The hydrogen bonding of cytosine with guanine: calorimetric and 1H-nmr analysis of the molecular interactions of nucleic acid bases.

Abstract

AbstractThe enthalpy of hydrogen‐bond formation between guanine (G) and cytosine (C) in o‐dichlorobenzene and in chloroform at 25°C has been determined by direct calorimetric measurement. We derivatized 2′‐deoxyguanosine and 2′‐deoxycytidine at the 5′‐ and 3′‐hydroxyls with triisopropylsilyl groups; these groups increase the solubility of the nucleic acid bases in nonaqueous solvents. Such derivatization also prevents the ribose hydroxyls from forming hydrogen bonds. Consequently, hydrogen‐bond formation in our system is primarily between the bases, and to a lesser extent, between base and solvent, and can be measured directly with calorimetry. To obtain the data on base‐pair formation, we first took into account the contributions from self‐association of each base, and where possible, have determined the ΔH of self‐association. From isoperibolic titration calorimetry, our measured ΔH of C2 formation in chloroform is −1.7 kcal/mol of C. Our measured ΔH of C:G base‐pair formation in o‐dichlorobenzene is −6.65 ± 0.32 kcal/mol. Since o‐dichlorobenzene does not form hydrogen bonds, the ΔH of C:G base‐pair formation in this solvent represents the ΔH of the hydrogen‐bonding interaction of C with G in a nonassociating solvent. In contrast, our measured ΔH of C:G base‐pair formation in chloroform is −5.77 ± 0.20 kcal/mol; thus, the absolute value of the enthalpy of hydrogen bonding in the C:G base pair is greater in o‐dichlorobenzene than in chloroform. Since chloroform is a solvent known to form hydrogen bonds, the decrease in enthalpic contribution to C:G base pairing in chloroform is due to the formation of hydrogen bonds between the bases and the solvent. The ΔH of hydrogen bonding of G with C reported here differs from previous indirect estimates: Our measurements indicate the ΔH is 50% less in magnitude than the ΔH based on spectroscopic measurements of the extent of interaction. We have also observed that the enthalpy of hydrogen bonding of C with G in chloroform is greater when G is in excess than when C is in excess. This increased heat is due to the formation of C:Gn > 1 complexes that we have observed using 1H‐nmr. Although C:G2 structures have previously been observed in triple‐stranded polymeric nucleic acids, higher order structures have not been observed between C and G monomers in nonaqueous solvents until now. By using monomers as a model system to investigate hydrogen‐bonding interactions in DNA and RNA, we have obtained the following results: A direct measurement of the ΔH of hydrogen bonding in the C:G complex in two nonaqueous solvents, and the first observation of C:Gn > 1 complexes between monomers. These results reinforce the importance of hydrogen bonding in the stabilization of various nucleic acid secondary and tertiary structures.