Unterstanding the interaction of DNA nanopores with lipid bilayers

Lead Research Organisation: University College London
Department Name: Chemistry


Chemistry is key to re-create synthetic versions of bio-macromolecular structures that are engineered for biomedical applications. The aim of this project is to generate synthetic versions of membrane pores and channels with DNA to achieve unprecedented molecular control for biosensing, targeted cell killing, and synthetic biology (Science 352, 890-891 (2016)). Synthetic DNA nanopores that mimic biological behaviour and insert into membranes have attracted considerable scientific interest. Recently, DNA pores have been described that selectively kill bacteria. This project will determine the molecular determinants of the interaction to obtain a more fundamental understanding. The insight will be exploited to develop new antibacterial approaches.


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509577/1 01/10/2016 30/09/2021
1849570 Studentship EP/N509577/1 20/02/2017 19/02/2020 Katya Ahmad
Description In the first phase of this study, we used ensemble-based coarse-grained molecular dynamics (CG-MD) simulations to investigate the long-time dynamics of TEG-cholesterol anchored DNA nanopores (DNP's) in a range of conditions; at high and low salt concentrations, in free solution and within a POPC bilayer. The comprehensive sampling of conformational states provided by this protocol allows us to predict and explain the changes in the structural properties of DNP's in different experimental conditions. Our simulations show that the DNP adopts a globular, bloated conformation in solution, and is prone to localised duplex fraying in high-salt conditions but maintains a tight barrel-like structure when spanning a membrane due to toroidal pore formation. Overall, the membrane-spanning DNP's barrel structure is stabilised in high salt conditions, where it maintains a stable, ellipsoidal lumen. Ensembles of microsecond-long membrane simulations reveal that, in low salt conditions, the DNP preferentially adopts a side-on membrane binding mode, whereas high salt conditions confer an increased propensity for transmembrane binding due to the higher electrostatic screening provided under these conditions. These findings align well with the principles of hydrophobic matching, and our computed values for the average pore dimensions, lumen width and ion conductance are in excellent agreement with experimental values, attesting to the reliability and precision of our ensemble-based CG - MD protocol.

We also investigated the conductance properties of the membrane spanning DNP's. I decided on using the GROMACS Computational Electrophysiology protocol (CompEl) to derive the current-voltage relationship of the membrane spanning pore in 1.0 M NaCl. A double-bilayer system was constructed by cloning the fully equilibrated 1.0 M NaCl TEG-C NP/POPC system in the z-direction, such that two distinct solvent compartments (A and B) were formed, corresponding to the cis and trans compartments in a traditional chip-based parallel bilayer recording instrument. Virtual cylinders were defined around the two parallel membrane-spanning pores, a point charge difference (q) was applied to the system in each simulation, and the net influx of cations and anions into each compartment was recorded every 0.1 ns. Whenever the ion count in each compartment differed from the reference count (defined by q), ions from one compartment were swapped with water molecules from the other compartment to restore the reference count. Each simulation was run for 100 ns, and each trajectory was split into 20 ns slices.For each trajectory slice, the potential difference (V) was calculated using the gmx potential tool, and the instantaneous current flowing through each pore (I) was calculated. Twenty-five replicas were performed in total; nine with q = 0, eight with q = 2, and eight with q = 4. The frequency of ion/water swaps caused the instantaneous potential difference to fluctuate sharply throughout each simulation, so some of the trajectory slices were omitted as the potential difference rose beyond the range of voltages that are typically employed in electrophysiology experiments on DNA nanopores ( 100 mV). A total of 138 usable data points were extracted from the full set of trajectories, and these were invoked to construct a current-voltage (I/V) curve. Bootstrap error analysis confirmed that twenty-five replicas yielded a converged value for the average conductance G, which was taken as the slope of the line fitted to the I/V curve. The computed average conductance was 1.48 ? 0.80 nS, which is in good agreement with the experimental average of 1.59 ? 0.07 ns. I noticed that when higher voltages (above 100 mV) were included in the I/V plots, this resulted in a marked decrease in the average conductance of the pore. Similar findings have been reported in experiments. The results from these simulations are still being investigated.

After equilibrating the three models (with fluorophores restrained in their position 2nm above the pore lumen, and the nanopore restrained within the membrane), I performed an ensemble of pulling simulations for each one. Two translocation pathways emerged for each model; one where the fluorophore translocated through the lumen, and one where translocation occurred around the pore (between the membrane-pore interface). I set up a series of umbrella sampling simulations (52 for each model) for each pathway, and from these I will extract the potential of mean force, allowing me to compare the free energy of translocation for each fluorophore, and each pathway.
Exploitation Route These findings can inform the design of future DNA nanopores, depending on their intended purpose. For increased rigidity, a 5-helix bundle may be more suitable, and small hydrophobic modifications on the interior of the pore may decrease inter-helix repulsion enough to stabilise the membrane-spanning orientation.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

Title Ensemble-Based Coarse-Grained Molecular Dynamics 
Description Combining MARTINI CG MD simulations of large bimolecular systems with an ensemble-based protocol, to yield reproducible results quickly and relatively cheaply. 
Type Of Material Improvements to research infrastructure 
Year Produced 2017 
Provided To Others? No  
Impact Individual MD trajectories are highly sensitive to their starting conditions, and the temporal evolution of an individual trajectory is stochastic, meaning that neighbouring trajectories with different initial velocities diverge quickly. One cannot assume that the conformational space has been sufficiently sampled from a handful of repeat simulations without performing appropriate error analyses on the computed data to determine whether or not the computed average is representative of the true ensemble average. Therefore, the reliability of the results produced in such studies is uncertain. In this work, we address this issue by employing an ensemble-based protocol, according to which a set of N concurrent "replicas" are run, producing a stable ensemble average and associated fluctuations such that running N+1 would not alter the behaviour significantly. Running ensembles of computationally inexpensive CG simulations enhances the sampling of conformational space, giving reliable results in a rapid and reproducible way. For uncertainty quantification, we have employed the bootstrap method for calculating the standard error associated with ensemble-averaged macroscopic properties of the CG TEG-C NP systems; namely the pore height (for all four models), and the bilayer thickness (for the membrane models). This statistical method has been utilised successfully in many ensemble-based all-atom MD studies, where the typical simulation duration is fairly short (4 - 20 ns), but its use in CG studies at longer timescales has not yet been reported. In this work, we have achieved similar success to previous AA studies, in terms of error control and reproducibility. 
Description UCL Postgraduate Open Day Poster Session 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I presented a poster on my research at the UCL Postgraduate open day, and engaged with prospective PhD students about the benefits of pursuing a PhD
Year(s) Of Engagement Activity 2019