Base-pair Stacking Interactions Research Articles

Single-molecule dissection of stacking forces in DNA.

We directly measured at the single-molecule level the forces and lifetimes of DNA base-pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual-beam optical tweezers with DNA origami components to allow positioning of blunt-end DNA helices so that the weak stacking force could be isolated. Base-pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free-energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from -0.8 to -3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA-based nanoscale devices according to user specifications.

Loop contributions to the folding thermodynamics of DNA straight hairpin loops and pseudoknots.

Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the effect of the loop length on the unfolding thermodynamics of three sets of DNA stem-loop motifs with the following sequences: (a) d(GCGCTnGCGC), where n = 3, 5, 7, 9; (b) d(CGCGCGT4GAAATTCGCGCGTnAATTTC), where n = 4, 6, and 8; and (c) d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where n = 5, 7, 9, and 11. The increase in loop length of the first set of hairpins yielded decreasing TM's and constant unfolding enthalpies, resulting in an entropy driven decrease in the stability of the hairpin (ΔG° = -7.5 to -6.1 kcal/mol). In the second set, the increase in the length of the loops yielded similar TM's and slight increases in the unfolding enthalpies. This translated into more stable pseudoknots with an increasing ΔG° from -13.2 to -17.1 kcal/mol. This effect can be rationalized in terms of the increased flexibility of the pseudoknot with larger loops optimizing base-pair stacking interactions. In the last set of molecules, the increase in the length of one of the loops yielded an increase in the TM's and larger increases in the enthalpies, which stabilize the pseudoknot significantly increasing the ΔG° from -8.5 to -16.6 kcal/mol. In this set, the thymine loop is complementary to the stem of A·T base pairs and the longer loops are able to form T*A·T base triplets due to the partial folding of the thymine loop into the ceiling of the major groove of the duplex, thus yielding a net formation of 1-3 T*AT/T*AT base-triplet stacks at the middle of its stem, depending on the loop length.

A new entropy model for RNA: part II. Persistence-related entropic contributions to RNA secondary structure free energy calculations

In previous work, we have shown that the entropy of a folded RNA molecule can be divided into local and global contributions using the cross-linking entropy (CLE) model, where, in the case of RNA, the cross- links are the base-pair stacking interactions. The local contribution to the CLE is revealed in the Kuhn length (a measure of the stiffness of the RNA). The Kuhn length acts as a scaling parameter. When the size of the system is rescaled, the relationship between local and global free energy must be renormalized to reflect this rescaling. In this renormalization process, the Kuhn length increases, the local entropy also increases due to freezing out of the local conformational degrees of freedom. At the same time, as the number of degrees of freedom decrease, there is a significant reduction in the global entropy. Here we present a method, based on the concepts of renormalization theory, to quantitatively estimate the size of the contribution from the local entropy as a function of the Kuhn length. The local entropy correction is used to predict the current empirically derived constant in the Jacobson-Stockmayer equation. The variation in the Kuhn length is shown to be largely influenced by the length of the double-stranded RNA stems formed in the secondary structure of folded RNA. This result is used to test the resulting entropy under a variable Kuhn length in stem-loop structures. Comparisons between a variable Kuhn length and a static Kuhn length on a short stem-loop of RNA are also examined. The model is quite general and is also directly applicable to protein structure and folding problems.

A new entropy model for RNA: part II. Persistence-related entropic contributions to RNA secondary structure free energy calculations

In previous work, we have shown that the entropy of a folded RNA molecule can be divided into local and global contributions using the cross-linking entropy (CLE) model, where, in the case of RNA, the cross- links are the base-pair stacking interactions. The local contribution to the CLE is revealed in the Kuhn length (a measure of the stiffness of the RNA). The Kuhn length acts as a scaling parameter. When the size of the system is rescaled, the relationship between local and global free energy must be renormalized to reflect this rescaling. In this renormalization process, the Kuhn length increases, the local entropy also increases due to freezing out of the local conformational degrees of freedom. At the same time, as the number of degrees of freedom decrease, there is a significant reduction in the global entropy. Here we present a method, based on the concepts of renormalization theory, to quantitatively estimate the size of the contribution from the local entropy as a function of the Kuhn length. The local entropy correction is used to predict the current empirically derived constant in the Jacobson-Stockmayer equation. The variation in the Kuhn length is shown to be largely influenced by the length of the double-stranded RNA stems formed in the secondary structure of folded RNA. This result is used to test the resulting entropy under a variable Kuhn length in stem-loop structures. Comparisons between a variable Kuhn length and a static Kuhn length on a short stem-loop of RNA are also examined. The model is quite general and is also directly applicable to protein structure and folding problems.

DNA Base Dimers Are Stabilized by Hydrogen-Bonding Interactions Including Non-Watson–Crick Pairing Near Graphite Surfaces

Single- and double-stranded DNA are increasingly being paired with surfaces and nanoparticles for numerous applications, such as sensing, imaging, and drug delivery. Unlike the majority of DNA structures in bulk that are stabilized by canonical Watson-Crick pairing between Ade-Thy and Gua-Cyt, those adsorbed on surfaces are often stabilized by noncanonical base pairing, quartet formation, and base-surface stacking. Not much is known about these kinds of interactions. To build an understanding of the role of non-Watson-Crick pairing on DNA behavior near surfaces, one requires basic information on DNA base pair stacking and hydrogen-bonding interactions. All-atom molecular simulations of DNA bases in two cases--in bulk water and strongly adsorbed on a graphite surface--are conducted to study the relative strengths of stacking and hydrogen bond interactions for each of the 10 possible combinations of base pairs. The key information obtained from these simulations is the free energy as a function of distance between two bases in a pair. We find that stacking interactions exert the dominant influence on the stability of DNA base pairs in bulk water as expected. The strength of stability for these stacking interactions is found to decrease in the order Gua-Gua > Ade-Gua > Ade-Ade > Gua-Thy > Gua-Cyt > Ade-Thy > Ade-Cyt > Thy-Thy > Cyt-Thy > Cyt-Cyt. On the other hand, mutual interactions of surface-adsorbed base pairs are stabilized mostly by hydrogen-bonding interactions in the order Gua-Cyt > Ade-Gua > Ade-Thy > Ade-Ade > Cyt-Thy > Gua-Gua > Cyt-Cyt > Ade-Cyt > Thy-Thy > Gua-Thy. Interestingly, several non-Watson-Crick base pairings, which are commonly ignored, have similar stabilization free energies due to interbase hydrogen bonding as Watson-Crick pairs. This clearly highlights the importance of non-Watson-Crick base pairing in the development of secondary structures of oligonucleotides near surfaces.

New homocamptothecins: Synthesis, antitumor activity, and molecular modeling

Homocamptothecins (hCPTs) represent a class of new emerging antitumor agents, which contains a seven-membered β-hydroxylactone in place of the conventional six-membered α-hydroxylactone ring (E ring) of camptothecins. Some novel 7-substituted hCPTs were designed and synthesized based on a newly developed synthetic route which couples ring A with ring C, E and D. Most of the synthesized compounds exhibit very high cytotoxic activity on tumor cell line A549. Some compounds, such as 9b, 9l, and 9y, show broad in vitro antitumor spectrum and are more potent than topotecan. Three-dimensional quantitative structure–activity relationship (3D-QSAR) methods, CoMFA and CoMSIA, were applied to explain the structure–activity relationship (SAR) of the synthesized compounds. Furthermore, molecular docking was used to clarify the binding mode of the synthesized compounds to human DNA topoisomerase I. The important hydrophobic, base-pair stacking, and hydrogen-bonding interactions were observed between the hCPT derivatives and their receptor. The results from molecular modeling will guide the design of novel hCPTs with higher antitumor activity.

Intra‐ and intermolecular interactions between cyclic‐AMP receptor protein and DNA: Ab initio fragment molecular orbital study

The ab initio fragment molecular orbital (FMO) calculations were performed for the cAMP receptor protein (CRP) complexed with a cAMP and DNA duplex to elucidate their sequence-specific binding and the stability of the DNA duplex, as determined by analysis of their inter- and intramolecular interactions. Calculations were performed with the AMBER94 force field and at the HF and MP2 levels with several basis sets. The interfragment interaction energies (IFIEs) were analyzed for interactions of CRP-cAMP with each base pair, DNA duplex with each amino acid residue, and each base pair with each residue. In addition, base-base interactions were analyzed including hydrogen bonding and stacking of DNA. In the interaction between DNA and CRP-cAMP, there was a significant charge transfer (CT) from the DNA to CRP, and this CT interaction played an important role as well as the electrostatic interactions. It is necessary to apply a quantum mechanical approach beyond the "classical" force-field approach to describe the sequence specificity. In the DNA intramolecular interaction, the dispersion interactions dominated the stabilization of the base-pair stacking interactions. Strong, attractive 1,2-stacking interactions and weak, repulsive 1,3-stacking interactions were observed. Comparison of the intramolecular interactions of free and complexed DNA revealed that the base-pairing interactions were stronger, and the stacking interactions were weaker, in the complexed structure. Therefore, the DNA duplex stability appears to change due to both the electrostatic and the CT interactions that take place under conditions of DNA-CRP binding.

THE ELASTIC THEORY OF SINGLE-MOLECULE DNA

Our recent work on the elastic responses of double- (ds) and single-stranded (ss) DNA at external force fields is reviewed. By constructing as elastic model of dsDNA in which the base-pair stacking interaction is included, we demonstrate that dsDNA entropic elasticity, cooperative extensibility, and supercoiling property can all be understood from a unified viewpoint. The base-pair stacking interaction is also found to determine the cooperativity of the stretch-induced hairpin-coil transition is ssDNA.

THE ELASTIC THEORY OF A SINGLE DNA MOLECULE

We review our recent efforts in understanding the elastic properties of double-stranded (ds) and single-stranded (ss) DNA macromolecules. A simple geometric model of dsDNA was constructed and solved by path integral methods. The good agreement with experiments on dsDNA's entropic elasticity, cooperative extensibility, and supercoiling property suggested that the short-ranged base-pair stacking interaction is crucial for the stability and the high deformability of dsDNA. For ssDNA at high ionic conditions, base-pairing and base-pair stacking interactions cause the polymer to fold into compact hairpin configurations. The force-induced hairpin-coil transition was studied with the generating function method. In accordance with experiment, this transition was found to be highly cooperative when the average stacking potential is higher than some critical level. At low ionic conditions, the electrostatic repulsive interaction along the ssDNA becomes dominant, and ssDNA can be regarded as model polyelectrolytes. Our MC simulation results suggested an exponential relation between external force and the total extension. This prediction was confirmed experimentally.

Stretching Single-Stranded DNA: Interplay of Electrostatic, Base-Pairing, and Base-Pair Stacking Interactions

Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.

Pulling hairpinned polynucleotide chains: Does base-pair stacking interaction matter?

Force-induced structural transitions both in relatively random and in designed single-stranded DNA (ssDNA) chains are studied theoretically. At high salt conditions, ssDNA forms compacted hairpin patterns stabilized by base pairing and base-pair stacking interactions, and a threshold external force is needed to pull the hairpinned structure into a random coiled one. The base-pair stacking interaction in the ssDNA chain makes this hairpin-coil conversion a discontinuous (first-order) phase transition process characterized by a force plateau in the force-extension curve, while lowering this potential below some critical level turns this transition into continuous (second-order) type, no matter how strong the base-pairing interaction is. The phase diagram (including hairpin-I, -II, and random coil) is discussed as a function of stacking potential and external force. These results are in quantitative agreement with recent experimental observations of different ssDNA sequences, and they reveal the necessity to consider the base-pair stacking interactions in order to understand the structural formation of RNA, a polymer designed by nature itself. The theoretical method used may be extended to study the long-range interaction along double-stranded DNA caused by the topological constraint of fixed linking number.

Effect of Neighboring Bases on Base-Pair Stacking Orientation: A Molecular Dynamics Study

It is generally believed that base-pair stacking interaction in DNA double helix is one of the strongest interactions that governs sequence directed structural variability. However, X-ray crystal structures of some base-paired doublet sequences have been seen to adopt different structures when flanked by different base-pairs. DNA crystal database, however, is still too small to make good statistical inference about effect of such flanking residues. Influence of neighboring residue on the local helical geometry of a base-paired doublet in B-DNA has been investigated here using molecular dynamics simulation. We have generated ensembles of structures for d(CA).d(TG) and d(AA).d(TT) base-paired doublets located at the centers of d(CGCGCAAAGCG).d(CGCTTTGCGCG) and d(CGC-GAAAACGCG). d(CGCGTTTTCGCG) sequences along with their analogs by varying the bases either at 5′- or 3′- position to the central doublet. Comparison of base paired doublet parameters for the ensembles of structures show that stacking geometry of d(CA).d(TG) doublet depends on some of the flanking base-pairs. On the other hand d(AA).d(TT) doublet remains nearly unperturbed when the flanking residues are altered.

A model for the binding of E. coli single-strand binding protein to supercoiled DNA

A model is proposed for the binding of E. coli single strand binding protein (SSB) to supercoiled DNA. The basic tetrameric binding units of SSB are assumed to bind in pairs to the complementary single strands of a locally melted region. The cooperativity of the binding includes contributions from both protein-protein and base-pair stacking interactions. Each bound SSB tetramer is assumed to unwind l = 34 bp, which implies an unwinding angle of 3.27 turns. The resulting loss of superhelical strain is the essential driving force for binding SSB to supercoiled DNAs. All molecular parameters entering into the theory are estimated from available data, except for the composite binding constant ( K a), which is adjusted to best-fit the theory to the fluorescence quenching (FQ) and diffusion coefficient ( D 0) data of Langowski et al. Very good fits are obtained with optimum values of K a that are consistent with estimates from other data. This binding model predicts several noteworthy features. (1) SSB binds essentially always in a single contiguous stack on a supercoiled plasmid, and relative fluctuations in stack length are quite small, in agreement with results of electron microscopy studies. (2) The progressive loss of superhelical strain with increasing bound ligand decreases the affinity of the DNA for SSB. This anti-cooperativity offsets the cooperativity of the binding and causes apparent saturation of the binding at rather low binding ratios. Consequently, over the limited span of the measurements, the FQ data can also be satisfactorily fitted by a non-cooperative model comprising a small number of independent sites. (3) When SSB binds to a population of different topoisomers, the distribution of linking differences of the resulting complexes is extremely narrow. Thus, SSB acts to level any differences in superhelical strain in a population of topoisomers. Finally, the effects of restricting binding to a region comprising only part of the plasmid are assessed.

DNA Polymorphism and Local Variation in Base-Pair Orientation: A Theoretical Rationale

Basepair stacking calculations have been carried out to understand the conformational polymorphism of DNA and its sequence dependence. The recently developed self-consistent parameter set, which is specially suitable for describing irregular DNA structures, has been used to describe the geometry of a basepair doublet. While for basepairs without any propeller, the favourable stacking patterns do not appear to have very strong features, much more noticeable sequence dependent stacking patterns emerge once a propeller is applied to the basepairs. The absolute minima for most sequences occurs for a doublet geometry close to the B-DNA fibre models. Hence in the B-DNA region, no strong sequence dependent features are found, but the range of doublet geometries observed in the crystal structures generally lie within the low energy contours, obtained from stacking energy calculations. The doublet geometry corresponding to the A-DNA fibre model is not energetically favourable for the purine-pyrimidine sequences, which prefer small roll angle values when the slide has a large negative value as in A-DNA. However positive roll with large negative slide is allowed for GG, GA, AG and the pyrimidine-purine steps. This is consistent with the observed geometries of various steps in A-DNA crystals. Thus the general features of the basepair doublets predicted from these theoretical studies agree very well with the results from crystal structure analysis. However, since most sequences show an overall preference for B-type doublet geometry, the B----A transition for random sequence DNA cannot be explained on the basis of basepair stacking interactions.

Local Variability and Base Sequence Effects in DNA Crystal Structures

The importance and usefulness of local doublet parameters in understanding sequence dependent effects has been described for A- and B-DNA oligonucleotide crystal structures. Each of the two sets of local parameters described by us in the NUPARM algorithm, namely the local doublet parameters, calculated with reference to the mean z-axis, and the local helical parameters, calculated with reference to the local helix axis, is sufficient to describe the oligonucleotide structures, with the local helical parameters giving a slightly magnified picture of the variations in the structures. The values of local doublet parameters calculated by NUPARM algorithm are similar to those calculated by NEWHELIX90 program, only if the oligonucleotide fragment is not too distorted. The mean values obtained using all the available data for B-DNA crystals are not significantly different from those obtained when a limited data set is used, consisting only of structures with a data resolution of better than 2.4 A and without any bound drug molecule. Thus the variation observed in the oligonucleotide crystals appears to be independent of the quality of their crystallinity. No strong correlation is seen between any pair of local doublet parameters but the local helical parameters are interrelated by geometric relationships. An interesting feature that emerges from this analysis is that the local rise along the z-axis is highly correlated with the difference in the buckle values of the two basepairs in the doublet, as suggested earlier for the dodecamer structures (Bansal and Bhattacharyya, in Structure & Methods: DNA & RNA, Vol. 3 (Eds., R.H. Sarma and M.H. Sarma), pp. 139-153 (1990)). In fact the local rise values become almost constant for both A- and B-forms, if a correction is applied for the buckling of the basepairs. In B-DNA the AA, AT, TA and GA basepair sequences generally have a smaller local rise (3.25 A) compared to the other sequences (3.4 A) and this seems to be an intrinsic feature of basepair stacking interaction and not related to any other local doublet parameter. The roll angles in B-DNA oligonucleotides have small values (less than +/- 8 degrees), while mean local twist varies from 24 degrees to 45 degrees. The CA/TG doublet sequences show two types of preferred geometries, one with positive roll, small positive slide and reduced twist and another with negative roll, large positive slide and increased twist.(ABSTRACT TRUNCATED AT 400 WORDS)

Sequence specific conformation of a DNA decamer containing an adenine tract studied in solution by 1H-NMR spectroscopy

The decanucleotide duplex d(AAAACGTTTT) 2 and a variety of phase-sensitive two-dimensional (2D) NMR experiments have been used to investigate the solution conformation of an adenine-tract and its junction with another DNA sequence. 2D nuclear Overhauser effect data confirm that the oligonucleotide has a general B-type DNA morphology but an array of unusual correlations implies that the adenine tract and the 5′-ApC junction have conformations more compatible with the modified X-ray structures recently reported for DNAs of similar sequence (Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221–226). The pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1′s of the complementary base to the 5′-neighbouring residue indicate that the A-T basepairs are highly propeller twisted and that the minor groove is narrowed, showing its greatest compression at the 3′-end of the tract at the 5′-ApC step. Quantifying spin-coupling interactions within the deoxyribose rings by analysing both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the purines is predominantly C2′-endo, with the pseudorotation phase angle P lying in the range 140–180°. For the pyrimidines, however, there are distortions away from this standard B-type geometry with the data being best described by P values lying in the range 90–130° (i.e., O4′-endo, C1′-exo). The sugar puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of their positions at, or close to, the ends of the duplex. Thus the conformation of the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are different with an abrupt change in sugar puckering occurring at the 5′-ApC (5′-GpT) step. Peculiar chemical shifts values for A4H2, T7CH 3 and sugar C5 H1′, H2′ and H2″, together with a number of interresidue NOEs with unusual intensities, imply that there are also substantial modifications to basepair stacking interactions at this step. Taken as a whole, our data are consistent with the view that the conformational dislocation at the 5′-ApC dinucleotide results from a combination of slide and roll manoeuvres and that the junction between the AAAA and CG sequences is a potential nucleation site for DNA bending.

Sequence-dependent effects in A-DNA double helices

To date, more than ten different A-DNA structures have been analyzed at near atomic resolution by X-ray methods. This structural information enables us for the first time, to begin to discover the rules which govern the conformation of the double helix in the A form. A detailed analysis of the various helical conformations show that local structural changes are induced by base-pair stacking interactions. Variations in stacking geometry are sequence dependent. For pyrimidine-purine sites, the stacking mode is determined by the nature of the base-pair doublet only, whereas for purine-pyrimidine and homopolymer sites the identity of neighboring base pairs is important as well.