Effect of Base Stacking on the Relative Thermodynamic Stability of Oligonucleotide Complexes: A Spectroscopic Study

Abstract

Three-strand oligonucleotide complexes are employed to assess the effect of base stacking and base pair mismatch on the relative thermodynamic stabilities of oligonucleotide duplexes. The melting behavior of three-strand oligonucleotide complexes incorporating nicks and gaps as well as internal single base mismatches is monitored using temperature-dependent optical absorption spectroscopy. A sequential three-state equilibrium model is used to analyze the measured melting profiles and evaluate thermodynamic parameters associated with dissociation of the complexes. The free-energy of stabilization of a nick complex compared to a gap complex due to base stacking is determined to be ?1.9 kcal/mol. The influence of a mispaired base in these systems is shown to destabilize a nick complex by 3.1 kcal/mol and a gap complex by 2.8 kcal/mol, respectively.     

Thermodynamic analysis of stacking hybridization of oligonucleotides with DNA template.

Contiguous stacking hybridization of oligodeoxyribonucleotides with DNA as template was investigated using three types of complexes: oligonucleotide contiguously stacked with the stem of the preformed minihairpin (complexes I), oligonucleotide tandems containing two (complexes II) or three (complexes III) short oligomers with a common DNA template. Enthalpy Delta H degrees and entropy Delta S degrees of the coaxial stacking of adjacent duplexes were determined for GC/G*pC, GT/A*pC, AC/G*pT, AT/A*pT, CT/A*pG, AG/C*pT, AA/T*pT and TT/A*pA nicked (*) dinucleotide base pairs. The maximal efficiency of co-operative interaction was found for the GC/G*pC interface (Delta G degrees(NN/N*pN)=-2.7 kcal/mol) and the minimal one for the AA/T*pT interface (Delta G degrees(NN/N*pN)=-1.2 kcal/mol) at 37 degrees C. As a whole, the efficiency of the base pairs interaction Delta G degrees(NN/N*pN) in the nick is not lower than that within the intact DNA helix (Delta G degrees(NN/NN)).These observed Delta G degrees(NN/N*pN) values are proposed may include the effect of the partial removal of fraying at the adjacent helix ends additionally to the effect of the direct stacking of the terminal base pairs in the duplex junction (Delta G degrees(NN/NN). The thermodynamic parameters have been found to describe adequately the formation of all tandem complexes of the II and III types with oligonucleotides of various length and hybridization properties. The performed thermodynamic analysis reveals features of stacking oligonucleotide hybridization which allow one to predict the temperature dependence of association of oligonucleotides and the DNA template within tandem complexes as well as to determine optimal concentration for formation of these complexes characterized by high co-operativity level.     

The thermodynamic advantage of DNA oligonucleotide 'stacking hybridization' reactions: energetics of a DNA nick.

'Stacking hybridization reactions' wherein two or more short DNA oligomers hybridize in a contiguous tandem orientation onto a longer complementary DNA single strand have been employed to enhance a variety of analytical oligonucleotide hybridization schemes. If the short oligomers anneal in perfect head-to-tail register the resulting duplex contains a nick at every boundary between hybridized oligomers. Alternatively, if the short oligomers do not hybridize precisely in register, i.e. single strand regions on the longer strand are left unbound, gaps are formed between regions where short oligomers bind. The resulting gapped DNA duplexes are considerably less stable than their nicked duplex analogs. Formation of base pair stacking interactions between neighboring oligomers at the nicks that do not occur in gapped duplexes has been proposed as the source of the observed added stability. However, quantitative evidence supporting this hypothesis for DNA has not been reported. Until now, a direct comparison of the thermodynamics of DNA nicks versus DNA gaps has not been performed. In this communication we report such a comparison. Analysis of optical melting experiments in a well defined molecular context enabled quantitative evaluations of the relative thermodynamic difference between nicked and gapped DNA duplexes. Results of the analysis reveal that a nick may be energetically favored over a gap by at least 1.4 kcal/mol and perhaps as much as 2.4 kcal/mol. The presence of a 5'phosphate at a nick or gap fails to significantly affect their stabilities.     

Synthesis and hybridization properties of the conjugates of oligonucleotides and stabilization agents. Part 3

New compounds having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5'-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleotide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response. The T(m) data and thermodynamic parameters for complex formation confirmed the ability of chromone (gamma-pyrone) derivatives to stabilize strongly the 7-mer/8-mer complementary complex. Moreover, benzochromone (naphthopyrane) and, surprisingly, tetrahydropyrimidinethanone derivatives showed the capacity of stabilizing this 7-mer/8-mer complementary complex. The effect of all these compounds on the stability of the oligonucleotide complexes (DeltaDeltaG at 37 degrees C ranged from -1.2 to -2.0 kcal/mol) was shown to be comparable to the effect of one nucleotide base pair and similar to the effect (DeltaDeltaG at 37 degrees C ranged from -1.5 to -2.0 kcal/mol) found for acridine-oligonucleotide conjugates, which served as a reference in this study.     

Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.     

Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'.

The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which T m values differed by 5 degrees C and Delta G 25 degrees C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5 degrees C and -Delta G 25 degrees C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4 degrees C when paired with G to 62.3 degrees C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10-20 degrees C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average Delta G = -4.8 kcal/mol) to the sequences containing an abasic site (average Delta G = -4.7 kcal/mol).     

Calorimetric unfolding of the bimolecular and i-motif complexes of the human telomere complementary strand, d(C(3)TA(2))(4)

A combination of spectroscopic and calorimetric techniques is used to determine the unfolding thermodynamics of the complexes formed by the complementary sequence of the human telomere, d(C(3)TA(2))(4), in the pH range of 4.2 to 6. Calorimetric melting curves show biphasic transitions; both transitions are shifted to higher temperatures as the pH is decreased, indicative of cytosine protonation, which favors the formation of C*C(+) base pairs. Furthermore, the transition temperature, T(M), of the lower transition depends on strand concentration, while the T(M) of the higher transition is independent of strand concentration, indicating the following sequential melting: bimolecular complex(s)-->intramolecular complex-->random coil. The thermodynamic profiles for the formation of each complex, bimolecular and i-motif reveals small favorable free energy terms resulting from favorable enthalpy-unfavorable entropy compensations, uptake of protons, marginal uptake of counterions (i-motif) and marginal release of water molecules (i-motif). Furthermore, an enthalpy of 3.2 kcal/mol (bimolecular complex) and 5.0 kcal/mol (i-motif) is estimated for a single C*C(+)/C*C(+) base-pair stack.     

The Influence of Nearest Neighbours on the Efficiency of Coaxial Stacking at Contiguous Stacking Hybridization of Oligodeoxyribonucleotides

Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV‐melting technique. It was shown that the free‐energy of the coaxial stacking interaction (ΔG°_ST at 37°C, 1 M NaCl, pH 7.4) at the complementary interface XA^*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA^*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA^*pTPu/PyATPu interface. A 5′‐phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A^*T/AT coaxial stacking varied from (? 0.97 kcal/mol) for unphosphorylated TA^*TA/TATA interface to three‐fold higher value (? 2.78 kcal/mol) for GA^*pTT/AATC interface.     

Cooperative nonenzymic base recognition--a study of interaction of oligoguanylic acid with poly C

The interaction between poly C and (Gp)_nG(n = 1,2) in dilute solution was investigated spectrophotometrically in 0.1M phosphate buffer pH 7.2 under conditions unfavorable for the formation of self-associated complexes of oligoguanylic acids. Two isosbestic points were observed when poly C was titrated gradually with GpGpG, one at 232–233 mμ(in the range of 0–33% poly C) and one around 238 mμ (in the range of 50–100% poly C). The melting temperature (T_m) of the 1:1 poly C: (Gp)nG complexes (n = 1,2) of varying concentration were determined. The equilibrium properties of the 1:1 complexes can be described by two interaction parameters, namely, (i) cooperative stacking interaction between the first nearest neighbor of the adsorbed oligomer, and (ii) intrinsic association constant of the adsorbed oligomer with its polymeric site, since the cooperative helix–coil transition particularly in the smaller oligonucleotide can be described by an “all or none” model. Based on such a model the enthalpy of stacking inteaction-dependent T_m values yielded directly the sum of the enthalpy of stacking interaction and of basepairing (which is dependent on the chain length of the oligomer) and the value of S, the stability constant of a G–C pair within a helix. The enthalpy of formation of G–C pair is then calculated as ?6.3 kcal/base pair either from the chain length dependent enthalpy term or from the temperature coefficient of S values. From the S value and the association constant of 1:1 GpGpGpC:GpCpCpC complex, other thermodynamic parameters such as nucleation parameter (β) and free energy of stacking interaction can be obtained.     

Thermodynamic contribution and nearest-neighbor parameters of pseudouridine-adenosine base pairs in oligoribonucleotides

Pseudouridine (Ψ) is the most common noncanonical nucleotide present in naturally occurring RNA and serves a variety of roles in the cell, typically appearing where structural stability is crucial to function. Ψ residues are isomerized from native uridine residues by a class of highly conserved enzymes known as pseudouridine synthases. In order to quantify the thermodynamic impact of pseudouridylation on U-A base pairs, 24 oligoribonucleotides, 16 internal and eight terminal Ψ-A oligoribonucleotides, were thermodynamically characterized via optical melting experiments. The thermodynamic parameters derived from two-state fits were used to generate linearly independent parameters for use in secondary structure prediction algorithms using the nearest-neighbor model. On average, internally pseudouridylated duplexes were 1.7 kcal/mol more stable than their U-A counterparts, and terminally pseudouridylated duplexes were 1.0 kcal/mol more stable than their U-A equivalents. Due to the fact that Ψ-A pairs maintain the same Watson-Crick hydrogen bonding capabilities as the parent U-A pair in A-form RNA, the difference in stability due to pseudouridylation was attributed to two possible sources: the novel hydrogen bonding capabilities of the newly relocated imino group as well as the novel stacking interactions afforded by the electronic configuration of the Ψ residue. The newly derived nearest-neighbor parameters for Ψ-A base pairs may be used in conjunction with other nearest-neighbor parameters for accurately predicting the most likely secondary structure of A-form RNA containing Ψ-A base pairs.     

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.     

The influence of nearest neighbours on the efficiency of coaxial stacking at contiguous stacking hybridization of oligodeoxyribonucleotides

Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV-melting technique. It was shown that the free-energy of the coaxial stacking interaction (deltaG degrees ST at 37 degrees C, 1 M NaCl, pH 7.4) at the complementary interface XA*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA*pTPu/PyATPu interface. A 5'-phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A*T/AT coaxial stacking varied from (- 0.97 kcal/mol) for unphosphorylated TA*TA/TATA interface to three-fold higher value (- 2.78 kcal/mol) for GA*pTT/AATC interface.     

Solution structure of the aminoacyl-capped oligodeoxyribonucleotide duplex (W-TGCGCAC)(2).

Reported here is the solution structure of the aminoacyl-DNA duplex (W-TGCGCAC)(2). This duplex forms a continuously pi-stacked helix consisting of both nucleobases and amino acid side chains. According to NMR and UV analyses, the duplex melts in a cooperative transition and with 1.3-1.8% greater hyperchromicity than the control duplex (TGCGCAC)(2). A van't Hoff analysis of UV melting points at different concentrations shows that the two tryptophan residues contribute 4.8 kcal/mol to the DeltaH degrees of complex formation at 10 mM salt concentration and less than 1 kcal/mol at 150 mM salt. The entropic cost for duplex association in the presence of the amino acid residues is 13 cal/molK greater than that for the control at 10 mM salt concentration, and 3 cal/molK lower than that of the control at 0.15 ionic strength. The conformation of W-TGCGCAC in duplex form, determined via restrained torsion angle molecular dynamics, shows an undisturbed B-form DNA duplex with dangling 3'-termini. The tryptophanyl residue at the 5'-terminus packs tightly against T2 and the proximal part of adenine, without engaging in hydrogen bonding. While not providing strong enthalpic net stabilization of the duplex, the tryptophan "cap" on the duplex does seem to reduce the fraying at the termini, indicating a subtle balance of entropic and enthalpic factors contributing to the molecular dynamics. The structure also shows that, at least in the present sequence context, stacking on the terminal base pair is more favorable than intercalation, probably because the enthalpic cost associated with breaking up the stacking between DNA base pairs cannot be paid for by favorable pi-stacking interactions with the indole ring of tryptophan. These results are of importance for understanding stacking interactions in protein-DNA complexes, particularly those in enzyme-substrate complexes involving exposed nucleobases.     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

A test of the model to predict unusually stable RNA hairpin loop stability

To investigate the accuracy of a model [Giese et al., 1998, Biochemistry37:1094-1100 and Mathews et al., 1999, JMol Biol 288:911-940] that predicts the stability of RNA hairpin loops, optical melting studies were conducted on sets of hairpins previously determined to have unusually stable thermodynamic parameters. Included were the tetraloops GNRA and UNCG (where N is any nucleotide and R is a purine), hexaloops with UU first mismatches, and the hairpin loop of the iron responsive element, CAGUGC. The experimental values for the GNRA loops are in excellent agreement (deltaG degrees 37 within 0.2 kcal/mol and melting temperature (TM) within 4 degrees C) with the values predicted by the model. When the UNCG hairpin loops are treated as tetraloops, and a bonus of 0.8 kcal/mol included in the prediction to account for the extra stable first mismatch (UG), the measured and predicted values are also in good agreement (deltaG degrees 37 within 0.7 kcal/mol and TM within 3 degrees C). Six hairpins with unusually stable UU first mismatches also gave good agreement with the predictions (deltaG degrees 37 within 0.5 kcal/mol and TM within 8 degrees C), except for hairpins closed by wobble base pairs. For these hairpins, exclusion of the additional stabilization term for UU first mismatches improved the prediction (AG degrees 37 within 0.1 kcal/mol and TM within 3 degrees C). Hairpins with the iron-responsive element loop were not predicted well by the model, as measured deltaG degrees 37 values were at least 1 kcal/mol greater than predicted.     

Substitution of an essential adenine in the U1A-RNA complex with a non-polar isostere

The RNA recognition motif (RRM) binds to single-stranded RNA target sites of diverse sequences and structures. A conserved mode of base recognition by the RRM involves the simultaneous formation of a network of hydrogen bonds with the base functional groups and a stacking interaction between the base and a highly conserved aromatic amino acid. We have investigated the energetic contribution of the functional groups involved in the recognition of an essential adenine, A6, in stem–loop 2 of U1 snRNA by the N-terminal RRM of the U1A protein. Previously, we found that elimination of individual hydrogen bond donors and acceptors on A6 destabilized the complex by 0.8–1.9 kcal/mol, while mutation of the aromatic amino acid (Phe56) that stacks with A6 to Ala destabilized the complex by 5.5 kcal/mol. Here we continue to probe the contribution of A6 to complex stability through mutation of both the RNA and protein. We have removed two hydrogen-bonding functional groups by introducing a U1A mutation, Ser91Ala, and replacing A6 with tubercidin, purine, or 1-deazaadenine. We find that the complex is destabilized an additional 1.2–2.6 kcal/mol by the elimination of the second hydrogen bond donor or acceptor. Surprisingly, deletion of all of the functional groups involved in hydrogen bonds with the U1A protein by substituting adenine with 4-methylindole reduced the binding free energy by only 2.0 kcal/mol. Experiments with U1A proteins containing mutations of Phe56 suggested that improved stacking interactions due to the greater hydrophobicity of 4-methylindole than adenine may be partly responsible for the small destabilization of the complex upon substitution of 4-methylindole for A6. The data imply that hydrophobic interactions can compensate energetically for the disruption of the complex hydrogen-bonding network between nucleotide and protein.     

Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates

Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in six 22-base pair deoxyoligonucleotides.

Effects of different end sequences on stability, circular dichroism spectra (CD), and enzyme binding properties were investigated for six 22-base pair, non-self-complementary duplex DNA oligomers. The center sequences of these deoxyoligonucleotides have 8-14 base pairs in common and are flanked on both sides by sequences differing in context and A-T content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming a two-state melting transition. Melting free energies (20 degrees C) of the six DNAs evaluated from DSC experiments ranged from -18.7 to -32.7 kcal/mol. van't Hoff estimates of the free energies ranged from -18.5 to -48.0 kcal/mol. With either method, the trends in free energy as a function of sequence were identical. Equilibrium binding by BamHI restriction endonuclease to the 22-base pair DNAs was also investigated. The central eight base pairs of all six molecules, 5'-A-GGATCC-A-3', contained a BamHI recognition sequence bounded by A-T base pairs. Magnesium free binding assays were performed by titering BamHI against a constant concentration of each of the deoxyoligonucleotide substrates and analyzing reaction products by gel retardation. Binding isotherms of the total amount of bound DNA versus protein concentration were constructed which provided semiquantitative estimates of the equilibrium dissociation constants for dissociation of BamHI from the six DNA oligomers. Dissociation constants ranged from 0.5 x 10(-)(9) to 12.0 x 10(-)(9) M with corresponding binding free energies of -12.5 to -10.6 (+/-0. 1) kcal/mol. An inverse relationship is found when binding and stability are compared.