James Clarke

Highly parallel direct RNA sequencing on an array of nanopores

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Degradative transport of cationic amphiphilic drugs across phospholipid bilayers

Drug molecules must cross multiple cell membrane barriers to reach their site of action. We present evidence that one of the largest classes of pharmaceutical drug molecules, the cationic amphiphilic drugs (CADs), does so via a catalytic reaction that degrades the phospholipid fabric of the membrane. We find that CADs partition rapidly to the polar–apolar region of the membrane. At physiological pH, the protonated groups on the CAD catalyse the acid hydrolysis of the ester linkage present in the phospholipid chains, producing a fatty acid and a single-chain lipid. The single-chain lipids rapidly destabilize the membrane, causing membranous fragments to separate and diffuse away from the host. These membrane fragments carry the drug molecules with them. The entire process, from drug adsorption to drug release within micelles, occurs on a time-scale of seconds, compatible with in vivo drug diffusion rates. Given the rate at which the reaction occurs, it is probable that this process is a significant mechanism for drug transport.

Pressure–temperature phase behaviour of natural sphingomyelin extracts

Sphingomyelin is the only sphingolipid occurring naturally in mammalian cells and can form up to 50% of the total phospholipid content of the myelin sheath which surrounds nerves. Having predominantly long, saturated acyl chains, it has a relatively high chain melting temperature and has been strongly associated with formation of lipid microdomains. Here, the lyotropic phase behaviour of sphingomyelin from three different natural sources (bovine brain, egg yolk and milk) in excess water is studied as a function of temperature and pressure by small- and wide-angle X-ray scattering, and solid state NMR. The different hydrocarbon chain length distributions of the three lipid extracts results in significant differences in their gel phase structure; both the bovine brain and egg yolk sphingomyelins can form a ripple gel phase but milk sphingomyelin forms an interdigitated gel phase due to the high degree of chain mismatch in its longer hydrocarbon chain components.

Using membrane stress to our advantage

The nature of the bilayer motif coupled with the ability of lipids and proteins to diffuse freely through this structure is crucial to the viability of cells and their ability to compartmentalize domains contained therein. It seems surprising to find then that biological as well as model membranes exist in a dynamic state of mechanical stress. The stresses within such membranes are surprisingly large, typically reaching up to 50 atm (1 atm=101.325 kPa) at the core of the membrane and vary as a function of depth. The uneven distribution of lateral pressures within monolayer leaflets causes them to bend away from or towards the water interface. This can result in the formation of complex, self-assembled mesophases, many of which occur in vivo. Our knowledge of the principles underlying membrane mechanics has reached the point where we are now able to manipulate them and create nano-structures with reasonable predictability. In addition, they can be used both to explain and control the partitioning of amphipathic proteins on to membranes. The dependence of the dynamics of membrane-bound proteins and the chemical reactivity of amphipathic drug molecules on membrane stresses suggests that Nature itself takes advantage of this. Understanding and manipulating these internal forces will be a key element in creating self-assembled, biocompatible, nanoscale cell-like systems.

Cholesterol containing model membranes studied by multinuclear solid state NMR spectroscopy

Ternary phase diagrams have been shown to be very useful in understanding the phenomena of lipid rafts. Recently a comprehensive ternary phase diagram for DPPC-d62/DOPC/Chol using static 2H-NMR has been reported [S. L. Veatch, O. Soubias, S. L. Keller, and K. Gawrisch Proceedings of the National Academy of Sciences of the United States of America, 2007, 104, 17650–17655]. However, it is often difficult to interpret these ternary phase diagrams as there are many possible ambiguities, in particular, the extent of the gel(so)–Lo–Lα(ld) three phase region and the clear demarcation between the gel(so) and Lo phase. Here we examine the canonical model lipid raft system by a multinuclear approach using 2H static and 13C, 31P Magic Angle Spinning (MAS) NMR. By using deuterated DPPC (DPPC-d62) it is possible to examine changes in the methylene chain order parameters of the saturated lipid whilst changing the ratio of saturated to unsaturated phospholipid. From these data it is possible to propose the existence at low temperatures (<20 °C) of a disrupted or disordered gel-type phase at ratios as high as 1:1:1 DOPC/DPPC/Chol, in equilibrium with fluid phases. The presence of a gel-type phase was also confirmed by 13C and 31P MAS NMR. This disrupted gel-type phase has different properties in terms of axial rotation than that of the liquid ordered phase (Lo). We have also shown that it is possible to observe different quadrupolar splittings for the inequivalent sn-1 and sn-2 methyl groups in a gel phase. We have concluded that this is not always indicative of a liquid-ordered lamellar Lo phase, as previously assumed. Above the melting point of the gel-type phase (∼20 °C), we observe a single fluid environment for both DPPC-d62 and Chol-d1 on the basis of the 2H-NMR spectra, which are an average of a fluid disordered (Lα) and an Lo phase. In this fluid regime, the order parameters for the 1:1:1 system DPPC-d62/DOPC/Chol are close to that of the binary (1:1) DPPC-d62:Chol system, indicating non-random distribution of the DPPC-d62. Increasing the ratio of DOPC to DPPC, from 1:1:1 to 1.5:1:1 to 2:1:1 in the ternary system, causes an increase in chain length of the DPPC-d62 in the fluid phase.

Epoxidation of bromoallenes connects red algae metabolites by an intersecting bromoallene oxide – Favorskii manifold

DMDO epoxidation of bromoallenes gives directly α,β-unsaturated carboxylic acids under the reaction conditions. Calculated (ωB97XD/6-311G(d,p)/SCRF = acetone) potential energy surfaces and (2)H- and (13)C-labeling experiments are consistent with bromoallene oxide intermediates which spontaneously rearrange via a bromocyclopropanone in an intersecting bromoallene oxide - Favorskii manifold.

Solid State NMR of Lipid Model Membranes

In this chapter we describe the use of solid state nuclear magnetic spectroscopy to study the structure of lyotropic phases and lipid model membranes and show its ability to probe, site specifically, at a sub-Ångstrom resolution. Here, we demonstrate the immense versatility of the technique and its ability to provide information on the different liquid crystalline phases present. A multinuclear for example (31)P, (1)H, and (13)C approach is able to elucidate both the structure and dynamics over a wide variety of timescales. This coupled with a non-perturbing label (2)H is able to provide information such as the order parameters for a wide variety of different liquid phases.