Free-energy perturbation calculations of DNA destabilization by base substitutions: The effect of neutral guanine thymine, adenine cytosine and adenine difluorotoluene mismatches

The ability to determine the stability of non-Watson-Crick base pairs in DN A by computer simulation is a necessary prerequisite for quantitative theor etical studies of DNA replication fidelity. Here we report calculations of the relative free energy of nucleotide mismatches in DNA. Our study evaluat ed the "solvation" free energy cost associated with thymine (T) --> difluor otoluene (F), T --> cytosine (C), and C --> T transformations in aqueous so lution and in a DNA duplex. The free energy differences were evaluated by t he free energy perturbation (FEP) method based on classical all-atom simula tions in a spherical surface-constraint water model. The dependence of the calculated free energies on the DNA sequence, the simulation length, size o f the water droplet, and phosphate charges was examined. The calculations w ere carried out for both the negatively charged DNA backbone neutralized by explicit Na+ counterions and the neutral backbone with no counterions. Alt hough the latter model provided overall free energy differences that were l ess sensitive to the radius of the simulation sphere and to the total simul ation time, the results obtained with short 150 ps simulations for the full y charged system containing counterions in 18 A water sphere showed the bes t overall agreement with the corresponding experimental results. The overal l Watson-Crick geometry of an adenine thymine (A.T) base pair has not chang ed during its transformation into an A.F base pair, where F is a nonpolar i sostere of thymine. The calculated change in the duplex binding free energy at 25 degreesC (Delta DeltaG degrees (bind)) for this "mutation" was 5.1 k cal/mol, compared to 3.6 +/- 1.7 kcal/nlol determined previously from the o bserved DNA melting thermodynamics. The transformation of C, forming a Wats on-Crick pair with guanine (G), into T yielded a wobble G.T mismatch analog ous to the one observed by X-ray crystallography. Delta DeltaG degrees (bin d) obtained for this mismatch (relative to the Watson-Crick G.C base pair) by FEP calculations were in a 1.0-3.4 kcal/mol range depending on DNA seque nce and simulation protocol used. This result is in reasonable agreement wi th the experimental estimate of about 3.6 kcal/mol. Starting from an A.T ba se pair, mutation of the thymine carbonyl group into the amino group led to a change of the overall geometry of the base pair from Watson-Crick to rev erse wobble. The Delta DeltaG degrees (bind) Of the resulting neutral A.C m ismatch (relative to the Watson-Crick A.T base pair) calculated using the n eutral and ionic DNA model was 10 and 7 kcal/mol, respectively. Using the o bserved binding affinity and pK(a) constants of the N1-protonated A(+).C ba se pair, the corresponding experimental Delta DeltaG degrees (bind) Of the neutral A.C base pair was determined to be 7.3 +/- 1.5 kcal/mol. Our abilit y to reproduce reasonably stabilities of non-Watson-Crick base pairs by "fi rst principle" calculations will be used in future calculations of DNA poly merase fidelity.