The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 degrees C to -10 degrees C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mol bp)(-1). Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4) J K(-1) (mol bp)(-1) and -5.0(+/-0.5) kJ (mol bp)(-1), respectively.     

End-to-end attraction of duplex DNA

Recent experiments [Nakata, M. et al., End-to-end stacking and liquid crystal condensation of 6 to 20 basepair DNA duplexes. Science 2007; 318:1276-1279] have demonstrated spontaneous end-to-end association of short duplex DNA fragments into long rod-like structures. By means of extensive all-atom molecular dynamic simulations, we characterized end-to-end interactions of duplex DNA, quantitatively describing the forces, free energy and kinetics of the end-to-end association process. We found short DNA duplexes to spontaneously aggregate end-to-end when axially aligned in a small volume of monovalent electrolyte. It was observed that electrostatic repulsion of 5'-phosphoryl groups promoted the formation of aggregates in a conformation similar to the B-form DNA double helix. Application of an external force revealed that rupture of the end-to-end assembly occurs by the shearing of the terminal base pairs. The standard binding free energy and the kinetic rates of end-to-end association and dissociation processes were estimated using two complementary methods: umbrella sampling simulations of two DNA fragments and direct observation of the aggregation process in a system containing 458 DNA fragments. We found the end-to-end force to be short range, attractive, hydrophobic and only weakly dependent on the ion concentration. The relation between the stacking free energy and end-to-end attraction is discussed as well as possible roles of the end-to-end interaction in biological and nanotechnological systems.     

2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects.

Metal-ion and sequence dependent changes in the stacking interactions of bases surrounding abasic (AB) sites in 10 different DNA duplexes were examined by incorporating the fluorescent nucleotide probe 2-aminopurine (2-AP), opposite to the site (AB-APopp) or adjacent to the site (AB-APadj) on either strand. A detailed study of the fluorescence emission and excitation spectra of these AB duplexes and their corresponding parent duplexes indicates that AB-APoppis significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-APadjon the AB strand is stacked, but AB-APadjon the opposite strand shows destabilized stacking interactions. The results also indicate that divalent cation binding to the AB duplexes contributes to destabilizaton of the base stacking interactions of AB-APopp, but has little or no effect on the stacking interactions of AB-APadj. Consistent with these results, the fluorescence of AB-APoppis 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-APoppin the presence of 2.5 mM MgCl2, a 3-fold decrease in fluorescence is observed ( K d = 400 +/- 90 nM) indicating that the unstacked 2-APoppbecomes more stacked upon binding. On the basis of these fluorescence studies a model for the local base stacking interactions at these AB sites is proposed.     

Development of dansyl-modified oligonucleotide probes responding to structural changes in a duplex

We have synthesized a nonnucleoside amidite block of dansyl fluorophore to prepare dansyl-modified oligonucleotides (ONTs). The fluorescence intensities of dansyl-ONT specifically increased by the presence of adjacent guanosine residues but, significantly reduced in a dansyl-flipping duplex. These changes were caused by solvatochromism effect due to the number of guanine which is hydrophobic functional group and the external environment of dansyl group. The fluorescence intensities could be plotted as a function of the ONTs concentrations and the increase in the fluorescence was observed to equimolar concentrations of target DNA. This duplex exhibited higher melting temperature relative to the corresponding duplexes containing other base pairs. Similar changes in fluorescence could be detected upon hybridization with complementary RNAs. Thus, the dansyl-modified ONTs provide sequence specific fluorescent probe of DNA and RNA.     

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

The interaction of MvaI restriction endonuclease with 14-membered deoxyribonucleotide duplexes containing modifications within the recognition site (CCA/TGG) has been studied. Substitution of m5dC for the internal dC residue, as well as substitution of fl5dU or rU for dT did not influence the initial rate of hydrolysis (v0) of modified strands, whereas the hydrolysis of unmodified strands was inhibited in some cases. Furthermore, the substitution of a pyrophosphate bond for a scissile phosphodiester bond in one strand completely inhibited digestion in this strand without any decrease of the rate of hydrolysis of the unmodified strand. In contrast to EcoRII endonuclease, which recognizes the same DNA sequence, in the case of MvaI endonuclease substrate recognition is possible in a wide range of conformational, electronic and hydrophobic alterations within the recognition site.     

Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin

The 3,3'-diethylthiacarbocyanine (DiSC(2)(5)) dye is able to aggregate on full matched PNA-DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-beta-cyclodextrin (Succ-beta-CyD) to the solutions containing PNA-DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC(2)(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.     

Computational studies on full-length Ku70 with DNA duplexes: base interactions and a helical path

The Ku70/80 heterodimer is among the first responding proteins to recognize and bind the DNA double strand breaks (DSBs). Once Ku is loaded at the DSB, it works as a scaffold to recruit other repair factors in non-homologous end joining thereby facilitates the following repair processes. In this work, we characterized the detailed interactions and binding free energies between a Ku70 subunit and several DNA duplexes, by using some well-established computational methods. The results reveal that the structure of the protein may suffer certain contractions without the company of Ku80, and may experience large conformational changes in the presence of different DNA duplexes. Notably, we observe the closest interactions between Ku70 and DNA can be easily strengthened to form H-bonds with the bases in the minor groove, which is unexpected. However, this finding is supported by the presence of a similar bond between Ku80 and DNA in the published crystal structure (PDB code 1JEY). We suggest that these interactions are responsible for the observed pausing sites when Ku translocates along DNA and the subtle difference in binding with AT- and GC-rich DNA ends. Additionally, simulations indicate the inner surface of the ring encircling the DNA is not flat, but contains a delicate clamp like structure, which is ideal to grip the two strands of DNA in the minor groove and confine the movement of the duplex in a unique helical path.     

Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of R and S stereochemistry.

Methyl phosphonate oligonucleotides have been used as antisense and antigene agents. Substitution of a methyl group for oxygen in the phosphate ester backbone introduces a new chiral center. Significant differences in physical properties and hybridization abilities are observed between the R(p) and S(p) diastereomers. Chirally pure methylphosphonate deoxyribooligonucleotides were synthesized, and the solution structures of duplexes formed between a single strand heptanucleotide methylphosphonate, d(Cp(Me)Cp(Me)Ap(Me)Ap(Me)Ap(Me)Cp(Me)A), hybridized to a complementary octanucleotide, d(TpGpTpTpTpGpGpC), were studied by NMR spectroscopy. Stereochemistry at the methylphosphonate center for the heptanucleotide was either RpRpRpRpRpRp (R(p) stereoisomer) or RpRpRpSpRpRp (S(p) stereoisomer, although only one of the six methylphosphonate centers has the S(p) stereochemistry). The results show that the methylphosphonate strands in the heteroduplexes exhibit increased dynamics relative to the DNA strand. Substitution of one chiral center from R(p) to S(p) has a profound effect on the hybridization ability of the methylphosphonate strand. Sugars in the phosphodiester strand exhibit C(2)(') endo sugar puckering while the sugars in the methyl phosphonate strand exhibit an intermediate C(4)(') endo puckering. Bases are well stacked on each other throughout the duplex. The hybridization of the methylphosphonate strand does not perturb the structure of the complementary DNA strand in the hetero duplexes. The sugar residue 5' to the S(p) chiral center shows A-form sugar puckering, with a C(3)(')-endo conformation. Minor groove width in the R(p) stereoisomer is considerably wider, particularly at the R(p) vs S(p) site and is attributed to either steric interactions across the minor groove or poorer metal ion coordination within the minor groove.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

Affinity photo-crosslinking study of the DNA base flipping pathway by HhaI methyltransferase.

HhaI DNA methyltransferase flips the inner cytosine in the recognition sequence 5'-GCGC-3' out of the DNA helix and into the catalytic site for methylation. To identify intermediate states on the base-flipping pathway, affinity photo-crosslinking experiments were performed with synthetic dodecamer duplexes containing modified bases 2-thiothymine (2sT) or 4-thiothymine (4sT) at the target base position. Here we show that the DNA strand containing 2sT, but not 4sT, covalently cross-links to the HhaI methyltransferase upon irradiation at 340-360 nm.     

Promoting DNA molecules association by amphiphilic derivatives of 1,3-diazaadamantanes containing hydrophobic side chains

Earlier, a new class of compounds, amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides, has been discovered. Longer hydrophobic side chains in 1,3-diazaadamantanes have been found to promote stronger acceleration of the reaction. In this study, the interaction of two 1,3-diazaadamantane derivatives containing different side chains with DNA was investigated using optical methods. Concentrations of micelle formation by the 1,3-diazaadamantanes, as well as the ranges of concentrations where the compounds/water mixtures exist in the form of true solutions, were determined based on the increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate probe. The affinities of 1,3-diazaadamantanes to DNA were determined with fluorescent intercalator displacement (FID) assay. A significant increase in the hydrodynamic volume of short DNA hairpins in complexes with 1,3-diazaadamantanes was revealed by the estimation of the fluorescence polarization of ethidium bromide probe bound in the hairpins. The intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantanes was confirmed by Förster resonance energy transfer in an equimolar mixture of hairpins fluorescently labeled with Cy-3 or Cy5. In the study, the number of positive charges on 1,3-diazaadamantane derivatives that contain side chains of different lengths was demonstrated to affect their affinity to DNA, while longer hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate DNA strand exchange.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Characterization of solute binding at human serum albumin site II and its geometry using a biochromatographic approach.

Chiral recognition mechanism relationships for binding at site II on human serum albumin (HSA) were investigated using D, L dansyl amino acids. Sodium phosphate salt was used as a solute-HSA interaction modifier. A new model was developed using a biochromatographic approach to describe the variation in the transfer equilibrium constant with the salt concentration, i.e., the nature of the interactions. The solute binding was divided into two salt concentration ranges c. For the low c values, below 0.03 M, the nonstereoselective interactions constituted the preponderant contribution to the variation in the solute binding with the salt concentration. For the high c values, above 0.03 M, the solute binding was governed by the hydrophobic effect and the stereoselective interactions. The different contributions implied in the binding process provided an estimation of both the surface charge density (sigma/F) and the surface area of the site II binding cavity accessible to solvent, which were found to be equal to around 10.10(-7) mol/m(2) and 2 nm(2). As well, the excess of sodium ions excluded by the solute transfer from the surface area of the pocket were about(-0.7) for dansyl norvaline and (-0.8) for dansyl tryptophan.     

Interaction of the MvaI restriction enzyme with synthetic DNA fragments

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.     

Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.     

Recognition of nucleic acid double helices by homopyrimidine 2', 5'-linked RNA.

We have studied the effect of a 2',5'-RNA third strand backbone on the stability of triple helices with a 'pyrimidine motif' targeting the polypurine strand of duplex DNA, duplex RNA and DNA/RNA hybrids. Comparative experiments were run in parallel with DNA and the regioisomeric RNA as third strands adopting the experimental design of Roberts and Crothers. The results reveal that 2',5'-RNA is indeed able to recognize double helical DNA (DD) and DNA (purine):RNA (pyrimidine) hybrids (DR). However, when the duplex purine strand is RNA and the duplex pyrimidine strand is DNA or RNA (i.e. RD or RR), triplex formation is not observed. These results exactly parallel what is observed for DNA third strands. Based on T m data, the affinities of 2',5'-RNA and DNA third strands towards DD and DR duplexes were similar. The RNA third strand formed triplexes with all four hairpins, as previously demonstrated. In analogy to the arabinose and 2'-deoxyribose third strands, the possible C2'- endo pucker of 2',5'-linked riboses together with the lack of an alpha-2'-OH group are believed to be responsible for the selective binding of 2',5'-RNA to DD and DR duplexes, over RR and RD duplexes. These studies indicate that the use of other oligonucleotide analogues will prove extremely useful in dissecting the contributions of backbone and/or sugar puckering to the recognition of nucleic acid duplexes.