Nanopore sensing of single molecules application to RNAP-DNA complexes, fabrication of graphene-nanopore devices and translocation algorithm analysis

Nanopore sensing is a single-molecule technique that allows to probe the nano-bio-world with accuracy. It is a non-invasive technique, which means that the molecule of interest does not need to be labeled to be detected by the nanopore. Most of the studies done with nanopores use electrophoresis. With an electric field applied across the nanopore, charged molecules move through this tiny hole. When a molecule passes through the nanopore it creates a dip in current, called current blockage, because it partially blocks the ions from going through and so it increases the nanopore resistance. This phenomenon is observed using a refined ammeter that detects changes in current in the range of pA. At the Laboratory of Nanoscale Biology (LBEN), we are interested in very small biological units, and this thesis is focused on the RNA polymerase, the enzyme that catalyzes the transcription reaction. Transcription is the first step in gene expression where DNA is copied into RNA. It is extensively studied at the bulk level especially the regulation mechanism, which in cancerous cells is impaired. We were interested in studying this enzyme at the single-molecule level for its functional as well as molecular motor properties. This work was done in close collaboration with Pascal Cousin at the Center for Integrative Genomics (CIG) in the group of Nouria Hernandez who helped to design the protein-DNA complexes and analyze the data. With nanopore sensing we were able to observe RNA polymerase-DNA complexes translocate through nanopores and capable to distinguish between individual complexes and bare RNA polymerase. We were also able to observe orientation of RNA polymerase in the nanopore whether flow or electric field predominates. The complexity of the signals from the protein-DNA complexes experiment motivated the development of a level detection software. This software was written in collaboration with Pierre Granjon from GIPSA-lab in Grenoble who had the expertise on a change detection method called the CUSUM algorithm. Our software was designed to analyze in details current blockages in nanopore signals with very little prior knowledge on the signal. With this work one can separate events according to their number of levels and study those sub-populations separately. This method also allows a global statistical view of all levels by plotting what we call a level histogram. We demonstrate that with such a technique one can identify populations that cannot be seen otherwise. As the theoretical literature has shown, it should be possible to sequence a ssDNA with a nanopore using transverse electronics for tunneling current measurement. In this thesis, we will discuss the fabrication work done on the first generation of graphene-nanopore devices and how we overcame the fabrication issues to produce reliable devices.