Temperature Dependence and Sequence Specificity of DNA Triplex Formation:  An Analysis Using Isothermal Titration Calorimetry

Abstract

We have investigated the thermodynamics and specificity of DNA triplex formation with isothermal titration calorimetry (ITC). The triplex formation between a 23-mer double-stranded homopurine-homopyrimidine and a 15-mer single-stranded homopyrimidine oligonucleotide forming T‚AT and C + ‚GC triads at pH 4.8 is driven by a large negative calorimetric enthalpy change, ∆H cal , of the order of -80 kcal/mol. ∆H cal is strongly temperature dependent, yielding a heat capacity change, ∆C p , of about -1 (kcal/mol)K -1 . The equilibrium association constant, K, obtained from the titration curve is about 9 × 10 7 M -1 at 25°C (binding free energy change, ∆G, is about -11 kcal/mol). Thus, the triplex formation is accompanied by a negative entropy change (∆S ) -245 (cal/mol)K -1 at 25°C). We found that K is insensitive to temperature near room temperature, leading to an apparently small van't Hoff enthalpy change (∆H vH ), in sharp contrast with the large negative ∆H cal . Together, the analyses of the observed temperature dependences of K and ∆H and the large negative ∆C p suggest that the triplex formation is a coupled process between conformational transitions in single-stranded DNA and its binding with double-stranded DNA. The examination of single mismatches in the triplex formation has shown that K and ∆G are not strongly affected by the particular combination of triad sequences (differences in ∆G are within 1.2 kcal/mol). In contrast, single mismatches affected ∆H cal to a greater extent (up to 7-kcal/mol differences). We discuss possible means to enhance specificity in triplex formation, implied by the present findings.

Chemical Modification of Oligonucleotides: A Novel Approach Towards Gene Targeting

“…The magnitudes of the negative ∆H and ∆S for the 2',4'-BNA NCmodified TFOs at pH 6.8 were smaller than those observed for Pyr15TM at pH 6.1 ( Table 4). The observed negative ∆H upon the triplex formation reflects major contributions from the hydrogen bonding and the base stacking involved in the triplex formation, the protonation of the cytosine bases upon the hydrogen bonding, and the accompanying deprotonation of the cacodylate buffer releasing the protons to bind with the cytosine bases [33][34][35]. The immobilization of electrostricted water molecules around polar atoms upon the triplex formation is also considered to be the major sources of the observed negative ∆H upon the triplex formation [33][34][35].…”

“…The observed negative ∆H upon the triplex formation reflects major contributions from the hydrogen bonding and the base stacking involved in the triplex formation, the protonation of the cytosine bases upon the hydrogen bonding, and the accompanying deprotonation of the cacodylate buffer releasing the protons to bind with the cytosine bases [33][34][35]. The immobilization of electrostricted water molecules around polar atoms upon the triplex formation is also considered to be the major sources of the observed negative ∆H upon the triplex formation [33][34][35]. Because the degree of the protonation may be similar between the 2',4'-BNA NC -modified TFOs at pH 6.8 and Pyr15TM at pH 6.1 due to the similar stoichiometry discussed above and the protons to bind with the cytosine bases are released from the same cacodylate buffer in both cases, the ∆H derived from the protonation of the cytosine bases and the accompanying deprotonation of the cacodylate buffer should be similar between the two cases.…”

“…The polar nitrogen atom in the aminomethylene chain to bridge 2'-O and 4'-C of the sugar moiety may possibly achieve such change. On the other hand, the observed ∆S upon the triplex formation is mainly contributed by the two factors, a negative conformational entropy change due to the conformational restraint of TFO involved in the triplex formation, and a positive dehydration entropy change from the release of structured water molecules surrounding the TFO and the target duplex upon the triplex formation [33][34][35]. Therefore, one of the reason for the smaller magnitudes of the negative ∆S for the 2',4'-BNA NC -modified TFOs at pH 6.8 in comparison with that for Pyr15TM at pH 6.1 (Table 4) may be based on the negative conformational entropy change.…”

“…The decrease in the kdissoc is a plausible kinetic reason to explain the remarkable gain in the Ka at neutral pH by the 2',4'-BNA NC modification ( Figure 4 and Tables 4 and 5). Both our group [35] and others [36] have previously proposed a model that triplexes form along nucleation-elongation processes: in a nucleation step only a few base contacts of the Hoogsteen hydrogen bonds may be formed between TFO and the target duplex, and this may be followed by an elongation step in which Hoogsteen base pairings progress to complete triplex formation. Both groups [35,36] have also suggested that the observed Ka, which is the ratio of kassoc to kdissoc, may mostly reflect a rapid equilibrium of the nucleation step, which is probably the rate-limiting process of the triplex formation.…”

“…Both our group [35] and others [36] have previously proposed a model that triplexes form along nucleation-elongation processes: in a nucleation step only a few base contacts of the Hoogsteen hydrogen bonds may be formed between TFO and the target duplex, and this may be followed by an elongation step in which Hoogsteen base pairings progress to complete triplex formation. Both groups [35,36] have also suggested that the observed Ka, which is the ratio of kassoc to kdissoc, may mostly reflect a rapid equilibrium of the nucleation step, which is probably the rate-limiting process of the triplex formation. In this sense, the 2',4'-BNA NC modification is considered to slow the collapse of the nucleation intermediate using the increased rigidity and the increased degree of hydration of 2',4'-BNA NC -modified TFO to increase the Ka of the pyrimidine motif triplex formation.…”