsic transition state can be described by the external transition state and q. (C) Thermodynamic parameters ΔH_TR,int and ΔS_TR,int are derived from Eyring analysis. Linear regression follows the equation depicted in the figure.

Figure 2

(A) Sequences of the DNA and RNA duplexes investigated. (B) CD spectra of the DNA duplex (solid line) and RNA duplex (dotted line). (C) Imino proton resonances of the DNA (upper) and RNA (lower) duplexes in 30 mM KCl and 15 mM K_xH_yPO₄, pH 6.3.

Figure 3

(A) Diagram showing ΔH_diss and TΔS_diss (293 K) values of basepair opening for the individual DNA and RNA nucleobases in high-salt buffer. (B) Diagram showing the ΔG_diss (293 K) values of the individual DNA and RNA nucleobases in high-salt buffer. (C) Same as for A, but in low-salt buffer. (D) Same as for B, but in low-salt buffer.

Figure 4

(A) ΔH_diss(ΔS_diss) correlation of individual nucleobases in the DNA duplex in low-salt buffer (50 mM K⁺; solid diamonds and solid line) and high-salt buffer (250 mM K⁺; open triangles and dotted line). For each salt concentration, the compensation temperature and the intercept of the fit with the y axis are indicated in the figure. (B) Same as in A but for RNA in low-salt buffer (50 mM K⁺; solid diamonds and solid line) and high-salt buffer (250 mM K⁺; open triangles and dotted line).

Figure 5

(A) Free-energy differences of DNA basepairs in 250 mM and 50 mM K⁺. (B) Same representation as in A for RNA basepairs.