The binding mode of the DNA bisintercalator luzopeptin investigated using atomic force microscopy

The luzopeptins are DNA bisintercalating antibiotics that contain a decadepsipeptide to which are attached two quinoline chromophores. We have used atomic force microscopy (AFM) to investigate the interaction between luzopeptin B and DNA in an attempt to shed light on the binding mode of this antibiotic. AFM images provided contour lengths which were used as a direct measure of bisintercalation. Binding of luzopeptin B was investigated using two different DNA sequences, one having a GC content of 42% and the other 59%, which revealed a higher degree of bisintercalation into the DNA sequences having the lower GC content. The measured increment in contour length was found to plateau at values corresponding to binding of one drug molecule every 40 and 72 bp to the 42 and 59% GC sequences, respectively. In addition to the length increase, a higher proportion of DNA molecules displaying complex morphology was observed as the concentration of luzopeptin was increased. Such molecules were not included in the measurements of contour length. We propose that the various manifestations of complex morphology arise from both inter- and intramolecular cross-linking of the DNA caused by binding of luzopeptin, providing direct evidence of cross-linked species by AFM imaging.     

Atomic force microscopy study of the structural effects induced by echinomycin binding to DNA

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer

We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA binding protein. Binding was evaluated using a variety of single and double-stranded DNA molecules. Mlh1-Pms1 bound short substrates with low affinity and showed a slight preference for single-stranded DNA. In contrast, Mlh1-Pms1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative. High affinity binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow. However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting that Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be relevant to its participation in DNA mismatch repair, recombination and cellular responses to DNA damage.     

Quantum dot binding to DNA: Single-molecule imaging with atomic force microscopy

The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309     

Binding of ethidium and bis(methidium)spermine to Z DNA by intercalation

The interaction of ethidium bromide, a DNA intercalating drug, and bis( methidium )spermine, a DNA bis-intercalating compound, with the left-handed Z form of poly(dG-dC) has been studied in 4.4 M NaCl. Spectrophotometric analysis using absorption, fluorescence and circular dichroism indicates that the complex formed between ethidium and Z DNA resembles very closely that formed with B DNA. This suggests that ethidium binds to Z DNA by intercalation. 31P NMR spectra are presented showing both the conversion of the Z form to the B form with increasing amounts of drug and the typical Z form spectrum at low binding densities. Data are also presented which show that the bifunctional intercalator bis( methidium )spermine binds to Z DNA in a manner similar to its binding to B DNA, i.e., by bis-intercalation. These results are important for our understanding the behavior of Z DNA and its biological significance.     

Application of atomic force microscopy to the study of micromechanical properties of biological materials

Atomic force microscopy (AFM) has been used to study the micromechanical properties of biological systems. Its unique ability to function both as an imaging device and force sensor with nanometer resolution in both gaseous and liquid environments has meant that AFM has provided unique insights into the mechanical behaviour of tissues, cells and single molecules. As a surface scanning device, AFM can map properties such as adhesion and the Young's modulus of surfaces. As a force sensor and nanoindentor AFM can directly measure properties such as the Young's modulus of surfaces or the binding forces of cells. As a stress-strain gauge AFM can study the stretching of single molecules or fibres and as a nanomanipulator it can dissect biological particles such as viruses or DNA strands. The present paper reviews key research that has demonstrated the versatility of AFM and how it can be exploited to study the micromechanical behaviour of biological materials.     

X-ray crystallographic study of DNA duplex cross-linking: simultaneous binding to two d(CGTACG)2 molecules by a bis(9-aminoacridine-4-carboxamide) derivative

Acridine-4-carboxamides form a class of known DNA mono-intercalating agents that exhibit cytotoxic activity against tumour cell lines due to their ability to inhibit topoisomerases. Previous studies of bis-acridine derivatives have yielded equivocal results regarding the minimum length of linker necessary between the two acridine chromophores to allow bis-intercalation of duplex DNA. We report here the 1.7 A resolution X-ray crystal structure of a six-carbon-linked bis(acridine-4-carboxamide) ligand bound to d(CGTACG)2 molecules by non-covalent duplex cross-linking. The asymmetric unit consists of one DNA duplex containing an intercalated acridine-4-carboxamide chromophore at each of the two CG steps. The other half of each ligand is bound to another DNA molecule in a symmetry-related manner, with the alkyl linker threading through the minor grooves. The two crystallographically independent ligand molecules adopt distinct side chain interactions, forming hydrogen bonds to either O6 or N7 on the major groove face of guanine, in contrast to the semi-disordered state of mono-intercalators bound to the same DNA molecule. The complex described here provides the first structural evidence for the non-covalent cross-linking of DNA by a small molecule ligand and suggests a possible explanation for the inconsistent behaviour of six-carbon linked bis-acridines in previous assays of DNA bis-intercalation.     

Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes.     

Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids.

Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.     

Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.     

Global architecture of human poly(A)-specific ribonuclease by atomic force microscopy in liquid and dynamic light scattering

Deadenylation is the initial and often rate-limiting step in the main pathways of eukaryotic mRNA decay. Poly(A)-specific ribonuclease (PARN) is a eukaryotic enzyme that efficiently degrades mRNA poly(A) tails. Structural and functional studies have shown that human PARN is composed of at least three functional domains, i.e. the catalytic nuclease domain and two RNA binding domains, the R3H and the RNA recognition motif (RRM), respectively. However, the complete structure of the full length protein is still unknown. We have investigated the global architecture of human PARN by atomic force microscopy (AFM) imaging in buffered milieu and report for the first time the dimensions of the full length protein at subnanometer resolution. The AFM images of single PARN molecules reveal compact ellipsoidal dimers (10.9 × 7.6 × 4.6nm). The dimeric form of PARN was confirmed by dynamic light scattering (DLS) measurements that rendered a molecular weight of 161 kDa, in accordance with previous crystal structures of PARN fragments showing a dimeric composition. We discuss a putative internal arrangement of three functional domains within the full length PARN dimer.     

DNA bis-intercalation: Theoretical calculation of binding curves

A statistical mechanical calculation of the binding properties of DNA bis-intercalators is presented, based on the sequence-generating function method of Lifson. The effects of binding by intercalation of one or both chromophores of a bifunctional intercalating agent are examined. The secular equation for a general model that includes the effects of neighbor (nearest and non-nearest) exclusion and/or cooperativity in the binding of both singly and doubly intercalated ligands is derived. Numerical results for binding curves are presented for a more restricted model in which each type of bound ligand rigorously excludes its nearest neighbor and the total number of sites covered by a doubly intercalated ligand is variable. At low values of free ligand concentration bis-intercalation dominates the binding process, while at high value of free ligand concentration, intercalation of only one chromophore per ligand becomes significant due to the unavailability of contiguous free sites required for bis-intercalation. Also, depending on the binding parameters, the free energy of the system can be lowered by a loss of doubly intercalated ligands in favor of singly intercalated ones. Corresponding to this transition in binding mode, the average number of sites occupied by a bound ligand decreases from that characteristic of bis-intercalation to that characteristic of mono-intercalation as free ligand concentration increases. An analysis of Scatchard plots describing bis-intercalation is presented.     

Structural changes of DNA induced by mono- and binuclear cancer drugs

The structural features of the drug-DNA adducts resulted from treatment of DNA with the platinum based mononuclear drug cisplatin and the binuclear drug [{trans-PtCl(NH3)2}2H2N(CH2)4NH2]Cl2 or bis(platin) have been investigated by atomic force microscopy (AFM). Reduction in the contour length of the DNA fragments has been observed after cisplatin treatment while, compaction and aggregation are found to be the primary structural modifications following treatment with the binuclear drug. The intermolecular interaction upon bis(platin) treatment leads to observation of highly condense aggregates without a distinct sight of single isolated DNA molecule. These differences in drug binding indicate that unlike the mononuclear drug cisplatin, bis(platin) causes extensive interhelical/intermolecular cross-linking through its multiple linking sites. To our knowledge, this is the first report of a comparative AFM study to monitor the effects of a mono- and a binuclear platinum anti-cancer drug on DNA structure. These observations should provide clues towards explaining the distinct biological activities of the two drugs.     

FM-AFM constant height imaging and force curves: high resolution study of DNA-tip interactions

Interaction of the atomic force microscopy (AFM) tip with the sample can be invasive for soft samples. Frequency Modulation (FM) AFM is gentler because it allows scanning in the non‐contact regime where only attractive forces exist between the tip and the sample, and there is no sample compression. Recently, FM‐AFM was used to resolve the atomic structure of single molecules of pentacene and of carbon nanotubes. We are testing similar FM‐AFM‐based approaches to study biological samples. We present FM‐AFM experiments on dsDNA deposited on 3‐aminopropyltriethoxysilane modified mica in ultra high vacuum. With flexible samples such as DNA, the substrate flatness is a sub‐molecular resolution limiting factor. Non‐contact topographic images of DNA show variations that have the periodicity of the right handed helix of B‐form DNA – this is an unexpected result as dehydrated DNA is thought to assume the A‐form structure. Frequency shift maps at constant height allow working in the non‐monotonic frequency shift range, show a rich contrast that changes significantly with the tip‐sample separation, and show 0.2 to 0.4 nm size details on DNA. Frequency shift versus distance curves acquired on DNA molecules and converted in force curves show that for small molecules (height < 2.5 nm), there is a contribution to the interaction force from the substrate when the tip is on top of the molecules. Our data shine a new light on dehydrated and adsorbed DNA behavior. They show a longer tip‐sample interaction distance. These experiments may have an impact on nanotechnological DNA applications in non‐physiological environments such as DNA based nanoelectronics and nanotemplating. Copyright © 2012 John Wiley & Sons, Ltd.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

PT-ACRAMTU, A Platinum–Acridine Anticancer Agent, Lengthens and Aggregates, but does not Stiffen or Soften DNA

We used atomic force microscopy (AFM) to study the dose-dependent change in conformational and mechanical properties of DNA treated with PT-ACRAMTU ([PtCl(en)(ACRAMTU-S)](NO₃)₂, (en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea. PT-ACRAMTU is the parent drug of a family of non-classical platinum-based agents that show potent activity in non-small cell lung cancer in vitro and in vivo. Its acridine moiety intercalates between DNA bases, while the platinum group forms mono-adducts with DNA bases. AFM images show that PT-ACRAMTU causes some DNA looping and aggregation at drug-to-base pair ratio (r _b) of 0.1 and higher. Very significant lengthening of the DNA was observed with increasing doses of PT-ACRAMTU, and reached saturation at an r _b of 0.15. At r _b of 0.1, lengthening was 0.6 nm per drug molecule, which is more than one fully stretched base pair stack can accommodate, indicating that ACRAMTU also disturbs the stacking of neighboring base pair stacks. Analysis of the AFM images based on the worm-like chain (WLC) model showed that PT-ACRAMTU did not change the flexibility of (non-aggregated) DNA, despite the extreme lengthening. The persistence length of untreated DNA and DNA treated with PT-ACRAMTU was in the range of 49–65 nm. Potential consequences of the perturbations caused by this agent for the recognition and processing of the DNA adducts it forms are discussed.     

Study of microorganism genome DNA by atomic force microscopy

The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM.     

DNA interaction with the bifunctional acridine dye

The comparative studies of the formation of DNA-complexes with the acridines containing one and two chromophores were accomplished. It was shown that both of acridines were bonded with DNA by means of intercalation irrespective of the ionic strength of medium (mu). When mu = 0.1 the diacridine (1,6-bis(9-acridylamino)-hexan) behaves as an mono-intercalator. Under these conditions both of the ligands exert equal influence of the molecular parameters of DNA. When mu = 0.001 the binding mode of the diacridine with DNA depends on its concentration in a complex. If a number of diacridine molecules on a pair of nucleotides (r) falls in a region 0 less than r less than 0.2 its binding with DNA is accomplished via the bis-intercalation mode and accompanied by the structure distortion of the monomer remnant of the macromolecule. As r increases from 0.2 to 0.4 the gradual change of the binding mode of the diacridine with DNA from bis-intercalation to mono-intercalation takes place. Moreover the structure of nucleotides is reduced. When mu = 0.001 the behaviour of DNA complexes with mono-acridine is analogous to the observed one when mu = 0.1.