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 y15) with a Watson-Crick 21-mer duplex composed ofone purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y2l). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum ofthe isolated 21-mer duplex and the CD spectrum of the 15-mer single strand. Spectroscopic and calorimetric measurements show that the triplex (y15u21y21) melts by two well-resolved sequential transitions. The first transition (melting temperature, Tm, =300C) is pH-dependent and involves the thermal expulsion of the 15-mer strand to form the free duplex u21y21 and the free single strand y15. The second transition (Tm 650C) is pH-independent between pH 6 and 7 and reflects the thermal disruption ofthe u21-y21 Watson-Crick duplex to form the component single strands. The thermal stability of the y15-u21y21 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 250C 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-S position forms a triplex with the u21y2I 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.More than three decades have passed since the first description of polynucleotide triple helices (1). In the ensuing years a small number of investigators interested in the fundamental properties of nucleic acids have studied the structure (2) and physical properties (3-6) of triple-helical nucleic acids. Most of the work in this area has focused on triple helices composed of one polypurine strand and two polypyrimidine strands; however, triplexes of (polypurine)2 polypyrimidine also are known (7-10).The widely accepted structural model for polypurine (polypyrimidine)2 triple helices is based on x-ray fiber diffraction studies on poly(A) poly(U)2 (2, 11) and poly(dA)...

The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.

We have analysed enzyme catalysis through a re-examination of the reaction coordinate. The ground state of the enzyme-substrate complex is shown to be related to the transition state through the mean force acting along the reaction path; as such, catalytic strategies cannot be resolved into ground state destabilization versus transition state stabilization. We compare the role of active-site residues in the chemical step with the analogous role played by solvent molecules in the environment of the noncatalysed reaction. We conclude that enzyme catalysis is significantly enhanced by the ability of the enzyme to preorganize the reaction environment. This complementation of the enzyme to the substrate's transition state geometry acts to eliminate the slow components of solvent reorganization required for reactions in aqueous solution. Dramatically strong binding of the transition state geometry is not required.

In this chapter, we review the current state of the thermodynamic database for triple helical oligonucleotide hybridization reactions and present a critical assessment of the methods used to obtain the relevant data. The thermodynamic stability of triple-helix oligonucleotide constructs is discussed in terms of its dependence on temperature, chain length, pH, salt, base sequence, base and backbone modifications, and ligand binding. In particular, we examine the coupling of hybridization equilibria to proton, cation, and drug-binding equilibria. Throughout the chapter, we emphasize that a detailed understanding of the endogenous and exogenous variables that control triplex stability is required for the rational design of oligonucleotides for specific therapeutic, diagnostic, and/or biotechnological applications, as well as for elucidating the potential cellular roles of these higher-order nucleic acid complexes.

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.