Plum, G. Eric and Park, Young-Whan and Singleton, Scott F. and Dervan, Peter B. and Breslauer, Kenneth J. (1990) Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: A spectroscopic and calorimetric study. Proceedings of the National Academy of Sciences of the United States of America, 87 (23). pp. 9436-9440. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:PLUpnas90
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We report a complete thermodynamic characterization of the stability and the melting behavior of an oligomeric DNA triplex. The triplex chosen for study forms by way of major-groove Hoogsteen association of an all-pyrimidine 15-mer single strand (termed y 15) with a Watson-Crick 21-mer duplex composed of one purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y21). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum of the isolated 21-mer duplex and the CD spectrum of the 15-mer single strand. Spectroscopic and calorimetric measurements show that the triplex (y15·u21·y21) melts by two well-resolved sequential transitions. The first transition (melting temperature, Tm, approximate to 30°C) is pH-dependent and involves the thermal expulsion of the 15-mer strand to form the free duplex u21·y21 and the free single strand y15. The second transition (Tm, approximate to 65°C) is pH-independent between pH 6 and 7 and reflects the thermal disruption of the u21·y21 Watson-Crick duplex to form the component single strands. The thermal stability of the y15·u21·y21 triplex increases with increasing Na+ concentration but is nearly independent of DNA strand concentration. Differential scanning calorimetric measurements at pH 6.5 show the triplex to be enthalpically stabilized by only 2.0 ± 0.1 kcal/mol of base triplets (1 cal = 4.184 J), whereas the duplex is stabilized by 6.3 ± 0.3 kcal/mol of base pairs. From the calorimetric data, we calculate that at 25C the y15·u21·y21 triplex is stabilized by a free energy of only 1.3 ± 0.1 kcal/mol relative to its component u21·y21 duplex and y15 single strand, whereas the 21-mer duplex is stabilized by a free energy of 17.2 ± 1.2 kcal/mol relative to its component single strands. The y15 single strand modified by methylation of cytosine at the C-5 position forms a triplex with the u21·y21 duplex, which exhibits enhanced thermal stability. The spectroscopic and calorimetric data reported here provide a quantitative measure of the influence of salt, temperature, pH, strand concentration, and base modification on the stability and the melting behavior of a DNA triplex. Such information should prove useful in designing third-strand oligonucleotides and in defining solution conditions for the effective use of triplex structure formation as a tool for modulating biochemical events.
|Additional Information:||Copyright © 1990 by the National Academy of Sciences. Contributed by Peter B. Dervan, September 12, 1990. K.J.B. wishes to dedicate this paper to Daniel Michael Breslau for teaching him the importance of adding a third strand to an already existing duplex. This work was supported by the National Institutes of Health Grants GM23509 (K.J.B.), GM34469 (K.J.B.), and GM35724 (P.B.D.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||triple helix; Hoogsteen hydrogen bonds; base triplet stability; triplex hybridization conditions; DNA recognition|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||25 Jan 2006|
|Last Modified:||26 Dec 2012 08:44|
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