Combined single-molecule fluorescence and optical single-channel recording for electro-optical protein nanopore sensing

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Over the past 30 years, nanopore sensing has emerged as a powerful single-molecule detection method capable of sequencing DNA and RNA, detecting proteins and metabolites, and studying chemical reactions at the single-molecule level. To further increase its sensing capabilities, as well as provide a deeper insight into its working mechanism a second, independent means of detection is required. In this context, electro-optical sensing, which couples the electrical readout to a simultaneous optical signal has attracted much interest. One example is single-molecule fluorescence, which can report on structural changes and provide spatial information that the current recording lacks. This has been implemented successfully in solidstate nanopores to quantitate CpG islands in DNA, provide a means to detect the presence of specific proteins and DNA sequences, as well as sequence DNA optically. However, closer examination of the literature shows that simultaneous electro-optical detection has yet to be achieved with protein nanopores. This is reminiscent of the lack of progress that has been made in successfully coupling single-molecule fluorescence to single-channel electrophysiology in the context of ion channel research, even though such a technique was touted to be the next quantum leap already in the 1990s. We and others have previously highlighted that several experimental difficulties lie at the basis for this, which we believe to also explain the lack of literature performing electro-optical detection with protein nanopores. Firstly, conventional black lipid membrane setups are fragile and do not readily allow optical interrogation with single-molecule resolution. Secondly, in custom-built setups that do allow single-molecule imaging of proteins embedded in lipid bilayers, it has been proven very difficult to link the optical and electrical signals in a meaningful way. The work performed in the context of this thesis seeks to overcome this by marrying single molecule fluorescence to optical single-channel recording to investigate DNA capture and unzipping by a protein nanopore embedded in a droplet-interface bilayer.

This thesis first discusses the development of the custom-built optical setup and image processing pipeline required to extract single-channel kinetics from optical single-channel recording before it is applied to investigate the capture and unzipping of a thrombin-binding aptamer inside a modied CsgG protein nanopore provided by Oxford Nanopore Technologies. Subsequently, the optimisation of DNA constructs designed for simultaneous electro-optical is discussed. Then, we demonstrate successful electro-optical detections of DNA capture and unzipping and highlight that it allows the identification of a population with the expected unzipping kinetics, thereby providing the first demonstration of electro-optical detection inside protein nanopores. A final chapter is dedicated to current limitations and improvements, as well as an outlook on follow-up investigations.
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMark Wallace (Supervisor) & Rivka Isaacson (Supervisor)

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