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Funding: Engineering and Physical Sciences Research Council [EP/M019195/1]; Engineering and Physical Sciences Research Council Studentship (to J.S.H.); Biotechnology and Biological Sciences Research Council [BB/J001694/2, BB/R021848/1]; ADTBio; University of Kentucky and NCI Cancer Center Support Grant [P30 CA177558]; The Carmen L. Buck Endowment; Emerging Fields Initiative of the Friedrich-Alexander-University of Erlangen-Nuremberg [Grant title ‘Chemistry in Live Cells’]; Wellcome Trust [099149/Z/12/Z]; Royal Society, University Research Fellowship (to J.E.L.). Funding for open access charge: University of Oxford. The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation. Publisher PDF

The mechanism by which 5-formylcytosine (fC) is recognised by enzymes involved in epigenetic modification and reading of DNA is not known, and recently an unusual DNA structure (F-DNA) was proposed as the basis for enzyme recognition of clusters of fC. We used NMR and X-ray crystallography to compare several modified DNA duplexes with the unmodified analogues and show that in the crystal state they all belong to the A-family, but in solution they are all members of the B-family. Contrary to the previous study, we find that 5-formylcytosine does not significantly affect the structure of DNA, though there are modest local differences at the modification sites. Hence, global conformation changes are unlikely to account for the recognition of this modified base, and our structural data favour a mechanism that operates at base-pair resolution for the recognition of 5-formylcytosine by epigenome-modifying enzymes.

To decode the function and molecular recognition of several recently discovered cytosine derivatives in the human genome - 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine - a detailed understanding of their effects on the structural, chemical, and biophysical properties of DNA is essential. Here, we review recent literature in this area, with particular emphasis on features that have been proposed to enable the specific recognition of modified cytosine bases by DNA-binding proteins. These include electronic factors, modulation of base-pair stability, flexibility, and radical changes in duplex conformation. We explore these proposals and assess whether or not they are supported by current biophysical data. This analysis is focused primarily on the properties of epigenetically modified DNA itself, which provides a basis for discussion of the mechanisms of recognition by different proteins.

Numerical simulation of hydrate dissociation in porous media is important to investigate future hydrate fuel extraction strategies and/or the impacts of climate change on the long-term stability of vulnerable near-surface hydrate deposits. The core-scale hydrate dissociation experiment of Masuda et al. (1999) represents an important experimental data set that can be used for benchmarking numerical simulators for this purpose. Data collected includes gas production, water production, boundary pressure and temperature from three internal observation points. At least six modeling studies exist within the literature seeking to simulate the gas production data and the temperature data. However, the pressure data and water production data are generally overlooked. In this article we present a set of numerical simulations capable of reconciling the Masuda et al. (1999) data set in its entirety. Improvements on existing modeling studies are achieved by: (1) using improved estimates of the initial hydrate saturation; (2) obtaining relative permeability parameters, a hydrate stability depression temperature and a convective heat transfer coefficient by calibration with the observed data; and (3) applying a new critical threshold permeability model, specifically to reconcile a relatively fast gas production with a relatively slow far-field boundary pressure response. A subsidiary finding is that permeability is significantly reduced in the presence of very low hydrate saturations. But more importantly, the multi-faceted effectiveness of the data set from Masuda’s experiment is clearly demonstrated for numerical simulation benchmarking in the future.

A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.