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Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel. NSM facilitates accurate determination of molecular weight from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the analysis of a complex biofluid, using conditioned cell culture medium containing extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomolecules, and to analyze single-cell secretomes.

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

Significance The main stabilizer of the DNA double helix is not the base-pair hydrogen bonds but coin-pile stacking of base pairs, whose hydrophobic cohesion, requiring abundant water, indirectly makes the DNA interior dry so that hydrogen bonds can exert full recognition power. We report that certain semihydrophobic agents depress the stacking energy (measurable in single-molecule experiments), leading to transiently occurring holes in the base-pair stack (monitorable via binding of threading intercalators). Similar structures observed in DNA complexes with RecA and Rad51, and previous observations of spontaneous strand exchange catalyzed in semihydrophobic model systems, make us propose that some hydrophobic protein residues may have roles in catalyzing homologous recombination. We speculate that hydrophobic catalysis is a general phenomenon in DNA enzymes., Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote “longitudinal breathing” and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an “open hole” population on the order of 10−2 compared to 10−4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.

Parkinson's disease (PD) is linked to aggregation of the protein α-synuclein (aS) into amyloid fibers. aS is proposed to regulate synaptic activity and may also play a role in gene regulation via interaction with DNA in the cell nucleus. Here, we address the role of the negatively-charged C-terminus in the interaction between aS and DNA using single-molecule techniques. Using nanofluidic channels, we demonstrate that truncation of the C-terminus of aS induces differential effects on DNA depending on the extent of the truncation. The DNA extension increases for full-length aS and the (1-119)aS variant, but decreases about 25% upon binding to the (1-97)aS variant. Atomic force microscopy imaging showed full protein coverage of the DNA at high aS concentration. The characterization of biophysical properties of DNA when in complex with aS variants may provide important insights into the role of such interactions in PD, especially since C-terminal aS truncations have been found in clinical samples from PD patients.

Carbapenem-resistant Klebsiella pneumoniae are a major global threat in healthcare facilities. The propagation of carbapenem resistance determinants can occur through vertical transmission, with genetic elements being transmitted by the host bacterium, or by horizontal transmission, with the same genetic elements being transferred among distinct bacterial hosts. This work aimed to track carbapenem resistance transmission by K. pneumoniae in a healthcare facility. The study involved a polyphasic approach based on conjugation assays, resistance phenotype and genotype analyses, whole genome sequencing, and plasmid characterization by pulsed field gel electrophoresis and optical DNA mapping. Out of 40 K. pneumoniae clinical isolates recovered over two years, five were carbapenem- and multidrug-resistant and belonged to multilocus sequence type ST147. These isolates harboured the carbapenemase encoding blaKPC-3 gene, integrated in conjugative plasmids of 140 kbp or 55 kbp, belonging to replicon types incFIA/incFIIK or incN/incFIIK, respectively. The two distinct plasmids encoding the blaKPC-3 gene were associated with distinct genetic lineages, as confirmed by optical DNA mapping and whole genome sequence analyses. These results suggested vertical (bacterial strain-based) transmission of the carbapenem-resistance genetic elements. Determination of the mode of transmission of antibiotic resistance in healthcare facilities, only possible based on polyphasic approaches as described here, is essential to control resistance propagation.

Published by Wiley-VCH Verlag GmbH & Co. KGaA. Fundamental research on Parkinson's disease (PD) most often focuses on the ability of α-synuclein (aS) to form oligomers and amyloids, and how such species promote brain cell death. However, there are indications that aS also plays a gene-regulatory role in the cell nucleus. Here, the interaction between monomeric aS and DNA in vitro has been investigated with single-molecule techniques. Using a nanofluidic channel system, it was discovered that aS binds to DNA and by studying the DNA–protein complexes at different confinements we determined that aS binding increases the persistence length of DNA from 70 to 90 nm at high coverage. By atomic force microscopy it was revealed that at low protein-to-DNA ratio, the aS binding occurs as small protein clusters scattered along the DNA; at high protein-to-DNA ratio, the DNA is fully covered by protein. As DNA-aS interactions may play roles in PD, it is of importance to characterize biophysical properties of such complexes in detail.

Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1.

Optical DNA mapping has over the last decade emerged as a very powerful tool for obtaining long range sequence information from single DNA molecules. In optical DNA mapping, intact large single DNA molecules are labeled, stretched out, and imaged using a fluorescence microscope. This means that sequence information ranging over hundreds of kilobasepairs (kbp) can be obtained in one single image. Nanochannels offer homogeneous and efficient stretching of DNA that is crucial to maximize the information that can be obtained from optical DNA maps. In this review, we highlight progress in the field of optical DNA mapping in nanochannels. We discuss the different protocols for sequence specific labeling and divide them into two main categories, enzymatic labeling and affinity-based labeling. Examples are highlighted where optical DNA mapping is used to gain information on length scales that would be inaccessible with traditional techniques. Enzymatic labeling has been commercialized and is mainly used in human genetics and assembly of complex genomes, while the affinity-based methods have primarily been applied in bacteriology, for example for rapid analysis of plasmids encoding antibiotic resistance. Next, we highlight how the design of nanofluidic channels can been altered in order to obtain the desired information and discuss how recent advances in the field make it possible to retrieve information beyond DNA sequence. In the outlook section, we discuss future directions of optical DNA mapping, such as fully integrated devices and portable microscopes.

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.

Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.

We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 μl sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.

Extracellular polymeric substances (EPS) produced by bacteria form a matrix supporting the complex three-dimensional architecture of biofilms. This EPS matrix is primarily composed of polysaccharides, proteins and extracellular DNA. In addition to supporting the community structure, the EPS matrix protects bacterial biofilms from the environment. Specifically, it shields the bacterial cells inside the biofilm, by preventing antimicrobial agents from getting in contact with them, thereby reducing their killing effect. New strategies for disrupting the formation of the EPS matrix can therefore lead to a more efficient use of existing antimicrobials. Here we examined the mechanism of the known effect of vitamin C (sodium ascorbate) on enhancing the activity of various antibacterial agents. Our quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development. As a result, the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted. Once the EPS content is reduced beyond a critical point, bacterial cells get fully exposed to the medium. At this stage, the cells are more susceptible to killing, either by vitamin C-induced oxidative stress as reported here, or by other antimicrobials or treatments.

The catalytic role of hydrophobic co-solutes in DNA strand exchange is demonstrated by FRET kinetics. Two mechanisms that contribute to this are base stacking destabilisation and nucleation-promoted DNA strand invasion. We propose that hydrophobic catalysis is involved in the strand-exchange activity of recombination enzymes.

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

Luminescent conjugated polyelectrolytes (LCPs) have emerged as novel stains to detect and distinguish between various amyloidogenic species, including prefibrillar aggregates and mature fibril deposits, both in vitro and in histological tissue samples, offering advantages over traditional amyloid stains. We here use linear dichroism (LD) spectroscopy under shear alignment to characterize interactions between the LCP poly(3-thiophene acetic acid) (PTAA) and amyloid fibrils. The positive signature in the LD spectrum of amyloid-bound PTAA suggests that it binds in the grooves between adjacent protein side-chains in the amyloid fibril core, parallel to the fibril axis, similar to thioflavin-T and congo red. Moreover, using LD we record the absorption spectrum of amylokl-bound PTAA in isolation from free dye showing a red-shift by ca 30 nm compared to in solution. This has important implications for the use of PTAA as an amyloid probe in situ and in vitro and we demonstrate how to obtain optimal amyloid-specific fluorescence read-outs using PTAA. We use the shift in maximum absorption to estimate the fraction of bound PTAA at a given concentration. PTAA binding reaches saturation when added in 36 times excess and at this concentration the PTAA density is 4-5 monomer units per insulin monomer in the fibril. Finally, we demonstrate that changes in LD intensity can be related to alterations in persistence length of amyloid fibrils resulting from changes in solution conditions, showing that this technique is useful to assess macroscopic properties of these biopolymers. (C) 2011 Elsevier Inc. All rights reserved.

The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard "foreign" contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.

Poly 2,6-[1,5-bis(dodecyloxy)naphthylene vinylene] is synthesized by microwave-assisted McMurry polymerization utilizing low-valent titanium generated from titanium tetrachloride and zinc. The obtained polymer is fluorescent with an average molecular weight of approximately 65,000 g/mol and a polydispersity of Mw/Mn ≈ 3. Absorption and fluorescence spectroscopy in solution and on spin-cast thin films reveal that the bis-alkoxy substituted PNV has a short effective conjugation length but a quite efficient exciton migration.

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the A beta(1-40) and A beta(1-42) variants of the Alzheimer's-related peptide amyloid-beta, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with A beta(1-40) compared to A beta(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with A beta(1-42) fibrils producing a longer ThT fluorescence lifetime compared to A beta(140). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed beta-sheet in the mature A beta fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation.

We demonstrate how nanofluidic channels can be used as a tool to rapidly determine the number and sizes of plasmids in bacterial isolates. Each step can be automated at low cost, opening up opportunities for general use in microbiology labs.

DNA-binding, green and yellow fluorescent probes with excellent brightness and high on/off ratios are reported. The probes are membrane permeable, live-cell compatible, and optimally matched to 405 nm and 514 nm laser lines, making them attractive alternatives to UV-excited and blue emissive Hoechst 33342 and DAPI nuclear stains. Their electronic structure was investigated by optical spectroscopy supported by TD-DFT calculations. DNA binding is accompanied by 27- to 75-fold emission enhancements, and linear dichroism demonstrates that one dye is a groove binder while the other intercalates into DNA.

DNA strand exchange is catalyzed by molecular crowding and hydrophobic interactions in concentrated aqueous solutions of polyethylene glycol, a discovery of relevance for understanding the function of recombination enzymes and with potential applications to DNA nanotechnology.

Binuclear ruthenium complexes that bind DNA by threading intercalation have recently been found to exhibit an exceptional kinetic selectivity for long polymeric adenine-thymine (AT) DNA. A series of oligonucleotide hairpin duplexes containing a central tract of 6-44 alternating AT base pairs have here been used to investigate the nature of the recognition mechanism. We find that, above a threshold AT tract length corresponding to one helix turn of B-DNA, a dramatic increase in threading intercalation rate occurs. In contrast, such length dependence is not observed for rates of unthreading. Intercalation by any mechanism that depends on the open end of the hairpin was found not to be important in the series of oligonucleotides used, as verified by including in the study a hairpin duplex cyclized by a copper-catalyzed "click" reaction. Our observations are interpreted in terms of a conformational pre-equilibrium, determined by the length of the AT tract. We finally find that mismatches or loops in the oligonucleotide facilitate the threading process, of interest for the development of mismatch-recognizing probes.

Whereas the emission from the ruthenium complex Delta Delta-[mu-bidppz(phen)(4)RU2](4+) (P) is five times larger when intercalated into poly(dAdT)(2) than when intercalated into ct-DNA, the homologue Delta Delta-[mu-bidppz(bipy)(4)RU2](4+) (B) has a smaller quantum yield and a red-shifted emission. The origin of this difference is here investigated by studying intercalation into oligonucleotides containing a central AT-tract. Increasing the length of the AT-tract increases the emission quantum yield for P but decreases it for B. However, not even four helix turns of AT base pairs is enough to mimic poly(dAdT)(2). B and P thus use the increased flexibility with increasing length of the AT-tract in opposite ways, whereas B gets more prone to quenching by water, P gets more protected from quenching. The earlier reported gradual increase of the intercalation rate with AT-stretch length is thus paralleled by a gradual change in the equilibrium properties of the intercalated state. (c) 2007 Elsevier B.V. All rights reserved.

The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.

Detergent sequestration using micelles as a hydrophobic sink for dissociated drug molecules is an established technique for determination of dissociation rates. The anionic surfactant molecules are generally assumed not to interact with the anionic DNA and thereby not to affect the rate of dissociation. By contrast, we here demonstrate that the surfactant molecules sodium dodecyl sulfate (SDS), sodium decyl sulfate, and sodium octyl sulfate all induce substantial rate enhancements of the dissociation of intercalators from DNA. Four different cationic DNA intercalators are studied with respect to surfactant-induced dissociation. Except for the smallest intercalator, ethidium, the dissociation rate constants increase monotonically with surfactant concentration both below cmc and (more strongly) above cmc, much more than expected from electrostatic effects of increased counterion concentration. The rate enhancement, most pronounced for the bulky, multicationic, hydrophobic DNA ligands in this study, indicates a reduction of the activation energy for the ligand to pass out from a deeply penetrating intercalation site of DNA. The discovery that surfactants enhance the rate of dissociation of cationic DNA-intercalators implies that rate constants previously determined by micelle-sequestered dissociation may have been overestimated. As an alternative, more reliable method, we suggest instead the addition of excess of dummy DNA as an absorbent for dissociated ligand.

The dumbbell shaped binuclear ruthenium complex ΔΔ-P requires transiently melted DNA in order to thread through the DNA bases and intercalate DNA. Because such fluctuations are rare at room temperature, the binding rates are extremely low in bulk experiments. Here, single DNA molecule stretching is used to lower the barrier to DNA melting, resulting in direct mechanical manipulation of the barrier to DNA binding by the ligand. The rate of DNA threading depends exponentially on force, consistent with theoretical predictions. From the observed force dependence of the binding rate, we demonstrate that only one base pair must be transiently melted for DNA threading to occur. Copyright © 2008 American Chemical Society.

We here report a remarkably slow rearrangement of binding modes for a binuclear ruthenium(II) complex upon interaction with DNA. It has been previously shown that Delta,Delta-[mu-(11,11'-bidppz)(phen)4Ru2]4+ binds to DNA in one of the grooves. However, we find that this is only an initial, metastable, binding mode, which is extremely slowly reorganized into an intercalative binding geometry. The slow rearrangement and dissociation, revealed by flow linear dichroism and fluorescence spectroscopy, are concluded to be a result from the complex being threaded through the DNA, with one of the bridging aromatic dppz ligands intercalated between the base pairs of the DNA, placing one metal center in the minor groove and one in the major groove. A negative LD, a high luminescence quantum yield, and long luminescence lifetimes, similar to the intercalating complex Delta-[Ru(phen)2dppz]2+, indicate intercalation of the bidppz moiety. The unique slow dissociation of the complex in its final DNA-binding mode suggests that this class of threading, partially intercalated binuclear complexes may be interesting in the context of cancer therapy. Also, their unique optical and photophysical properties could make such complexes, either alone or scaffolded by DNA structures, of interest for the development of nanometer-sized molecular optoelectronic devices.

We demonstrate that the detected emission intensity from YOYO-labeled DNA molecules confined in 180 nm deep Si/SiO2 nanofunnels changes significantly and not monotonically with the width of the funnel. This effect may be of importance for quantitative fluorescence microscopy and for experiments with a tight photon budget.

The mechanism of DNA strand exchange, performed in vivo by proteins in the RecA family, is, despite extensive studies, still not understood in detail. We are therefore investigating a model system, using the surface of a lipid vesicle, to systematically study the effect of individual parameters on the rate of the strand exchange reaction. DNA oligonucleotides are accumulated on the surface of cationic liposomes and the strand exchange is monitored using FRET. We show that in the presence of lipid vesicles containing 35% cationic lipid the strand exchange takes place with a significantly enhanced rate and improved yield, compared to in bulk without liposomes. The highest rate is observed at a charge ratio where the positive surface charge is matched by the negative charges from DNA, and thus all double- and single-stranded DNA can be bound to the liposome surface. The DNA duplex is destabilized on the positively charged surface and with the accumulation of complementary single strands in close proximity a fast exchange is facilitated. We also show that when aggregation of liposomes is minimized by incorporation of a PEG-modified lipid, the strand exchange is still efficient, although with a lower rate than for the unmodified system. We conclude that the liposome model system, with the possibility to systematically vary the surface composition, is a promising platform for further studies of DNA strand exchange reactions.

Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and ‘clicked’ using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the ‘Tri-Click’ (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques—including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes—the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(II)–TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles.