Table 1. Tm values (°C) determined for the melting of the triplexes shown in Figure 2A.
| pH | N = | A | G | C | T | BAU |
|---|---|---|---|---|---|---|
| 5.0 | N.AT | 57.3 | 54.7 | 55.3 | 63.8 | 70.8a |
| N.TA | 54.7 | 58.8 | 53.4 | 53.8 (10.0) | 55.9 (14.9) | |
| N.GC | 61.8 | 58.1 | 67.4 | 57.8 (6.0) | 62.8 (8.0) | |
| N.CG | 55.7 | 56.8 | 57.6 | 58.1 (5.7) | 56.7 (14.1) | |
| 5.5 | N.AT | 44.4 | 39.0 | 39.8 | 54.5 | 59.5 |
| N.TA | 40.7 | 47.3 | 38.7 | 39.2 (15.3) | 41.8 (17.7) | |
| N.GC | 48.4 | 44.1 | 56.6 | 43.7 (10.8) | 51.8 (7.7) | |
| N.CG | 40.7 | 41.9 | 43.7 | 44.5 (10.0) | 42.0 (17.5) | |
| 6.0 | N.AT | — | — | — | 34.8 | 41.2 |
| N.TA | — | — | — | — | — | |
| N.GC | — | — | 34.8 | — | 30.0 (11.2) | |
| N.CG | — | — | — | — | — |
The Tms were determined by the fluorescence melting technique at pH 5.0, 5.5 and 6.0 as indicated. The duplex concentration was 0.25 μM, while the third strand was 3 μM. Each value is the average of three separate determinations. The Tms shown obtained from melting profiles with a temperature change of 0.2°C/min indicates that the Tm was too low to measure (<25°C). For T and BAU, the figures in parentheses show the difference in Tm between the correct triplet (i.e. T.AT or BAU.AT) and the mismatched triplet. Tm values are reproducible to within less than 0.5°C.
aThis complex showed hysteresis between the melting and annealing profiles and the Tm for annealing was 64.4°C. The melting and annealing Tms for the BAU.AT complex were 69.0 and 66.0°C when the rate of temperature change was reduced to 0.067°C/min.