Base Pair Opening Research Articles

Bending and circularization of site-specific and stereoisomeric carcinogen-DNA adducts.

The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S), 10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino group of guanine residues in DNA in vivo and in vitro. While the (-)-7S,8R,9R,10Senantiomer, (-)-anti-BPDE, also reacts with DNA to form similar covalent N2-deoxyguanosyl adducts, this diol epoxide is nontumorigenic and its mutagenic activities are different from those of (+)-anti-BPDE. In this work, T4 ligase-induced cyclization methods have been employed to demonstrate that the (+)-anti-[BP]-N2-dG lesions (G*) cause significantly greater amounts of bending and circularization of the one-base overhang undecamer duplex 5'-d(CACAT[G*]TACAC).d(TGTACATGTGG) than the stereoisomeric oligonucleotide duplex with G* = (-)-anti-[BP]-N2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotides, the ratio of circular to linear DNA multimers reaches values of 8-9 for circle contour sizes of 99-121 base pairs, while for the (-)-anti-[BP]-N2-dG-modified DNA this ratio reaches a maximum value of only approximately 1 at 154-176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90-92% of the observed circular ligation products range from 30 to 51 degrees per (+)-trans-anti-[BP]-N2-dG lesion and from 20 to 40 degrees per (-)-trans-anti-[BP]-N2-dG lesion. In the case of unmodified DNA, the probability of circular product formation is at least 1 order of magnitude less efficient than in the BPDE-modified sequences and about 90% of the circular products exhibit bending angles in the range of 14 -19 degrees . In the most abundant circular products observed experimentally, the bending angles are 40 degrees and 26 +/- 2 degrees per (+)-anti-[BP]- or (-)-anti-[BP]-modified 11-mer; these values correspond to a net contribution of 21-26 degrees and 5-19 degrees , respectively, to the observed overall bending per lesion. The coexistence of circular DNA molecules of different sizes and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends, which permit efficient cyclization, especially in the case of (+)-trans-[BP]-N2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N2-dG lesion in the 11-mer duplex 5'-d(CACAT[G*]TACAC).d(GTGTACATGTG) indicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the duplex on the 5'-side of the modified guanosine residue, which may be due to bending enhanced base pair opening and bending induced by the bulky carcinogen residue. The implications of base sequence-dependent flexibilities and conformational mobilities of anti-[BP]-N2-dG lesions on DNA replication and mutation are discussed.

Internal motions of nucleic acid structures and the determination of base-pair lifetimes

Over the years, imino proton exchange measurements have provided a description of base-pair opening and of properties of internal motions of nucleic acids such as the individual opening of base pairs and the insensitivity of base-pair lifetimes to the nature and stability of neighboring pairs. A recent determination of base-pair lifetimes in d(CGCGATCGCG) conflicts with the original measurements and their interpretation. This question is analyzed in the present work. We emphasize the importance of high concentrations of exchange catalyst ( eg 1 M NH 3) for the accurate determination of base-pair lifetimes. These concentrations entail a high ionic strength, which can lead to aggregation, enhanced magnetic relaxation and underestimation of base-pair lifetimes if exchange is measured by its effect on the proton relaxation rate. Magnetization transfer which provides a more direct method for the measurement of proton exchange rates is therefore preferred. We show that the lack of measurements at high catalyst concentration is responsible for the discrepancy mentioned above. Measurements by the magnetization transfer method validate the original interpretation and inversion recovery experiments illustrate the effect of the ionic strength on the relaxation rate of the imino protons.

Quantum-chemical ab initio Study on the Adenine-Difluorotoluene Complex - A Mimic for the Adenine-Thymine Base Pair

Recent experiments have shown that difluorotoluene (F), a nonpolar isostere for thymine (T), codes efficiently and specifically for adenine (A) in DNA replication. F has almost the same shape as thymine but it is unable to form conventional hydrogen bonds with adenine. Therefore, it has been claimed that not hydrogen bonding but shape complementary may be important for the selection of the correct bases by DNA-replicating enzymes. In order to gain deeper insight into structure, charge distribution and energetics of the A-F and A-T base pairs we have performed quantum-chemical ab initio and density functional calculations at the HF, MP2 and B3LYP levels. The interaction energy of the A-F complex amounts to -3.8 kcal/mol (MP2) and is thus substantially smaller than typical ab initio interaction energies for Watson-Crick or non-canonical base pairs. The A-T and A-F complexes are planar and their overall geometries are similar (root-mean-square deviation: 0.4 Å). The calculated donor acceptor atom distances in A-T are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. However, A-F shows a base pair opening as compared to A-T. Even though the interaction energy in the A-F base pair is small, the distances for the N6-H…F and N1…H-C3 contacts are still below the sum of the van-der-Waals radii, which means that the interaction is not governed by van-der- Waals forces alone. If the experimental findings can be confirmed, then our results indicate that DNA polymerase is able to retain high fidelity with base pairs of much smaller interaction energies than found for the conventional Watson-Crick and non-canonical base pairs.

Solution structure and base pair opening kinetics of the i-motif dimer of d(5mCCTTTACC): a noncanonical structure with possible roles in chromosome stability

Repetitive cytosine-rich DNA sequences have been identified in telomeres and centromeres of eukaryotic chromosomes. These sequences play a role in maintaining chromosome stability during replication and may be involved in chromosome pairing during meiosis. The C-rich repeats can fold into an 'i-motif' structure, in which two parallel-stranded duplexes with hemiprotonated C.C+ pairs are intercalated. Previous NMR studies of naturally occurring repeats have produced poor NMR spectra. This led us to investigate oligonucleotides, based on natural sequences, to produce higher quality spectra and thus provide further information as to the structure and possible biological function of the i-motif. NMR spectroscopy has shown that d(5mCCTTTACC) forms an i-motif dimer of symmetry-related and intercalated folded strands. The high-definition structure is computed on the basis of the build-up rates of 29 intraresidue and 35 interresidue nuclear Overhauser effect (NOE) connectivities. The i-motif core includes intercalated interstrand C.C+ pairs stacked in the order 2*.8/1.7*/1*.7/2.8* (where one strand is distinguished by an asterisk and the numbers relate to the base positions within the repeat). The TTTA sequences form two loops which span the two wide grooves on opposite sides of the i-motif core; the i-motif core is extended at both ends by the stacking of A6 onto C2.C8+. The lifetimes of pairs C2.C8+ and 5mC1.C7+ are 1 ms and 1 s, respectively, at 15 degrees C. Anomalous exchange properties of the T3 imino proton indicate hydrogen bonding to A6 N7 via a water bridge. The d(5mCCTTTTCC) deoxyoligonucleotide, in which position 6 is occupied by a thymidine instead of an adenine, also forms a symmetric i-motif dimer. However, in this structure the two TTTT loops are located on the same side of the i-motif core and the C.C+ pairs are formed by equivalent cytidines stacked in the order 8*.8/1.1*/7*.7/2.2*. Oligodeoxynucleotides containing two C-rich repeats can fold and dimerize into an i-motif. The change of folding topology resulting from the substitution of a single nucleoside emphasizes the influence of the loop residues on the i-motif structure formed by two folded strands.

Unique base-pair breathing dynamics in PNA-DNA hybrids

Kinetic and thermodynamic parameters, derived from 1H-NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia, are reported for two mixed-sequence peptide nucleic acid (PNA)-DNA hybrids and their counterpart DNA duplex. The exchange times of the imino protons in the PNA strands extrapolate to very short base-pair lifetimes in the limit of infinite exchange catalyst concentration. This is not due to generally less stable base-pairs in PNA-DNA hybrids, since the lifetimes, apparent dissociation constants and thermodynamic stability (ΔG°) of the innermost DNA guanine imino protons are similar in the hybrid duplexes and in the DNA duplex. In addition, the apparent dissociation constants determined for PNA bases of the hybrids are of the same order as those of the corresponding bases in the DNA duplex. An exchange process from the closed state was found to be inconsistent with the experimental data. From these results, we conclude that opening and closing rates of the PNA guanine and thymine bases are at least two orders of magnitude higher than those of the corresponding bases in the DNA duplex. Unusual kinetics in the hybrids is also evident from the destabilization of the complementary DNA strand thymine bases, which exhibit base-pair dissociation constants increased by approximately two orders of magnitude compared to what is observed in the DNA duplex, while the DNA strand guanine bases are largely unaffected.The general pattern of the base-pair dynamics in the hybrids obtained when using trimethylamine as an exchange catalyst is the same as when using ammonia. However, the long base-pair lifetimes i.e. those of the DNA duplex and the guanine bases of the DNA strands in the hybrids, are approximately three to five times longer than when using ammonia. Thus, all opening events sensed by ammonia are not accessible to trimethylamine. These observations are discussed in regard to the mechanism of base-pair opening and the nature of the open state.

Nanosecond Molecular Dynamics Study of a Polycation Ribonucleic Guanidine (RNG) Duplexed with a Complementary DNA Oligomer Strand

Replacement of the phosphodiester linkages of the polyanion RNA with guanidinium linkers provides the polycation ribonucleic guanidine (RNG). Molecular dynamics studies to 1080 ps of the hybrid duplex formed from an octameric RNG strand r(Ag)8 with a complementary DNA oligomer strand d(Tp)8 have been carried out with explicit water solvent and Na+Cl- counterions under periodic boundary conditions with use of the CHARMM force field and Ewald summation method. The Watson−Crick hydrogen bonding pattern of the A/T tracts was maintained intact through the simulation without any structural restraints. Even for the two ends, only one end was partially frayed. The overall structure is equilibrated at a B-DNA conformation, and the RNG strand takes on the general conformation of the DNA backbone. The structural history of the RNG·DNA duplex was determined by examining histograms from the dynamics run. The histograms are of the hydrogen bonding pattern (sequence recognition) and base pair opening occurrences, minor groove width narrowing (3 Å), and bending of tracts. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 33° for the averaged structure. Propeller twist (associated with three-centered hydrogen bonding) up to −30°, native to DNA AT base pairing, was also observed. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3‘-endo domain. However, for the RNG strand the pseudorotation angles converted to O4‘-endo.

Molecular Dynamics Simulations Find That 3‘ Phosphoramidate Modified DNA Duplexes Undergo a B to A Transition and Normal DNA Duplexes an A to B Transition

We present unrestrained molecular dynamics studies on a deoxyribose dodecamer duplex d(CGCGAATTCGCG)2 and its phosphoramidate (replacing O3‘ by NH) analog using particle mesh Ewald electrostatics1 and the Cornell et al. force field.2 The simulations were carried out beginning in both canonical A and B forms, which differ in RMS position by ∼6.5 A. These simulations, which were carried out for ∼1−1.5 ns, are consistent with experiment in that the unmodified dodecamer, whether started in A or B DNA, converges to a B structure which is closer to the observed X-ray structure (9bna) than canonical B. On the other hand, the phosphoramidate modified duplex, whether started in A or B forms, converges to an A form. In this duplex, however, the structure from the simulation begun in the B form has a dependence on the initial location of the N−H hydrogen. In one of our simulations, we find base pair opening and closing at the end of the duplex. Specifically, in the simulation of d(CGCGAATTCGCG)2 begun in the A form,...

Modelling base pair opening: the role of helical twist

Molecular modelling with the JUMNA program has been used to study the conformational and energetic aspects of base pair opening within a B-DNA double helix. Using new geometrical restraints, it has been possible to map base opening into the minor and major grooves of the double helix, without fixing an axis of rotation or the plane in which the base moves. The results show notably that minor groove opening is favoured by locally unwound DNA. Although the modelling is carried out with a simple distance-dependent dielectric function, the DelPhi program is also used to check that more realistic Poisson-Boltzmann electrostatics yields similar results.

Design and analysis of molecular motifs for specific recognition of RNA

Selective targeting of RNA has become a recent priority in drug design strategies due to the emergence of retroviruses, the need for new antibiotics to counter drug resistance, and our increased awareness of the essential role RNA and RNA structures play in the progression of disease. Most organic compounds known to specifically target RNA are complex, naturally occurring antibiotics that are difficult to synthesize or derivatize and modification of these compounds to optimize interactions with structurally unique RNAs is difficult. The de novo design of synthetically accessible analogues is one possible alternative; however, little is known about the RNA recognition principles on which to design new compounds and limited information on RNA structure in general is available. To contribute to the growing body of knowledge on RNA recognition principles, we have prepared two series of polycationic RNA-binding agents, one with a linear scaffold, the other with a macrocyclic scaffold. We evaluated these compounds for their ability to bind to DNA and RNA, as well as to a specific RNA, the regulatory sequence, RRE, derived from HIV-1, by using thermal melting, circular dichroism, and electrophoresis gel shift methods. Our results suggest that cationic charge centers of high p K a that are displayed along a scaffold of limited flexibility bind preferentially to RNA, most likely within the major groove. Related derivatives that bind more strongly to DNA more closely mimic classical DNA minor-groove binding agents. Several of the macrocyclic polycations expand on a new binding motif where purine bases in duplex RNA are complexed within the macrocyclic cavity, enhancing base-pair opening processes and ultimately destabilizing the RNA duplex. The results in this report should prove a helpful addition to the growing information on molecular motifs that specifically bind to RNA.

Imino proton exchange and base-pair kinetics in the AMP-RNA aptamer complex

We report on the dynamics of base-pair opening in the ATP-binding asymmetric internal loop and flanking base-pairs of the AMP-RNA aptamer complex by monitoring the exchange characteristics of the extremely well resolved imino protons in the NMR spectrum of the complex. The kinetics of imino proton exchange as a function of basic pH or added ammonia catalyst are used to measure the apparent base-pair dissociation constants and lifetimes of Watson-Crick and mismatched base-pairs, as well as the solvent accessibility of the unpaired imino protons in the complex. The exchange characteristics of the imino protons identify the existence of four additional hydrogen bonds stabilizing the conformation of the asymmetric ATP-binding internal loop that were not detected by NOEs and coupling constants alone, but are readily accommodated in the previously reported solution structure of the AMP-RNA aptamer complex published from our laboratory. The hydrogen exchange kinetics of the non-Watson-Crick pairs in the asymmetric internal loop of the AMP-RNA aptamer complex have been characterized and yield apparent dissociation constants (αKd) that range from 10−2 to 10−7. Surprisingly, three of these αKd values are amongst the lowest measured for all base-pairs in the AMP-RNA aptamer complex. Comparative studies of hydrogen exchange of the imino protons in the free RNA aptamer and the AMP-RNA aptamer complex establish that complexation stabilizes not only the bases within the ATP-binding asymmetric internal loop, but also the flanking stem base-pairs (two pairs on either side) of the binding site. We also outline some preliminary results related to the exchange properties of a sugar 2′-hydroxyl proton of a guanosine residue involved in a novel hydrogen bond that has been shown to contribute to the immobilization of the bound AMP by the RNA aptamer, and whose resonance is narrow and downfield shifted in the spectrum.

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Sequence and temperature effect on hydrogen bond disruption in DNA determined by a statistical analysis.

We use the modified self-consistent phonon approximation theory to calculate temperature dependent interbase hydrogen bond disruption profiles for a number of six base pair repeating sequence infinite B-DNA polymers with various guanine-cytosine/adenine-thymine ratios. For comparison we also include results we have obtained in our earlier work on several B-DNA homopolymers, copolymers and a four-base-pair repeating sequence polymer. Our theory gives a statistical estimate of thermal fluctuational disruption probability of individual hydrogen bonds in individual base pairs in DNA as a function of temperature. The calculated probabilities show no sequence dependence at premelting temperatures, in agreement with proton exchange measurements. These probabilities however become very sensitive to base sequence at temperatures close to the observed melting temperatures. Multiphasic critical transitions are found in which a portion of base pairs are disrupted at temperature below the final disruption temperature. These transitions include localized as well as non-localized base pair opening. The localized transitions involve disruption of a few base-pairs at every other location without large scale base unstacking, and they may not appear in the observed UV curves with current resolution. On the other hand the overall disruption behavior is consistent with observations. The midpoint transition temperatures are close to the observed melting temperatures and these temperatures show the observed linear dependence on guanine-cytosine content. Our calculations indicate that our theory can be used effectively to calculate H-bond disruption behavior of different DNA sequences.

Melting profile and temperature dependent binding constant of an anticancer drug daunomycin-DNA complex.

We calculate thermal fluctuational base pair opening probability and the drug binding constant of a daunomycin-bound Poly d(CGTA).Poly d(TACG) at temperatures from room temperature to its melting temperature. For comparison we also carry out a calculation on a drug-free DNA with the same sequence. Our calculations are carried out by means of a statistical approach using microscopic structures and established force fields and with cooperative effects incorporated into the algorithm. Both hydrogen bond disruption probabilities and drug unstacking probability are determined self-consistently. These probabilities are then used to determine temperature dependent base pair opening probabilities and the drug binding constant. The calculated base pair opening probabilities and drug binding constant are found to be in fair agreement with experiments carried out at room temperature. Our calculation shows cooperative base pair disruption and drug dissociation at certain critical temperatures close to the observed melting temperatures for similar helices. We find that the temperature dependence of the drug binding constant fits well to the van't Hoff relation, in agreement with observations. Our calculation indicates the occurrence of a premelting transition in the drug-bound DNA helix. Some comments are made about this premelting transition.

Description of RNA Folding by "Simulated Annealing">

An algorithm is proposed which describes the thermodynamically as well as the kinetically controlled folding process of RNA. The algorithm, based on a special Monte Carlo procedure known as “Simulated Annealing”, takes into account the probabilities for opening and closing of single base-pairs. Thus, the algorithm is able to reach structures and structure distributions near the global minimum of structure space, and is not restricted by the tendency to halt in local minima. Three types of structural folding processes may be analysed by this algorithm. Firstly, using thermodynamic data, structure ensembles comparable to those obtained by dynamic programming are achieved. Secondly, using kinetic data, the processes of structure formation and structural rearrangement may be simulated. Thirdly, additionally taking into account RNA polymerase chain elongation rates, the process of “sequential folding” during transcription may be described. Analysis of all types of structural folding and refolding is performed for RNA sequences related to potato spindle tuber viroid (PSTVd). The computed results are in accordance with experimental data and biological functions known for PSTVd.

A Cationic Cyclophane That Forms a Base-Pair Open Complex with RNA Duplexes

It is now well-established that synthetic organic cations can interact with the helical conformations of DNA and RNA and can stabilize these structures. Such interactions can also perturb the funct...

P-B1-35 PNA/DNA hybrids exhibit unique breathing dynamics: Evidence for nonsymmetric basepair opening: Sehlstedt, U., 1 Leijon, M., 1 Nielsen, P.E., 2 Gräslund, A. 1 1 Dept. of Biophys., Stockholm Univ. (SWE), 2 DMBG, Panum Inst., Copenhagen (DK)

P-B1-35 PNA/DNA hybrids exhibit unique breathing dynamics: Evidence for nonsymmetric basepair opening: Sehlstedt, U., 1 Leijon, M., 1 Nielsen, P.E., 2 Gräslund, A. 1 1 Dept. of Biophys., Stockholm Univ. (SWE), 2 DMBG, Panum Inst., Copenhagen (DK)

Base-catalysis of imino proton exchange in DNA: effects of catalyst upon DNA structure and dynamics.

Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon measurements of the rates of exchange of imino protons with water protons at high concentrations of the exchange catalyst. Under these conditions, the exchange catalyst may affect structural or dynamic properties of the nucleic acid molecule and thus, limit the significance of the exchange data. To address this problem, we have used NMR spectroscopy to characterize the effects of a catalyst of imino proton exchange, namely, ammonia upon the structure and dynamics of the self-complementary DNA dodecamer [d(CGCAGATCTGCG)]2. The changes in structure were monitored in proton NOESY and DQF-COSY experiments and in phosphorus spectra at 15 degrees C and at ammonia concentrations ranging from 0.002 to 0.5 M. The results indicate that ammonia induces subtle changes in the solution conformation of the dodecamer, but the overall structure is maintained close to the B-type DNA structure. However, the relaxation rates (i.e., transverse, longitudinal, and cross-relaxation rates) of several non-exchangeable protons were found to increase by approximately 50% upon changing ammonia concentration from 0.002 to 0.5 M. The increases were comparable for all protons investigated suggesting that they originate from an ammonia-induced increase in the overall correlation time of the DNA dodecamer. Numerical analysis revealed that the catalyst-induced enhancements in proton relaxation can alter significantly the calculated values of the exchange rates of imino protons, especially those obtained from measurements of the line widths of these proton resonances.

Monte Carlo analysis of conformational transitions in superhelical DNA

Metropolis–Monte Carlo algorithms are developed to analyze the strand separation transition in circular superhelical DNA molecules. Moves that randomize the locations of unpaired regions are required in order to diminish correlations among the sampled states. This approach enables accurate simulations to be performed in reasonable computational times. Sufficient conditions to guarantee the formal correctness of the complete algorithm are proven to hold. The computation time required scales at most quadratically with molecular length, and is approximately independent of linking difference. Techniques are developed to estimate the sample size and other calculation parameters needed to achieve a specified accuracy. When the results of Monte Carlo calculations that use shuffling operations are compared with those from statistical mechanical calculations, excellent agreement is found. The Monte Carlo methodology makes possible calculations of transition behavior in cases where alternative approaches are intractable, such as in long molecules under circumstances where several runs of open base pairs occur simultaneously. It also allows the analysis of transitions in cases where the base pair separation energies vary in complex manners, such as through near-neighbor interactions, or the presence of modified bases, abasic sites, or bound molecules.

Calculating the Thermodynamics of Weakly Hydrogen‐Bonded Complexes from Heteronuclear NMR Data: Base‐pairing stabilities of a 5‐methyl(15N2)[O2, O4‐17O2]uridine (= (15N2)[O2,O4‐17O2]ribosylthymine) derivative, and structural implications

Abstract2′,3′‐O‐Isopropylidene‐5‐methyl(15N2)[O2,O4‐17O2]uridine (= 2′,3′‐O‐isopropylidene (15N2)[O2,O4‐17O2]‐ribosylthymine; 1) was analyzed by 15N‐and 17O‐NMR spectroscopy. The 15N and 17O chemical shifts revealed, in the absence and presence of unlabelled 2′,3′‐O‐isopropylideneadenosine (2), the formation of thymine‐thymine and thymine‐adenine base pairs in CHCl3. As expected, cyclic complexes stabilized by two H‐bonds occurred at low temperatures, but at elevated temperatures, the data suggest that open complexes involving only one H‐bond prevailed. The 17O‐NMR data showed the cyclic thymine‐adenine pair in a reverse base pair geometry. The open base pair involved contacts to the urea‐derived carbonyl O‐atom of thymine. The thermodynamics of complex formation of the cyclic and open forms in both homo and hetero pairs were calculated from the temperature and concentration dependence of the 15N‐NMR data using a new method. It involves a fitting procedure onto the experimental isotherms using a theoretically derived function with the standard Gibbs free energy as a parameter to be optimized. ΔH° and ΔS° were derived from a linear regression of ΔG°(T) vs. T. The fitting procedure circumvents the baseline problem and could be automated and used to calculate correct thermodynamics from UV‐monitored melting curves of oligonucleotides. Since titrations are not involved, this dilution method should also be a useful alternative for stability studies of supramolecular complexes in H2O and in organic solvents.

Energetics of base pair opening in a DNA dodecamer containing an A3T3 tract.

Nuclear magnetic resonance spectroscopy has been used to characterize the kinetics and energetics of opening of base pairs in the DNA dodecamer [d(CGCAAATTTGCG)]2. The dodecamer contains an A3T3 tract that induces intrinsic curvature of the helix axis. Previous studies from this and other laboratories have shown that the kinetics of base pair opening in AnTn tracts is unique: the opening rates of the A.T base pairs in the interior of the tract are much lower than that of the A.T base pair at the 5'-end of the tract. In the present work, we have investigated the energetics of the pathways for opening of the A.T base pairs in the A3T3 tract. The energetic parameters of the activated state(s) are obtained from the temperature dependence of the opening rate constants. The lower opening rates for the A.T base pairs situated in the interior of the tract are shown to originate from higher activation enthalpies which are compensated, in part, by increases in the activation entropies. We have also obtained an energetic characterization of the open state(s) of the A.T base pairs in the dodecamer by measuring the equilibrium constants for base pair opening and their temperature dependence. The results suggest that the transitions from closed to open state(s) in the A.T base pairs of the A3T3 tract are energetically similar.