Thermodynamic Characterization of the Stability and the Melting Behavior of a DNA Triplex: A Spectroscopic and Calorimetric Study

• Published in: Proceedings of the National Academy of Sciences - PNAS 1990-12, Vol.87 (23), p.9436-9440
• Main Authors: Plum, G. Eric, Park, Young-Whan, Singleton, Scott F., Dervan, Peter B., Breslauer, Kenneth J.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biochemistry; Biological and medical sciences; Calorimetry, Differential Scanning; Chemical bases; Circular Dichroism; DNA; DNA - chemistry; Dna, deoxyribonucleoproteins; Enthalpy; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Kinetics; Melting; Methylation; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Denaturation; Nucleic acids; Oligodeoxyribonucleotides - chemical synthesis; Oligodeoxyribonucleotides - chemistry; Spectroscopy; Thermal stability; Thermodynamics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.23.9436

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, ≈ 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, ≈ 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 25⚬C 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.