Identification of binding mechanisms in single molecule-DNA complexes

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.     

Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.     

Interaction of Oxazole Yellow Dyes with DNA Studied with Hybrid Optical Tweezers and Fluorescence Microscopy

We have integrated single molecule fluorescence microscopy imaging into an optical tweezers set-up and studied the force extension behavior of individual DNA molecules in the presence of various YOYO-1 and YO-PRO-1 concentrations. The fluorescence modality was used to record fluorescent images during the stretching and relaxation cycle. Force extension curves recorded in the presence of either dye did not show the overstretching transition that is characteristic for bare DNA. Using the modified wormlike chain model to curve-fit the force extension data revealed a contour length increase of 6% and 30%, respectively, in the presence of YO-PRO-1 and YOYO-1 at 100 nM. The fluorescence images recorded simultaneously showed that the number of bound dye molecules increased as the DNA molecule was stretched and decreased again as the force on the complex was lowered. The binding constants and binding site sizes for YO-PRO-1 and YOYO-1 were determined as a function of the force. The rate of YO-PRO-1 binding and unbinding was found to be 2 orders of magnitude larger than that for YOYO-1. A kinetic model is proposed to explain this observation.     

Flexible docking of DNA fragments and actinocin derivatives

We have applied molecular docking methods to systems containing nucleic acids as targets and biologically active substances as ligands. The complexes of DNA fragments and actinocin derivatives with different lengths of aminoalkyl side chains were obtained by molecular docking. It was observed that actinocin derivatives could form energetically favourable complexes with DNA both as intercalators and minor groove binders. It was shown that small changes in the binding energy (～1?kcal/mol) could result in complexes with substantially different structure. The complexes of actinocin derivatives and DNA fragments were stabilized by hydrogen bonding upon intercalation and minor groove binding. It was found that the change of solvent-accessible surface area upon binding of the actinocin derivative to DNA linear increased with the growth of methylene groups' number in ligand side chains. The solvation energy change upon binding of actinocin derivatives to DNA calculated by the WSAS method was favourable in the case of small uncharged ligands and unfavourable for positively charged ligands.     

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.     

On the importance of van der Waals interaction in the groove binding of DNA with ligands: restrained molecular dynamics study

Comparison of interaction energy between an oligonucleotide and a DNA-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics. Distortion in DNA was found for the major groove mode whereas less significant changes for both ligand and DNA were detected for the minor groove binding after molecular dynamics simulation. The conformation of the ligand obtained from the major groove mode resembles that computed with the ligand soaked in water. The van der Waals contact energy was found to be as significant as electrostatic energy and more important for difference in binding energy between these two binding modes. The importance of van der Waals force in groove binding was supported by computations on the complex formed by the repressor peptide fragment from the bacteriophage 434 and its operator oligonucleotide.     

The Hoechst 33258 covalent dimer covers a total turn of the double-stranded DNA

With the goal to design ligands recognizing extended regions on dsDNA, a covalent dimer of the fluorescent dye Hoechst 33258 [bis-HT(NMe)] composed of two dye molecules linked via the phenol oxygen atoms with a (CH2)3-N+ H(CH3)-(CH2)3 fragment was constructed using computer modeling and then synthesized. Its interactions with the double-stranded DNA (dsDNA) were studied by fluorescent and UV-Vis spectroscopy and circular (CD) and linear dichroism (LD). Based on variations in the affinity to the dsDNA, it was shown that complexes of three types are formed. The first type complexes result from binding of a bis-HT(NMe) monomer in the open conformation; in this case the ligand covers the total dsDNA turn and is located in the minor groove according to the positive value of CD at 370 nm. In addition, the ability to form bis-HT(NMe)-bridges between two dsDNA molecules, i.e., each of the two bis-HT(NMe) ends binds to two different dsDNA molecules, was demonstrated for the first type complexes. Spectral characteristics (maximal absorption at 362 nm, positive sign, and maximal value of CD at 370 nm) of the first type complexes conform to those of the specific Hoechst 33258 complex with poly[d(A-T)] x poly[d(A-T]. The second type complexes correspond to the bis-HT(NMe) sandwich (as an inter- or intramolecular) binding to dsDNA with stoichiometry > or = 5 bp. Thereby, a negative LD at 360 nm and the location of bis-HT(NMe) sandwiches in the minor groove of B form dsDNA seems contradictory. Spectral characteristics (maximal positive CD at 345 nm, a dramatic decrease in fluorescence intensity and the shift of its maximum to 490 nm) of these complexes favor a suggestion that this binding correlates to the formation of nonspecific dimeric Hoechst 33258 complex with dsDNA. The third type complexes are characterized by stoichiometry of one bis-HT(NMe) molecule per approximately 2 bp and the tendency to zero of LD values at 270 and 360 nm. We assume that in these complexes bis-HT(NMe) sandwich dimers are formed on dsDNA. The complexes of this type conform to the aggregation type complex of Hoechst 33258 with dsDNA. The ability of bis-HT(NMe) to cover the whole dsDNA turn or form bridges with two dsDNA upon the formation of the first type complexes essentially distinguishes it from Hoechst 33258, which can only occupy 5 bp and does not form such bridges. This specific property of bis-HT(NMe) may support new biological activities.     

Intercalating fluorescence dye YOYO-1 prevents the folding transition in giant duplex DNA

Recently, it has become clear that with the addition of polyamines, giant DNA molecules of size greater than 10 kbp exhibit all-or-none switching between elongated coil and folded compact states. Here the effects of the intercalating fluorescent labeling dye, YOYO-1, and the minor-groove binding fluorescent labeling dye, DAPI, on the folding transition of single giant T4 DNA (166 kbp) induced by spermidine(3+) were examined, by use of the experimental technique of single molecular chain observation with fluorescence microscopy. It is found that the intercalating dye, YOYO-1, markedly prevents the folding transition, whereas the minor-groove binding dye, DAPI, exhibits negligible effect on the folding transition. This action of YOYO-1 is discussed in relation to the biological effect of intercalators.     

Molecular modelling methods for prediction of sequence-selectivity in DNA recognition

We describe how one can apply molecular modelling methods, based on the molecular mechanics/generalised Born (MM/GB) approach, to the prediction of the relative affinity of DNA minor groove binding ligands for different DNA sequences. We discuss the theoretical background to the technique, some variations in the methodology that can be employed, and illustrate its application through a case study: analysis of the energetics of binding of Hoechst 33258 to the minor groove of various A/T-rich DNA duplexes. We show how the underpinning molecular dynamics (MD) simulations can be set up, how they can be analysed for satisfactory behaviour, and various approaches to extracting thermodynamics of drug binding from them. We find that while certain elaborations to the basic MM/GB method can improve the agreement with experimental data (e.g., calculating the DNA perturbation energy), others have to be analysed with more caution (e.g., calculating configurational entropy changes). Overall, these methodologies can rank the affinity of a ligand for the minor groove of different DNA sequences fairly well, but the calculation of absolute binding affinities is not very reliable.     

Molecular dynamics investigation of the interaction between DNA and distamycin

The complex of the minor groove binding drug distamycin and the B-DNA oligomer d-(CGCAAATTTGCG) was investigated by molecular dynamics simulations. For this purpose, accurate atomic partial charges of distamycin were determined by extended quantum chemical calculations. The complex was simulated without water but with hydrated counterions. The oligomer without the drug was simulated in the same fashion and also with 1713 water molecules and sodium counterions. The simulations revealed that the binding of distamycin in the minor groove induces a stiffening of the DNA helix. The drug also prevents a transition from B-DNA to A-DNA that was found to occur rapidly (30 ps) in the segment without bound distamycin in a water-free environment but not in simulations including water. In other simulations, we investigated the relaxation processes after distamycin was moved from its preferred binding site, either radially or along the minor groove. Binding in the major groove was simulated as well and resulted in a bound configuration with the guanidinium end of distamycin close to two phosphate groups. We suggest that, in an aqueous environment, tight hydration shells covering the DNA backbone prevent such an arrangement and thus lead to distamycin's propensity for minor groove binding.     

Recognition of nine base pair sequences in the minor groove of DNA at subpicomolar concentrations by a novel microgonotropen.

The dsDNA interactions of the novel microgonotropen L1 have been characterized via spectrofluorometric titrations and thermal melting studies. A microgonotropen consists of a DNA minor groove binding moiety attached to a basic side chain capable of reaching out of the minor groove and grasping the acidic DNA phosphodiester backbone. L1 was synthesized employing solid-phase chemistry. L1 is shown to distinguish nine base pair A/T rich binding sites from sites possessing fewer than nine contiguous A/T base pairs. Further, L1 binds its preferred dsDNA sequences at subpicomolar concentrations. The equilibrium constant for complexation (K(1)) of a nine base pair A/T rich dsDNA binding site by L1 is roughly 10(13) M(-1). Single base pair A/T --> G/C substitutions within the nine base pair A/T rich binding site of L1 decreases the equilibrium constant for DNA binding by 1-2 orders of magnitude. The three proplyamine side chains of L1 enhance the agents free energy of binding by more than 5 kcal. Molecular modeling suggests that L1 adopts a 'spiral-like' conformation which fits almost a full turn of the DNA helix.     

DNA-templated click chemistry for creation of novel DNA binding molecules

We have developed a new methodology for producing new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by the addition of dsDNA. Furthermore, the resulting peptide–acridine conjugate showed a slightly stronger binding to dsDNA. These results indicate that the DNA-templated click chemistry is applicable for screening new binding molecules.     

Analysing DNA complexes by circular and linear dichroism

The application of linear and circular dichroism (LD and CD) in nucleic acid research id illustrated by recent results aimed at answering specific structural problem in the interaction of DNA with molecules of biological importance. We first consider the circumstances under which ligands, such as DAPI (4′, 6-diamidino-2-phenylindole), change their preferred binding mode in the minor groove to major groove binding or intercalation. As an extension of this problem we refer to the switch between groove binding and intercalation of structurally similar ligands such as ellipticines and trigonal ruthenium complexes. We also explore the use of LD and CD in the determination of the structure of the complex formed between the polynucleotide poly(dA) and the novel ‘peptide nucleic acid’, consisting of nucleic acid bases joined by a polyamide homomorphous with the deoxyribose-phosphate backbone of DNA. Finally, the structure and interaction of the recombination enzyme RecA with DNA is discussed, in particular the influence of the presence of the intercalators, groove binders or covalent DNA adducts.     

Molecular basis for potentiation of bleomycin-mediated degradation of DNA by polyamines. Experimental and molecular mechanical studies

The bleomycin-mediated degradation of DNA is stimulated (amplified) by certain DNA binding compounds, such as polyamines, that distort the double helix. Computer modelling studies suggest that putrescine (1), spermidine (2), and spermine (3) bind preferentially on the floor of the major groove of (dGdC)5.(dGdC)5. This interaction results in a bend of the oligomer helix toward the major groove and enlargement of the minor groove, both effects being in the order 1 less than 2 less than 3. These polyamine-induced distortions, as obtained from theoretical studies, parallel the experimental values of the amplification activities of 1-3 in the bleomycin-mediated degradation of poly(dGdC).poly(dGdC). The amplification mechanism of non-competitive binding of amplifier molecules in the major groove, and bleomycin in the minor groove, is proposed. It is suggested that the amplifier-induced conformational changes of the DNA helix increase affinity of the activated bleomycin complex toward the DNA minor groove and, consequently, result in an increased efficiency of the bleomycin-mediated degradation of the helix.     

Influence of surface shape on DNA binding of bimetallo helicates

In order to probe the DNA-helicate interactions responsible for the DNA binding and remarkable changes of the DNA secondary structure induced by a tetracationic bi-metallo helicate [Fe(2)(L(1))(3)](4+) (L(1)=C(25)H(20)N(4)), we have designed and synthesised derivatives with hydrophobic methyl groups at different positions on the ligand backbone. Two dimetallo helicates [Fe(2)(L(i))(3)](4+) were prepared using ligands L(3) and L(5) with the methyl substituent on, respectively, the 3 and 5 positions of the pyridyl ring thus producing a wider or slightly longer tetracationic DNA binder. UV/visible absorbance, circular and linear dichroism spectroscopies have been used to characterize the interactions of the cylinders with DNA with the aim of investigating any sequence preference or selectivity upon binding. Competitive binding studies using fluorescent dyes Hoechst 33258 (a minor groove binder), ethidium bromide (an intercalator) and a major groove binding cation (cobalt (III) hexammine) which induces the B-->Z transition have been employed to determine the binding geometries of the enantiomers of two methylated helicates (L(3) and L(5)) to DNA and compare with the data obtained previously for the unmethylated analogue (L(1)). The results demonstrate that the racemic mixtures and the resolved enantiomers of all helicates bind to DNA inducing structural changes. The overall conclusion from the effect of adding these groups to the surface of the parent helicate is that increasing the width (L(3)) reduces the DNA binding strength, the bending and coiling effect and the groove selectivity of the enantiomers compared with the parent compound. There is limited evidence to suggest a slight GC sequence preference. Lengthening the helicate (L(5)) results in DNA interactions similar to those of the parent compounds, with an increased preference of the P enantiomer for the minor groove indicating an enhancement of mode selectivity.     

Quinoxaline antibiotics enhance peptide nucleic acid binding to double-stranded DNA

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.