Supercoiled Structure Research Articles

Synthesis, calf-thymus DNA interaction with trimethyl-/triphenyl-tin(IV) derivatives of ɑ-lipoic acid and their mixed ligand complexes with N-donor (1,10-phenanthroline/2,2′-bipyridyl/benzimidazole)

The mixed ligand complexes of trimethyl-/triphenyl-tin(IV) derivatives of ɑ-lipoic acid (HLA), viz. R3Sn(LA) {R = Me (1), and Ph (2)}with 1,10-phenanthroline (phen), viz. [Me3Sn(LA).phen] (3), 2,2′-bipyridyl, viz. [Ph3Sn(LA).by] (4) and benzimidazole (bz), viz. [R3Sn(LA).bz] {R = Me (5), and Ph (6)} were synthesised and satisfactorily characterized by analytical and spectroscopic techniques, especially FT-IR, NMR (1H, 13C, and 119Sn NMR) spectroscopy and ESI-MS spectrometry. The ∠C-Sn-C bond angle calculated by optimization of their gas-phase geometry revealed that complexes (1) and (2) have distorted tetrahedral geometry and complexes (3) and (4) exhibited distorted octahedral geometry while complexes (5) and (6) exhibited trigonal bipyramidal environment around the tin, which agreed with the data obtained from NMR. Spectroscopic titrations (UV–visible absorption, fluorescence titration, and circular dichroism) were performed to ascertain the binding mode of the complex with calf thymus DNA (CT-DNA). The obtained values of Kb in the order of 105 M−1 suggested partial intercalative interactions of complexes with DNA grooves/base pairs. The complexes efficiently cleaved plasmid DNA (pBR322) into supercoiled (Form I), nicked/circular (From II), and linear structure (Form III). The fascinating biophysical data suggests the potential of these complexes as anticancer agents.

Compaction, Relaxation, and Linearization of Megadalton-Sized DNA Plasmids: DNA Structures Probed by CD-MS.

High purity plasmid DNA is a raw material for recombinant protein production as well as an active ingredient in DNA vaccines. There are four primary plasmid structures that can be observed in a typical plasmid formulation: supercoiled, relaxed (circular), linearized, and condensed. Determining what structures are present in a sample is important, as the structure can affect activity; the supercoiled structure has the highest activity, and >90% supercoiled is desired for industry standards. Recently, charge detection mass spectrometry (CD-MS) was used to distinguish two of the structures, supercoiled and condensed, by measuring the charge deposited on the ions by positive mode electrospray. Here, CD-MS is used to probe the structures of DNA plasmids during compaction with polycations, and through enzymatic treatment to relax and linearize plasmids. We find that all four structural types for plasmid DNA have unique charging profiles that can be distinguished using CD-MS. The extent of mechanical shearing of the DNA plasmids during electrospray is strongly influenced by the structural type.

Trimethyltin(IV) (p-fluoro-/p‑hydroxy-/p-nitro-)benzoates and their benzimidazole complexes: X-ray structures of [Me3Sn(p-NO2C6H4CO2)]n, and [Me3Sn(p-NO2C6H4CO2). bz], DFT calculations, DNA binding, and DNA cleavage studies

A series of trimethyltin(IV) carboxylates with Sn‒O bonds: [Me3Sn(p-FC6H4CO2)] (1), [Me3Sn(p-HOC6H4CO2)] (2), [Me3Sn(p-NO2C6H4CO2)]n (3), and their benzimidazole complexes with Sn‒N bond: [Me3Sn(p-FC6H4CO2).bz] (4), [Me3Sn(p-HOC6H4CO2).bz] (5), and [Me3Sn(p-NO2C6H4CO2).bz] (6) (bz = benzimidazole) have been synthesized and characterized by elemental analysis, FT-IR, 1H, 13C, 119Sn NMR, and electrospray ionization mass spectrometric (ESI-MS) studies. The crystal structures for (3) and (6) have been authenticated by X-ray diffraction. In the crystal structure, polymeric coordination compound (3) and the monocrystal system (6) have trigonal bipyramidal geometry around the Sn centre, where most electronegative atoms occupied the axial positions, and three methyl groups occupied the equatorial positions. The gas-phase tetrahedral geometries of (1) and (2), asymmetric unit (Sc-XRD) of (3), and trigonal bipyramidal geometries of (4–6) have been optimized using the B3LYP function and LanL2Z basis, and obtained ∠C-Sn-C bond angles agree with those obtained from NMR/X-ray studies. The interaction of (1–6) with calf thymus DNA (ctDNA) has been investigated by spectroscopic titrations such as UV–visible, fluorescence, and circular dichroism (CD) titrations, and calculated binding constant values found in the 104–105 M−1 range which suggest a groove binding or electrostatic interaction between complexes and ctDNA. The gel electrophoresis studies suggest that (1–6) effectively cleaved the plasmid pBR322 DNA into three bands: the supercoiled, nicked/circular, and linear structures. The intriguing biophysical findings from the study indicate that these complexes have properties that make them strong contenders for additional research as potential anti-cancer agents.

Spatio-temporal organization of the E. coli chromosome from base to cellular length scales.

Escherichia coli has been a vital model organism for studying chromosomal structure, thanks, in part, to its small and circular genome (4.6 million base pairs) and well-characterized biochemical pathways. Over the last several decades, we have made considerable progress in understanding the intricacies of the structure and subsequent function of the E. coli nucleoid. At the smallest scale, DNA, with no physical constraints, takes on a shape reminiscent of a randomly twisted cable, forming mostly random coils but partly affected by its stiffness. This ball-of-spaghetti-like shape forms a structure several times too large to fit into the cell. Once the physiological constraints of the cell are added, the DNA takes on overtwisted (negatively supercoiled) structures, which are shaped by an intricate interplay of many proteins carrying out essential biological processes. At shorter length scales (up to about 1 kb), nucleoid-associated proteins organize and condense the chromosome by inducing loops, bends, and forming bridges. Zooming out further and including cellular processes, topological domains are formed, which are flanked by supercoiling barriers. At the megabase-scale both large, highly self-interacting regions (macrodomains) and strong contacts between distant but co-regulated genes have been observed. At the largest scale, the nucleoid forms a helical ellipsoid. In this review, we will explore the history and recent advances that pave the way for a better understanding of E. coli chromosome organization and structure, discussing the cellular processes that drive changes in DNA shape, and what contributes to compaction and formation of dynamic structures, and in turn how bacterial chromatin affects key processes such as transcription and replication.

A Method for Calculating the Sign and Degree of Chirality of Supercoiled Protein Structures

Chirality plays an important role in studies of natural protein structures. Therefore, much attention is paid to solving the problems associated with the development of criteria and methods for assessing the chirality of biomolecules. In this paper, a new method for calculating the sign and degree of chirality of superhelices is proposed. The method makes it possible to characterize the chirality sign and to quantify coiled-coils and collagen superhelices. The degree of chirality is understood as a value indicating the intensity of twisting of individual helices around the axis of the superhelix. The calculation requires information about the relative spatial arrangement of the alpha carbon of the amino acid residues of the helices that make up the superhelix. The use of a small amount of raw data makes the method easy to apply, and the validity of the results of this study is confirmed through the analysis of real protein structures.

Coiled-coil domain-containing 154 promotes colorectal cancer proliferation and metastasis via interacting with minichromosome maintenance complex component 2

Coiled-Coil Domain-Containing (CCDC) is a large class of structural proteins containing left-handed supercoiled structure. The clinical value and the functional implication of CCDC in colorectal cancer (CRC) remain unknown. Based on the genetic, transcriptional, and clinical data from The Cancer Genome Atlas, five of thirty-six CCDC proteins were differentially expressed in the CRC and associated with the survival of patients with CRC. A CCDC-score model was established to evaluate the prognosis of patients. The potential function of Coiled-Coil Domain-Containing 154 (CCDC154) was investigated using bioinformatical methods, which unveiled that high expression of CCDC154 indicates poor survival for patients with CRC and correlates with low infiltration of CD8+ T cells and high infiltration of neutrophils, indicating that CCDC154 enhances tumor growth and metastasis. CCDC154 interacts with Minichromosome Maintenance Complex Component 2 (MCM2) protein and promotes malignant phenotype via MCM2. We validated the expression level and survival prediction value of CCDC154 in clinical samples, and analyzed its co-expression of MCM2, Ki-67 and p53. This work discloses the role of CCDC in clinical setting and CCDC154 functions in CRC.

Piperazin incorporated Schiff Base derivatives: Assessment of in vitro biological activities, metabolic enzyme inhibition properties, and molecular docking calculations.

The cytotoxic activities of the compounds were determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) method in human breast cancer (MCF-7), human cervical cancer (HeLa), and mouse fibroblast (L929) cell lines. The compounds MAAS-5 and four modified the supercoiled tertiary structure of pBR322 plasmid DNA. MAAS-5 showed the highest cytotoxic activity in HeLa, MCF-7, and L929 cells with IC50 values of 16.76 ± 3.22, 28.83 ± 5.61, and 2.18 ± 1.22 µM, respectively. MAAS-3 was found to have almost the lowest cytotoxic activities with the IC50 values of 93.17 ± 9.28, 181.07 ± 11.54, and 16.86 ± 6.42 µM in HeLa, MCF-7, and L929 cells respectively at 24 h. Moreover, the antiepileptic potentials of these compounds were investigated in this study. To this end, the effect of newly synthesized Schiff base derivatives on the enzyme activities of carbonic anhydrase I and II isozymes (human carbonic anhydrase [hCA] I and hCA II) was evaluated spectrophotometrically. The target compounds demonstrated high inhibitory activities compared with standard inhibitors with Ki values in the range of 4.54 ± 0.86-15.46 ± 8.65 nM for hCA I (Ki value for standard inhibitor = 12.08 ± 2.00 nM), 1.09 ± 0.32-29.94 ± 0.82 nM for hCA II (Ki value for standard inhibitor = 18.22 ± 4.90 nM). Finally, the activities of the compounds were compared with the Gaussian programme in the B3lyp, HF, M062X base sets with 6-31++G (d,p) levels. In addition, the activities of five compounds against various breast cancer proteins and hCA I and II were compared with molecular docking calculations. Also, absorption, distribution, metabolism, excretion, and toxicity analysis was performed to investigate the possibility of using five compounds as drug candidates.

The evolution of archaeal flagellar filaments

Eukaryotes, archaea, and bacteria each independently evolved flagellar-based motility systems allowing cells to swim in solution. The two prokaryotic flagellar systems involve the motor-driven rotation of supercoiled flagellar filaments, which function as propellers providing the thrust needed to swim. Despite being unrelated to each other, archaeal and bacterial flagellar filaments have convergently evolved to form similar supercoils with pitches around 1 to 2.5 µm and diameters around 0.2 to 0.6 µm. The recent discovery of 11 protofilament states in the supercoiled bacterial flagella and 10 in archaeal flagella [Kreutzberger et al., Cell (2022)] further highlights the analogous mechanism of supercoiling shared by these two structures. It is known that archaeal flagellar filaments are homologous to archaeal type IV pili (T4P), and the N-terminal domains in these archaeal T4P and flagellar filaments are homologous to the N-terminal domains in bacterial T4P. However, the features which distinguish archaeal flagellar filaments from archaeal T4P remained unclear. Here, we report several new structures of archaeal T4Ps and archaeal flagellar filaments and compare these with existing structures. We find that archaeal T4Ps have highly variable symmetries with nearly all of their helical twists varying by about 1-5 degrees and having either 7 or no protofilaments. All archaeal flagellar filament structures have helical twists within ∼0.2 degrees of each other and have 10 protofilaments. Strikingly, one of our new structures from Pyrobaculum calidifontis is of a flagellar-like filament with a pilin-like sequence which has a symmetry most similar to archaeal flagellar filaments assembling into a 10-protofilament structure. This provides the basis for proposing that archaeal flagellar filaments arose from such P.calidifontis-like T4P with symmetries very similar to those of the extant archaeal flagellar filaments, with supercoiled structures arising following the emergence of the filaments with 10 protofilaments.

Purification and CryoEM Image Analysis of the Bacterial Flagellar Filament.

The bacterial flagellum is a large assembly of about 30 different proteins and is divided into three parts: the filament that acts as a screw propeller, the hook as a universal joint, and the basal body as a rotary motor. In the case of Salmonella, the filament length is 10-15μm, which is more than ten times longer than the size of the cell. The filament is composed of only one component protein, flagellin, and is made of 11 protofilaments. The filament can form 12 different supercoiled structures as polymorphic forms. Each protofilament can take either the L (left-handed) or R (right-handed) state, and the number ratio of the protofilaments in these two states determines the shape of the supercoil. Some point mutations in flagellin make the filament straight by making all the protofilaments in one of the two states. The straight filaments enable us to use their helical symmetries for structural analysis by electron cryomicroscopy (cryoEM) and single particle image analysis. Here, we describe the methods for the purification of the flagellar filament and cryoEM data collection and image analysis.

Supercoiled filaments propel them all

In this issue of Cell, Kreutzberger and colleagues report the near-atomic-resolution, cryo-EM structures of the supercoiled filaments of both bacterial and archaeal motility machines. Despite the lack of homology, the supercoiled filament structures reveal shared fundamental features of prokaryotic locomotion and represent a prime example of convergent evolution.

Degradation and inactivation of chromosomal and plasmid encoded resistance genes/ARBs and the impact of different matrices on UV and UV/H2O2 based advanced oxidation process

This study reports a structured investigation on the degradation kinetics of different types (gyrAR,tetAR, qnrSR) and conformational forms (chromosomal, plasmids) of ARGs and mobile genetic elements (intl-1, plasmids) as a function of water matrix (DI water, phosphate buffer, wastewater) with UV and UV/H2O2 treatments. Extracellular, intracellular and the free-ARGs fate were tracked to infer the impact of various parameters on the degradation efficacy of the treatment process. The degradation profile of e-ARGs (118–454 bp) showed 1–4 log reductions but did not correlate strongly to amplicon size indicating the importance of active sites distribution and/or types of ARGs for UV induced gene damage. The i-ARGs showed similar degradation rates compared to e-ARGs for UV in phosphate buffer (PBS) but showed (1.3–2 times) slower rates for i-ARGs with UV/H2O2 due to scavenging of OH radicals by the cellular components. While the ARB inactivation was effective, but ARG damage was not supplemental as i-ARGs and f-ARGs persisted. In the wastewater matrix, generation of radical species was contributing to improved degradation rates from UV/H2O2 treatment, specifically for f-ARGs resulting in significantly improved degradation (p<0.05) compared to PBS. These indicates a non-selective nature of attack from radical species generated from UV irradiation on the effluent organic matter (EfOM) than sequenced based damage to the genes from UV. For the plasmid degradation, conformational differences pertaining to the supercoiled structures and intracellular forms influenced slower (1.2–2.8 times) UV mediated gene damage rate as opposed to chromosomal ARGs. These results can be useful for better assessing UV based treatment processes for effective ARG removal.

The interplay of supercoiling and thymine dimers in DNA.

Thymine dimers are a major mutagenic photoproduct induced by UV radiation. While they have been the subject of extensive theoretical and experimental investigations, questions of how DNA supercoiling affects local defect properties, or, conversely, how the presence of such defects changes global supercoiled structure, are largely unexplored. Here, we introduce a model of thymine dimers in the oxDNA forcefield, parametrized by comparison to melting experiments and structural measurements of the thymine dimer induced bend angle. We performed extensive molecular dynamics simulations of double-stranded DNA as a function of external twist and force. Compared to undamaged DNA, the presence of a thymine dimer lowers the supercoiling densities at which plectonemes and bubbles occur. For biologically relevant supercoiling densities and forces, thymine dimers can preferentially segregate to the tips of the plectonemes, where they enhance the probability of a localized tip-bubble. This mechanism increases the probability of highly bent and denatured states at the thymine dimer site, which may facilitate repair enzyme binding. Thymine dimer-induced tip-bubbles also pin plectonemes, which may help repair enzymes to locate damage. We hypothesize that the interplay of supercoiling and local defects plays an important role for a wider set of DNA damage repair systems.

The interaction of proanthocyanidins with DNA molecules studied by atomic force microscopy and spectroscopic method

The interactions of the naturally available botanicals with DNA molecules have received considerable attention owing to the potential to develop for medicinal agents. In this study, the interaction of proanthocyanidins with DNA molecules was studied by atomic force microscopy (AFM) and spectroscopic method. The AFM observation indicated that proanthocyanidins induced DNA molecules from double helix chains to the thick rope and toroids. The heights of the formed DNA structures are more than eight times than that of DNA double helix. Spectroscopic measurement results revealed that proanthocyanidins intercalated between the base pairs of DNA in the intercalative binding mode, which resulted in unwinding the DNA helix, twisting the DNA strands and finally transforming into the supercoiled DNA structures. All these results implied that DNA molecule is an important interaction target of proanthocyanidins, and the formed compact DNA structures have biological significance on the gene expression and regulation.

Halogen Bonding Mediated Hierarchical Supramolecular Chirality

As a highly directional force, halogen bonds based on σ-holes have potential to manipulate supramolecular chirality and build functional chiral systems, which however are largely unexplored. In this work, we report the manipulation of supramolecular chirality in hierarchically self-assembled systems via intracomponent halogen bonds. Cholesteryl cyanostilbene conjugates and 1,3,5-trifluoro-2,4,6-triiodobenzene formed a C3 symmetrical supramolecular complex with ultrahigh binding affinity and binding constants at 1011 order of magnitude. The halogen bonded propeller geometries exhibited inversed chirality as well as chiroptical activity compared to the pristine helically orientated aggregates. Halogen bonds enabled the engineering of nanoarchitectures, affording supercoiled helical structures and highly aligned nanotubes. This work unveils the role of halogen bonds in controlling supramolecular chirality, establishing a protocol to build functional chiroptical materials from species containing σ-holes and halogenated domains.

Supercoiling Structure-Based Design of a Trimeric Coiled-Coil Peptide with High Potency against HIV-1 and Human β-Coronavirus Infection.

Hexameric structure formation through packing of three C-terminal helices and an N-terminal trimeric coiled-coil core has been proposed as a general mechanism of class I enveloped virus entry. In this process, the C-terminal helical repeat (HR2) region of viral membrane fusion proteins becomes transiently exposed and accessible to N-terminal helical repeat (HR1) trimer-based fusion inhibitors. Herein, we describe a mimetic of the HIV-1 gp41 HR1 trimer, N3G, as a promising therapeutic against HIV-1 infection. Surprisingly, we found that in addition to protection against HIV-1 infection, N3G was also highly effective in inhibiting infection of human β-coronaviruses, including MERS-CoV, HCoV-OC43, and SARS-CoV-2, possibly by binding the HR2 region in the spike protein of β-coronaviruses to block their hexameric structure formation. These studies demonstrate the potential utility of anti-HIV-1 HR1 peptides in inhibiting human β-coronavirus infection. Moreover, this strategy could be extended to the design of broad-spectrum antivirals based on the supercoiling structure of peptides.

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Morphological Determinants of Carbon Nanomaterial-Induced Amyloid Peptide Self-Assembly.

Hybridizing carbon nanomaterials (CNMs) with amyloid fibrils—the ordered nanostructures self-assembled by amyloidogenic peptides—has found promising applications in bionanotechology. Understanding fundamental interactions of CNMs with amyloid peptides and uncovering the determinants of their self-assembly structures and dynamics are, therefore, pivotal for enriching and enabling this novel class of hybrid nanomaterials. Here, we applied atomistic molecular dynamics simulations to investigate the self-assembly of two amyloid peptides—the amyloidogenic core residues 16-22 of amyloid-β (Aβ16−22) and the non-amyloid-β core of α-synuclein (NACore68−78)—on the surface of carbon nanotubes (CNT) with different sizes and chirality. Our computational results showed that with small radial CNTs, both types of peptides could form β-sheets wrapping around the nanotube surface into a supercoiled morphology. The angle between β-strands and nanotube axes in the supercoil structure depended mainly on the peptide sequence and CNT radius, but also weakly on the CNT chirality. Large radial CNTs and the extreme case of the flat graphene nanosheet, on the other hand, could nucleate amyloid fibrils perpendicular to the surface. Our results provided new insights of hybridizing CNMs with amyloid peptides and also offered a novel approach to manipulate the morphology of CNM-induced amyloid assembly by tuning the surface curvature, peptide sequence, and molecular ratio between peptides and available CNM surface area, which may be useful in engineering nanocomposites with high-order structures.

Characterizing Writhed Geometries in Open and Closed Supercoiled DNA Structures

Linking number, twist, and writhe are common quantities used in the construction and analysis of supercoiled DNA structures. While direct evaluation of writhe via the Gaussian integral formulation proves useful in analyzing cases of closed DNA structures such as minicircles, this method does not hold for all cases of open curve geometries such as linear DNA helices. Additionally, methods of artificial closure for evaluation of the Gaussian integral on open curves are non-trivial and may lead to geometric misrepresentation of the helical axis. Furthermore, direct evaluation of the Gaussian integral does yield distinct information with respect to the local and non-local contributions to the writhe, which may distinguish between buckling and extension type deformations to the DNA helix. Here, we discuss extensions of writhe and the Călugăreanu-White-Fuller theorem to open curve geometries independent of artificial closure and their applications to the analysis of supercoiled DNA conformations. Applications of decomposing writhe and its extensions into local and non-local components in the analysis of DNA conformations are discussed along with relations between the varying writhe methods in the context of magnetic/optical tweezers experiments and plectonemic linear DNA helices. These plectonemes are generated using coarse-grained molecular dynamics techniques. Finally, we present a user-friendly program for evaluating these methods directed towards applications in analyzing molecular dynamics trajectories that includes support for use with multiple existing MD softwares.

Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.

Supercoiling DNA optically

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.