Understanding and Designing Novel NanoPores

Nanopores are essential for the transport of molecules into and out of our cells. We are trying to learn how nature builds and uses nanopores by creating our won designer nanopores. A two-pronged approach is used. In one half of the project, we are building larger versions of these nanopores – still more 100 times thinner than a human hair – to understand the design principles. With the identified design rules we build nanopores from DNA to mimic functions and possibly outperform natural nanopores. During the project we have built microfluidic model systems that allowed us to test the physical principles that govern the transport of molecules on the nanoscale. In order to visualise molecules we recreated model systems that were 1000 times larger that the molecular systems. Working on the micrometre-scale we could employ optical microscopy and particle tracking to investigate the forces that influence the transport through confinement. The main outcomes of the project include the successful validation of model prediction that can help to reveal the unknown energy landscapes in ion-channels, nanopores and during protein folding. In addition, our microfluidic systems yield unprecedented data for theoreticians to develop novel ideas to better understand and hence improve models that describe molecular systems and their dynamics. In the future, our developments will allow insights into systems driven out-of-equilibrium.

In parallel to the statistical physics focus, we used DNA nanotechnology to create three-dimensional assemblies that can be used for the analysis of biomolecules using the above mentioned nanopores. DNA nanotechnology uses small DNA molecules as a building material for self-assembly, akin to molecular Lego bricks, that can self-assemble into any shape. On the nanoscale form defines function and hence DNA nanotechnology is ideally suited to investigate physics on this length scale. During the project we answered fundamental questions on the role of polymer dynamics for the transport of molecules through nanopores. The findings will help to improve the functionality of biosensors that may rival the resolution of optical microscopes by all-electrical measurements.

We have made major contributions in three areas with only major breakthroughs highlighted. In total the project generated almost 50 publications many in major international journals.
Statistical mechanics in confinement: In our Science Advances paper from 2020, the exquisite experimental control of our microfluidic model systems allowed the direct measurement of first-passage times. We uncovered key details of the energy landscapes that underpin a range of experimental systems through quantitative analysis of first-passage time distributions. By combined study of colloidal dynamics in confinement, transport through a biological pore, and the folding kinetics of DNA hairpins, we demonstrate conclusively how a short-time, power-law regime of the first-passage time distribution reflects the number of intermediate states associated with each of these processes, despite their differing length scales, time scales, and interactions. We thereby establish a powerful method for investigating the underlying mechanisms of complex molecular processes. In our Physical Review X paper from 2021 we investigated transition paths. Any experimentally observed trajectory of a diffusing particle is just one realization of the innumerable possible trajectories that could occur. While it is possible to mathematically quantify the relative likelihood for two such trajectories, these predictions cannot be tested directly because, strictly speaking, the probability of any single trajectory is zero. We provide the missing link between theory and experiment, by establishing a protocol to extract ratios of path probabilities from measured time series.

DNA membrane nanopores: We demonstrated the largest and smallest DNA nanopores and clarified the ion pathway through the lipid membrane. This is a major step to understand and establish design rules for DNA nanopores. With the DNA based protein detection we are currently finalising an agreement with a start up company to exploit the results. Based on these early successes we collaborated with the group of Prof Aksimentiev in Urbana-Champaign and discovered that our DNA nanopores act as artificial scramblases that connect the leaflets in lipid membranes. Our systems outperform natural enzymes by several orders of magnitude in transport rates. We are now able to build DNA scramblases can be controlled by external stimuli like salt concentration and through adaptations of the structure we can also change the lipid flipping rates. All these experiments were guided by intensive collaboration with molecular dynamics models and lead to more than 10 publications on all aspects of these DNA-based ion channels. The work on DNA-based scramblases is also filed as an US patent.

Nanopores sensing: At the start of the project we invented DNA carriers that allow for the multiplexed identification of proteins in complex mixtures. The work published in Nature nanotechnology 2016 was awarded with the Helmholtz Price for Applied Metrology and lead to a number of major breakthroughs in the nanopore field. Most notably we used the structured DNA carriers to measure the velocity of a molecule during translocation and discovered a two stage process with the DNA molecule slowing down and speeding up during the process. The work was finally published in Nature Physics 2021. The results are remarkable as we compare two different nanopore systems, namely glass nanopores and membrane nanopore. Both yield qualitatively similar results showing that polymer dynamics can be investigated with nanopores and present generic principles in the translocation process. In addition to these fundamental work, we used the DNA carriers for a multitude of applications including the detection of single nucleotide variations in small oligos, DNA data storage application and DNA hard drives, and the investigation of weak binders with a combination of DNA nanotechnology and strand displacement. We are now using these ideas for the analysis of RNA molecules with two patents recently filed and a start-up founded by one of the postdoctoral researchers and the PI.

After the end of the project we will continue the work with a proof-of-concept grant that will help translate the work from the lab to the application stages.

Periodic Reporting for period 4 - DesignerPores (Understanding and Designing Novel NanoPores)

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