Nucleic Acids Research, 1990, Vol. 18, No. 12 3557-3564
© 1990
CHEMISTRY |
Spectroscopic and calorimetric investigation on the DNA triplex formed by d(CTCTTCTTTCTTTTCTTTCTTCTC) and d(GAGAAGAAAGA) at acidic pH
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste 34127 Trieste 1Institute of Biology, Faculty of Medicine, University of Udine 33100 Udine, Italy
Received March 9, 1990. Revised May 18, 1990. Accepted May 18, 1990.
The equimolar mixture of d(CTCTTCTTTCTTTTCTTT-CTTCTC) (dY24) and d(GAGAAGAAAGA) (dR11 [designated (dY24).(dR11)], forms at pH = 5 a DNA triplex, which mimicks the H-DNA structure. The DNA triplex was identified by the following criteria: (i) dY24 and dR11co-migrate in a poly-acrylamide gel, with a mobility and a retardation coefficient comparable to those observed for an 11-triad DNA triplex, previously characterized in our laboratories (1); (ii) the intercalator ethidium bromide shows a poor affinity for (dR11).(dY24) at pH = 5, and a high affinity at pH = 8; (iii) the (dR11).(dY24) mixture is not a substrate for DNase I at pH = 5; (iv) the CD spectrum of (dR11).(dY24), at pH = 5, is consistent with those previously reported for triple-stranded DNA.
The (dR11).(dY24) mixture exhibits a thermally induced co-operative transition, which appears to be monophasic, reversible and concentration dependent. This transition is attributed to the disruption of the DNA triplex into single strands. The enthalpy change of the triplex-coil transition was measured by DSC (
Hcal = 129±6 kcal/mol) and, assuming a two-state model, by analysis of UV-denaturation curves (average of two methods HUV = 137 ± 13 kcal/mol). Subtracting from Hcal of triplex formation the contributions due to the Watson-Crick helix and to the protonation of the C-residues, we found that each pyrimidine binding into the major groove of the duplex, through a Hoogsteen base pair, is accompanied by an average H = –5.8 ± 0.6 kcal/mol. The effect on the stability of the (dR11).(dY24) triplex due to the substitution of a T: A: T triad with a T: T: T one was also investigated.
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