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B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. \"Bubble\" templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context.

Sensitivity of cell-free circulating tumour DNA (ctDNA) assays is often hampered by the limited quantity of intact mutant nucleotide fragments. To overcome the issue of substrate limitation in clinical applications, we developed an enrichment method utilizing pyrrole-imidazole (PI) polyamides and their ability to bind the minor groove of B-DNA. We present here a proof-of-concept experiment to enrich specific mutant KRAS alleles with biotinylated PI polyamides. We investigated the clinical feasibility of incorporating PI polyamides to detect KRAS mutations in ctDNA from 40 colorectal cancer (CRC) patients, of whom 17 carried mutations in KRAS . After enriching ctDNA with those polyamides, we used digital PCR to detect several common KRAS codon 12 mutations. Enrichment by biotinylated PI polyamides improved the sensitivity of ctDNA analysis (88.9% vs. 11.1%, P  < 0.01) in 9 non-metastatic mutation-positive patients. We observed no differences in performance for the 8 metastatic subjects (100% vs. 75%, P  = 0.47). In the remaining 23/40 patients with wild type KRAS codon 12, no mutant alleles were detected with or without polyamide-facilitated enrichment. Enriching B-form of ctDNA with PI polyamides significantly improved the assay sensitivity in detecting KRAS mutations in non-metastatic CRC patient samples.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterised by antibodies to DNA (anti-DNA) and other nuclear macromolecules. Anti-DNA antibodies are markers for classification and disease activity and promote pathogenesis by forming immune complexes that deposit in the tissue or stimulate cytokine production. Studies on the antibody response to DNA have focused primarily on a conformation of DNA known as B-DNA, the classic right-handed double helix. Among other conformations of DNA, Z-DNA is a left-handed helix with a zig-zag backbone; hence, the term Z-DNA. Z-DNA formation is favoured by certain base sequences, with the energetically unfavourable flip from B-DNA to Z-DNA dependent on conditions. Z-DNA differs from B-DNA in its immunogenicity in animal models. Furthermore, anti-Z-DNA antibodies, but not anti-B-DNA antibodies, can be present in otherwise healthy individuals. In SLE, antibodies to Z-DNA can occur in association with antibodies to B-DNA as a cross-reactive response, rising and falling together. While formed transiently in chromosomal DNA, Z-DNA is stably present in bacterial biofilms; biofilms can provide protection against antibiotics and other challenges including elements of host defence. The high GC content of certain bacterial DNA also favours Z-DNA formation as do DNA-binding proteins of bacterial or host origin. Together, these findings suggest that sources of Z-DNA can enhance the immunogenicity of DNA and, in SLE, stimulate the production of cross-reactive antibodies that bind both B-DNA and Z-DNA. As such, DNA can act as a molecular chameleon that, when stabilised in the Z-DNA conformation, can drive autoimmunity.

Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a ‘cogwheel’ mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA–DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme. DNA sliding clamps are ring-shaped proteins that encircle DNA and harbour polymerases and other factors that promote processive DNA replication. Here the authors use X-ray crystallography, NMR and MD simulations to propose a model for a PCNA sliding mechanism that relies on short-lived polar interactions.

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA–protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T–G, T–T, or T–C) in a double-stranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.

Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B–B (or A–A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B–A (or A–B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible—and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5′ and 3′ ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions—all readily testable—as will be described in this review.

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-Hg II -T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-Hg II -(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-Hg II -(N4)C base pairs and greater A-form characteristics. Ten-fold different 1 J coupling constants ( 15 N, 199 Hg) are observed for (N3)C-Hg II (114 Hz) versus (N4)C-Hg II (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-Hg II -T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding Hg II , demonstrating that C-Hg II -T has unique switching potential in DNA-based materials and devices. Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here, the authors prepared a metallo-DNA duplex including two C-Hg(II)-T base pairs separated by six normal Watson-Crick base pairs and investigated its solution structure and dynamics using NMR spectroscopy.

Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody’s incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.

GapR is a nucleoid-associated protein required for the cell cycle of Caulobacter cresentus . We have determined new crystal structures of GapR to high resolution. As in a recently published structure, a GapR monomer folds into one long N-terminal α helix and two shorter α helices, and assembles into a tetrameric ring with a closed, positively charged, central channel. In contrast to the conclusions drawn from the published structures, we observe that the central channel of the tetramer presented here could freely accommodate B-DNA. Mutation of six conserved lysine residues lining the cavity and electrophoretic mobility gel shift experiments confirmed their role in DNA binding and the channel as the site of DNA binding. Although present in our crystals, DNA could not be observed in the electron density maps, suggesting that DNA binding is non-specific, which could be important for tetramer-ring translocation along the chromosome. In conjunction with previous GapR structures we propose a model for DNA binding and translocation that explains key published observations on GapR and its biological functions.

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s−1, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.