Melting temperature measurement and mesoscopic evaluation of single, double and triple DNA mismatches

Abstract

Unlike the canonical base pairs AT and GC, the molecular properties of mismatches such as hydrogen bonding and stacking interactions are strongly dependent on the identity of the neighbouring base pairs. As a result, due to the sheer number of possible combinations of mismatches and flanking base pairs, only a fraction of these have been studied in varying experiments or theoretical models. Here, we report on the melting temperature measurement and mesoscopic analysis of contiguous DNA mismatches in nearest-neighbours and next-nearest neighbour contexts. A total of 4032 different mismatch combinations, including single, double and triple mismatches were covered. These were compared with 64 sequences containing all combinations of canonical base pairs in the same location under the same conditions. For a substantial number of single mismatch configurations, 15%, the measured melting temperatures were higher than the least stable AT base pair. The mesoscopic calculation, using the Peyrard-Bishop model, was performed on the set of 4096 sequences, and resulted in estimates of on-site and nearest-neighbour interactions that can be correlated to hydrogen bonding and base stacking. Our results confirm many of the known properties of mismatches, including the peculiar sheared stacking of tandem GA mismatches. More intriguingly, it also reveals that a number of mismatches present strong hydrogen bonding when flanked on both sites by other mismatches. To highlight the applicability of our results, we discuss a number of practical situations such as enzyme binding affinities, thymine DNA glycosylase repair activity, and trinucleotide repeat expansions.

Fig. 1. Schematic diagram of the intramolecular interactions in the PB model, exemplified for a double mismatch (shaded area). The hydrogen bonds are represented here by the Morse potential depth D for each base pair (coils) and the stacking interactions are represented by the elastic constant k for each nearest neighbour (zigzag lines).

Fig. 2. Schematic diagram of context groups, exemplified for the same double mismatch of Fig. 1. BP context groups are shown in the upper part and respective Morse potentials are displayed in the same colour. Similarly, NN context groups are shown in the lower part and their associated stacking potential constants k are colour coded accordingly.

Fig. 3. Work-flow of the optimization procedure.

Fig. 4. Calculated Morse potentials for CI (red bullets) type base pairs. The dashed grey line is the value of the Morse potential of the canonical AT base pair. See also ESI Table S4.

Fig. 5. Heat map of stacking interactions k of CI nearest-neighbours in form BP1–BP2, that is, first base pair followed by second base pair. Lower case letters refer to mismatched base pairs. The matrix was ordered by row and column such that the highest values are clustered in the top-right corner of the map, represented by the dashed box. Note that the matrix is symmetrical towards the antidiagonal (bottom-right to top-left), for instance aa–gg is the same as gg–aa, therefore we left the lower part empty for clarity. The actual values are shown in ESI Table S5.

Fig. 6. Context dependent (CD) Morse potentials for (a) AA, (b) CC, (c) GG and (d) TT mismatches. Shown are only those Morse potentials that deviate by more than 30% from the seed CI potentials, shown as dashed grey lines. Also shown are the transition/transversion characteristic and the BP context group α. Colour coding is as follows: vvv (black); tvv and vtv (brown); tvt and ttv (blue); Tvt, Tvv and TTv (red to orange); TvT and TtT (green to lime). The complete set is shown in ESI Fig. S1–S4 and ESI Table S6, and the full context groups are given in ESI Table S2.

Fig. 7. Context dependent (CD) Morse potentials for (a) AC, (b) CT, (c) AG and (d) GT mismatches. Shown are only those Morse potentials that deviate by more than 30% (panels a–c), or 50% for panel (d), from the seed CI potentials, shown as dashed grey lines. Colour coding for the transition/transversion characteristic is as follows: vvv (black); tvv and vtv (brown); tvt and ttv (blue); Tvt, Tvv and TTv (red to orange); TvT and TtT (green to lime). The complete set is shown in ESI Fig. S5–S8 and ESI Table S6.

Fig. 8. Heat map of the average stacking interactions 〈k〉 of CD nearest-neighbours in form BP1–BP2. The matrix was ordered by row and column, such that the highest values are clustered in the top-right corner of the map, represented by the dashed box. Note that the matrix is symmetrical towards the antidiagonal, for instance aa–gg is the same as gg–aa, therefore we left the lower part empty for clarity. Grey boxes refer to canonical base pairs that were not included in the CD-type optimization. Boxes with black or white border represent the cases where the standard deviation exceeds 4.0 eV nm⁻².

Fig. 9. Heat map of the standard deviation of stacking interactions std(k) of CD nearest-neighbours in form BP1–BP2. The matrix was ordered by row and column, such that the highest values are clustered in the top-right corner of the map, represented by the dashed box. Note that the matrix is symmetrical towards the antidiagonal, for instance ca–ct is the same as tc–ac, therefore we left the lower part empty for clarity. Grey boxes refer to canonical base pairs that were not included in the CD-type optimization.

Fig. 10. Average displacements for sequences with GT^al mismatches {Ag̲C/Tt̲G} (red curves) and corresponding canonical base pairs (dark grey curves). The calculation was carried out at 180 K, which has no relation to the melting temperatures. Sequences from ref. 97.

Fig. 11. Average displacements of the sequences with central triple mismatches, ccc/ccc (red bullets), ttt/ttt (blue squares), tat/tat (green boxes) and their reported Rad4 binding specificities. For comparison, a sequence without a mismatch is also shown (grey bullets). The calculation was carried out at 150 K, which has no relation to the melting temperatures. Sequences are TGACTCGACATCCMMMGCTACAA/ACTGAGCTGTAGGCMMMGATGTT based on ref. 11 and, only the central part around the mismatched region MMM/MMM is shown.

Fig. 12. Average displacements of the sequences with triple mismatches, aaa/ccc (red bullets), aaa/aaa (blue squares), aaa/ggg (green boxes) and their reported MutS recognition. For comparison, a sequence without a mismatch is also shown (grey bullets). The calculation was carried out at 150 K, which has no relation to the melting temperatures. Sequences used are K (red bullets), L (blue squares), M (green boxes) and WT (grey bullets), from Table 1 of ref. 14.

Fig. 13. Average displacements of tandem trinucleotide repeats of type (a) (CNG)₅ and (b) (GNC)₅, with central mismatches AA (blue boxes), CC (red bullets), GG (green boxes) and TT (black circles). Panel (c) shows repeats of type (GAA)₅ formed solely of mismatches calculated with CI parameters. The calculation was carried out at 150 K, which has no relation to the melting temperatures.