Nano-scale gene delivery systems; current technology, obstacles, and future directions.

Current medicinal chemistry (2018)

A Garcia-Guerra, TL Dunwell, S Trigueros

Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review.

Structural Mechanisms of Mechanosensitivity in the TREK-2 K2P Potassium Channel


SJ Tucker

The structural movement of the TM4 segment during pore gating in TREK1 channels


F Schulz, M Rapedius, SJ Tucker, T Baukrowitz

A conserved drug-binding site controls the selectivity filter gate in K2P K+ channels


M Schewer, F Schulz, U Mert, H Sun, T Koehler, M Tegtmeier, M Musinszki, H Belabed, M Nazare, SJ Tucker, T Baukrowitz

The effects of stretch activation on ionic selectivity of the TREK-2 K2P K+ channel.

Channels (Austin, Tex.) 11 (2017) 482-486

E Nematian-Ardestani, V Jarerattanachat, P Aryal, MSP Sansom, SJ Tucker

The TREK-2 (KCNK10) K2P potassium channel can be regulated by variety of polymodal stimuli including pressure. In a recent study, we demonstrated that this mechanosensitive K+ channel responds to changes in membrane tension by undergoing a major structural change from its 'down' state to the more expanded 'up' state conformation. These changes are mostly restricted to the lower part of the protein within the bilayer, but are allosterically coupled to the primary gating mechanism located within the selectivity filter. However, any such structural changes within the filter also have the potential to alter ionic selectivity and there are reports that some K2Ps, including TREK channels, exhibit a dynamic ionic selectivity. In this addendum to our previous study we have therefore examined whether the selectivity of TREK-2 is altered by stretch activation. Our results reveal that the filter remains stable and highly selective for K+ over Na+ during stretch activation, and that permeability to a range of other cations (Rb+, Cs+ and NH4+) also does not change. The asymmetric structural changes that occur during stretch activation therefore allow the channel to respond to changes in membrane tension without a loss of K+ selectivity.

Dynamic role of the tether helix in PIP2-dependent gating of a G protein-gated potassium channel.

The Journal of general physiology (2017)

E Lacin, P Aryal, IW Glaaser, K Bodhinathan, E Tsai, N Marsh, SJ Tucker, MSP Sansom, PA Slesinger

G protein-gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the "tether helix" (C-linker) that interact with the negatively charged PIP2 However, these crystal structures of neuronal GIRK channels in complex with PIP2 provide only snapshots of PIP2's interaction with the channel and thus lack details about the gating transitions triggered by PIP2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6'K), supports a complex and dynamic interaction with PIP2 When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP2 Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6' substitution reveal an open GIRK2 channel with PIP2 molecules adopting novel positions. This dynamic interaction with PIP2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gβγ subunits and ethanol.

Asymmetric mechanosensitivity in a eukaryotic ion channel.

Proceedings of the National Academy of Sciences of the United States of America 114 (2017) E8343-E8351

MV Clausen, V Jarerattanachat, EP Carpenter, MSP Sansom, SJ Tucker

Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli.

Speed of the bacterial flagellar motor near zero load depends on the number of stator units.

Proceedings of the National Academy of Sciences of the United States of America 114 (2017) 11603-11608

AL Nord, Y Sowa, BC Steel, C-J Lo, RM Berry

The bacterial flagellar motor (BFM) rotates hundreds of times per second to propel bacteria driven by an electrochemical ion gradient. The motor consists of a rotor 50 nm in diameter surrounded by up to 11 ion-conducting stator units, which exchange between motors and a membrane-bound pool. Measurements of the torque-speed relationship guide the development of models of the motor mechanism. In contrast to previous reports that speed near zero torque is independent of the number of stator units, we observe multiple speeds that we attribute to different numbers of units near zero torque in both Na+- and H+-driven motors. We measure the full torque-speed relationship of one and two H+ units in Escherichia coli by selecting the number of H+ units and controlling the number of Na+ units in hybrid motors. These experiments confirm that speed near zero torque in H+-driven motors increases with the stator number. We also measured 75 torque-speed curves for Na+-driven chimeric motors at different ion-motive force and stator number. Torque and speed were proportional to ion-motive force and number of stator units at all loads, allowing all 77 measured torque-speed curves to be collapsed onto a single curve by simple rescaling.

The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery.

Accounts of chemical research 50 (2017) 2496-2509

RK O'Reilly, AJ Turberfield, TR Wilks

Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 108 distinct molecules. To put this in context, there are 1013 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure-a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery-imagine being able to evolve molecules for light harvesting, or catalysts for CO2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous "DNA robots" have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution.

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

Nucleic Acids Research 45 (2017) 926-937

A Plochowietz, I Farrell, Z Smilansky, BS Cooperman, AN Kapanidis

Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel.

Structure (London, England : 1993) 25 (2017) 708-718.e2

P Aryal, V Jarerattanachat, MV Clausen, M Schewe, C McClenaghan, L Argent, LJ Conrad, YY Dong, ACW Pike, EP Carpenter, T Baukrowitz, MSP Sansom, SJ Tucker

The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.

A BEST example of channel structure annotation by molecular simulation.

Channels (Austin, Tex.) 11 (2017) 347-353

S Rao, G Klesse, PJ Stansfeld, SJ Tucker, MSP Sansom

An increasing number of ion channel structures are being determined. This generates a need for computational tools to enable functional annotation of channel structures. However, several studies of ion channel and model pores have indicated that the physical dimensions of a pore are not always a reliable indicator of its conductive status. This is due to the unusual behavior of water within nano-confined spaces, resulting in a phenomenon referred to as "hydrophobic gating". We have recently demonstrated how simulating the behavior of water within an ion channel pore can be used to predict its conductive status. In this addendum to our study, we apply this method to compare the recently solved structure of a mutant of the bestrophin chloride channel BEST1 with that of the wild-type channel. Our results support the hypothesis of a hydrophobic gate within the narrow neck of BEST1. This provides further validation that this simulation approach provides the basis for an accurate and computationally efficient tool for the functional annotation of ion channel structures.

Regulation of Two-pore Domain K plus Channels by Natural Effectors and Pharmacological Agents


M Schewe, F Schulz, U Mert, H Sun, H Belabed, M Musinszki, T Koehler, M Tegtmeier, M Nazare, EP Carpenter, SJ Tucker, T Baukrowitz

Amphiphilic DNA tiles for controlled insertion and 2D assembly on fluid lipid membranes: the effect on mechanical properties

Nanoscale 9 (2017) 3051-3058

C Dohno, S Makishi, K Nakatani, S Contera

Structural and Functional Response of a Mechanosensitive K2P K+ Channel to Asymmetric Membrane Tension


V Jarerattanachat, MV Clausen, P Aryal, EP Carpenter, MSP Sansom, SJ Tucker

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes.

Nature communications 7 (2016) 13025-

RR Ishmukhametov, AN Russell, RM Berry

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Magneto-electrical orientation of lipid-coated graphitic micro-particles in solution

RSC Adv. 6 (2016) 46643-46653

J Nguyen, S Contera, I Llorente García

Bilayer-Mediated Structural Transitions in the TREK-2 Mechanosensitive K2P Channel


P Aryal, V Jarerattanachat, SJ Tucker, MSP Sansom

Mutations targeting the plug-domain of the Shewanella oneidensis proton-driven stator allow swimming at increased viscosity and under anaerobic conditions.

Molecular microbiology 102 (2016) 925-938

S Brenzinger, L Dewenter, NJ Delalez, O Leicht, V Berndt, A Paulick, RM Berry, M Thanbichler, JP Armitage, B Maier, KM Thormann

Shewanella oneidensis MR-1 possesses two different stator units to drive flagellar rotation, the Na+ -dependent PomAB stator and the H+ -driven MotAB stator, the latter possibly acquired by lateral gene transfer. Although either stator can independently drive swimming through liquid, MotAB-driven motors cannot support efficient motility in structured environments or swimming under anaerobic conditions. Using ΔpomAB cells we isolated spontaneous mutants able to move through soft agar. We show that a mutation that alters the structure of the plug domain in MotB affects motor functions and allows cells to swim through media of increased viscosity and under anaerobic conditions. The number and exchange rates of the mutant stator around the rotor were not significantly different from wild-type stators, suggesting that the number of stators engaged is not the cause of increased swimming efficiency. The swimming speeds of planktonic mutant MotAB-driven cells was reduced, and overexpression of some of these stators caused reduced growth rates, implying that mutant stators not engaged with the rotor allow some proton leakage. The results suggest that the mutations in the MotB plug domain alter the proton interactions with the stator ion channel in a way that both increases torque output and allows swimming at decreased pmf values.

Designer cantilevers for even more accurate quantitative measurements of biological systems with multifrequency AFM

Nanotechnology 27 (2016) 132501-132501

S Contera