Scott L. Cockroft

A Single-Molecule Nanopore Device Detects DNA Polymerase Activity with Single-Nucleotide Resolution

The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first observation of the base-by-base DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single-nucleotide primer extensions with high sensitivity and spatial resolution (<or=2.4 A). The device is assembled in a suspended lipid membrane by threading and mechanically capturing a single strand of DNA-PEG copolymer inside an alpha-hemolysin protein pore. Single-nucleotide primer extensions result in successive displacements of the template DNA strand within the protein pore, which can be monitored by the corresponding stepped changes in the ion current flowing through the pore under an applied transmembrane potential. The system described thus represents a promising advance toward nanopore-mediated single-molecule DNA sequencing concept and, in addition, might be applicable to studying a number of other biopolymer-protein interactions and dynamics.

Chemical double-mutant cycles: dissecting non-covalent interactions

Thermodynamic double-mutant cycles and triple-mutant boxes are widely employed for the experimental quantification of non-covalent interactions and cooperative effects in proteins. This review describes the application of these powerful methodologies to the study of non-covalent interactions in synthetic systems.

Substituent effects on aromatic stacking interactions

Synthetic supramolecular zipper complexes have been used to quantify substituent effects on the free energies of aromatic stacking interactions. The conformational properties of the complexes have been characterised using NMR spectroscopy in CDCl(3), and by comparison with the solid state structures of model compounds. The structural similarity of the complexes makes it possible to apply the double mutant cycle method to evaluate the magnitudes of 24 different aromatic stacking interactions. The major trends in the interaction energy can be rationalised using a simple model based on electrostatic interactions between the pi-faces of the two aromatic rings. However, electrostatic interactions between the substituents of one ring and the pi-face of the other make an additional contribution, due to the slight offset in the stacking geometry. This property makes aromatic stacking interactions particularly sensitive to changes in orientation as well as the nature and location of substituents.

How much do van der Waals dispersion forces contribute to molecular recognition in solution

The emergent properties that arise from self-assembly and molecular recognition phenomena are a direct consequence of non-covalent interactions. Gas-phase measurements and computational methods point to the dominance of dispersion forces in molecular association, but solvent effects complicate the unambiguous quantification of these forces in solution. Here, we have used synthetic molecular balances to measure interactions between apolar alkyl chains in 31 organic, fluorous and aqueous solvent environments. The experimental interaction energies are an order of magnitude smaller than estimates of dispersion forces between alkyl chains that have been derived from vaporization enthalpies and dispersion-corrected calculations. Instead, it was found that cohesive solvent-solvent interactions are the major driving force behind apolar association in solution. The results suggest that theoretical models that implicate important roles for dispersion forces in molecular recognition events should be interpreted with caution in solvent-accessible systems.

Biological Nanopores for Single‐Molecule Biophysics

Financial support is graciously acknowledged from the Chinese Scholarship Council and the MTEM International Studentship scheme (scholarship to L. M), The Royal Society of Chemistry and The Leverhulme Trust. We thank J. Chu of the Ghadiri laboratory (The Scripps Research Institute) for helpful discussions.

Experimental Measurement of Noncovalent Interactions Between Halogens and Aromatic Rings

Chemical double mutant cycles have been used to quantify the interactions of halogens with the faces of aromatic rings in chloroform. The halogens are forced over the face of an aromatic ring by an array of hydrogen‐bonding interactions that lock the complexes in a single, well‐defined conformation. These interactions can also be engineered into the crystal structures of simpler model compounds, but experiments in solution show that the halogen–aromatic interactions observed in the solid state are all unfavourable, regardless of whether the aromatic rings contain electron‐withdrawing or electron‐donating substituents. The halogen–aromatic interactions are repulsive by 1–3 kJ mol−1. The interactions with fluorine are slightly less favourable than with chlorine and bromine.

Desolvation and substituent effects in edge-to-face aromatic interactions

Experimental measurements of aromatic edge-to-face interaction energies in both molecular torsion balances and supramolecular zipper complexes can be reliably estimated using a simple electrostatic solvation model and alpha/beta H-bond constants.

Man-made molecular machines: membrane bound

Natures molecular machines are a constant source of inspiration to the chemist. Many of these molecular machines function within lipid membranes, allowing them to exploit potential gradients between spatially close, but chemically distinct environments to fuel their work cycle. Indeed, the realisation of such principles in synthetic transmembrane systems remains a tantalising goal. This tutorial review opens by highlighting seminal examples of synthetic molecular machines. We illustrate the importance of surfaces for facilitating the extraction of work from molecular switches and motors. We chart the development of man-made transmembrane systems; from passive to machine-like stimuli-responsive channels, to fully autonomous transmembrane molecular machines. Finally, we highlight higher-order compartmentalised systems that exhibit emergent properties. We suggest that such higher-order architectures could serve as platforms for sophisticated devices that co-ordinate the activity of numerous transmembrane molecular machines.

Quantifying Solvophobic Effects in Nonpolar Cohesive Interactions

The hydrophobic effect plays a central role in determining the structure, activity, and properties of biomolecules and materials. In contrast, the general manifestation of this phenomenon in other solvents—the solvophobic effect—although widely invoked, is currently poorly defined because of the lack of a universally accepted descriptor. Here we have used synthetic molecular balances to measure solvent effects on aromatic, aliphatic, and fluorous nonpolar interactions. Our solvent screening data combined with independent experimental measurements of supramolecular association, single-molecule folding, and bulk phase transfer energies were all found to correlate well with the cohesive energy density (ced) of the solvent. Meanwhile, other measures of solvent cohesion, such as surface tension and internal pressure, gave inferior correlations. Thus, we establish ced as a readily accessible, quantitative descriptor of solvophobic association in a range of chemical contexts.

The Origin of Chalcogen-Bonding Interactions

Favorable molecular interactions between group 16 elements have been implicated in catalysis, biological processes, and materials and medicinal chemistry. Such interactions have since become known as chalcogen bonds by analogy to hydrogen and halogen bonds. Although the prevalence and applications of chalcogen-bonding interactions continues to develop, debate still surrounds the energetic significance and physicochemical origins of this class of σ-hole interaction. Here, synthetic molecular balances were used to perform a quantitative experimental investigation of chalcogen-bonding interactions. Over 160 experimental conformational free energies were measured in 13 different solvents to examine the energetics of O···S, O···Se, S···S, O···HC, and S···HC contacts and the associated substituent and solvent effects. The strongest chalcogen-bonding interactions were found to be at least as strong as conventional H-bonds, but unlike H-bonds, surprisingly independent of the solvent. The independence of the conformational free energies on solvent polarity, polarizability, and H-bonding characteristics showed that electrostatic, solvophobic, and van der Waals dispersion forces did not account for the observed experimental trends. Instead, a quantitative relationship between the experimental conformational free energies and computed molecular orbital energies was consistent with the chalcogen-bonding interactions being dominated by n → σ* orbital delocalization between a lone pair (n) of a (thio)amide donor and the antibonding σ* orbital of an acceptor thiophene or selenophene. Interestingly, stabilization was manifested through the same acceptor molecular orbital irrespective of whether a direct chalcogen···chalcogen or chalcogen···H-C contact was made. Our results underline the importance of often-overlooked orbital delocalization effects in conformational control and molecular recognition phenomena.