Measurement and Theory of Hydrogen Bonding Contribution to Isosteric DNA Base Pairs

Abstract

We address the recent debate surrounding the ability of 2,4-difluorotoluene (F), a low-polarity mimic of thymine (T), to form a hydrogen-bonded complex with adenine in DNA. The hydrogen bonding ability of F has been characterized as small to zero in various experimental studies, and moderate to small in computational studies. However, recent X-ray crystallographic studies of difluorotoluene in DNA/RNA have indicated, based on interatomic distances, possible hydrogen bonding interactions between F and natural bases in nucleic acid duplexes and in a DNA polymerase active site. Since F is widely used to measure electrostatic contributions to pairing and replication, it is important to quantify the impact of this isostere on DNA stability. Here we studied the pairing stability and selectivity of this compound and a closely related variant, dichlorotoluene deoxyriboside (L), in DNA, using both experimental and computational approaches. We measured the thermodynamics of duplex formation in three sequence contexts and with all possible pairing partners by thermal melting studies using the van’t Hoff approach, and for selected cases by isothermal titration calorimetry (ITC). Experimental results showed that internal F-A pairing in DNA is destabilizing by 3.8 kcal/mol (van’t Hoff, 37 °C) as compared with T-A pairing. At the end of a duplex, base-base interactions are considerably smaller; however, the net F-A interaction remains repulsive while T-A pairing is attractive. As for selectivity, F is found to be slightly selective for adenine over C, G, T by 0.5 kcal mol, as compared with thymine’s selectivity of 2.4 kcal/mol. Interestingly, dichlorotoluene in DNA is slightly less destabilizing and slightly more selective than F, despite the lack of strongly electronegative fluorine atoms. Experimental data were complemented by computational results, evaluated at the M06-2X/6-31+G(d) and MP2/cc-pVTZ levels of theory. These computations suggest that the pairing energy of F to A is ~28% of that of T-A, and most of this interaction does not arise from the F⋯HN interaction, but rather from the CH⋯N interaction. The nucleobase analog shows no inherent selectivity for adenine over other bases, and L-A pairing energies are slightly weaker than for F-A. Overall the results are consistent with a small favorable non-covalent interaction of F with A offset by a large desolvation cost for the polar partner. We discuss the findings in light of recent structural studies and of DNA replication experiments involving these analogs.

DNA Base Pair Mismatches Induce Structural Changes and Alter the Free-Energy Landscape of Base Flip

“…Mismatched base pairs contain a consistently lower number of hydrogen bonds than their matched counterparts. Because hydrogen bonding between opposing bases determines DNA stability, (35)(36)(37)(38)(39)(40) these results indicate decreased stability in the presence of mismatches; a result well-known experimentally. (41-44)…”

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

“…Nonpolar thymidine analogues were synthesized to mimic the shape of thymine nucleoside with overall 1.0 Å increment in size from the smallest dH to the largest dI (Figure 5a) (Kim et al, 2005; Kim and Kool, 2005). This system allows us to measure the effects of the loss of hydrogen bonding at the individual “checkpoint” steps (Khakshoor et al, 2012). In addition, the size effect of the template nucleobase can be evaluated by comparing among the five analogues.…”

Backbone FCH⋅⋅⋅O Hydrogen Bonds in 2′F‐Substituted Nucleic Acids

“…[3c,d, 4] Backbone C À H···O hydrogen bonds have received less attention, although they have been proposed to play a role in the stabilization of four stranded i-motif structures, [5] and in tight packing of RNA and DNA elements. [8] The prevalence of 2'-fluorinated oligonucleotides in nucleic acids research motivated us to examine the role of enhanced nonconventional hydrogen bonding in duplex stability. Fluorine substitutions are particularly relevant, since fluorine provokes significant charge polarization effects in geminal and vicinal protons, an effect that has been well-characterized in small molecular systems.…”

“…[7] Recently, Kakshoor et al have observed fluorineenhanced aromatic C À H···N interactions that significantly affect the strength of 2,4-difluorotoluene-adenine base pairs. [8] The prevalence of 2'-fluorinated oligonucleotides in nucleic acids research motivated us to examine the role of enhanced nonconventional hydrogen bonding in duplex stability.…”