Abstract
dc:description.abstractOver two decades ago, scientists created the first nanopore- an α-hemolysin channel inserted into a lipid bilayer separating two buffered KCl-filled compartments. Today, nanopores are often associated with sequencing – likely due to the immense success of companies like Oxford Nanopore Technologies. However, nanopores can also be made of inorganic materials with tuneable diameters, presenting new opportunities in the fields of biosensing and diagnostics. In the nanopore sensing technique, molecules are driven through the nanosized opening between two chambers of salt solution using forces induced by electric fields. Upon applying an electric field across the nanopore, the voltage drives electrically charged molecules, like DNA, through the nanopore. As molecules move through the pore, the liquid containing the salt ions is displaced. The drop in liquid volume in the nanopore correlates to an increase in resistance and thus a drop in current. This current drop can provide information about the charge, molecular weight, and conformation of the analyte. Because of the this, nanopores can also act as an ideal platform for sensing proteins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated (Cas) protein system is sequence-specific RNA-guided protein duo that enables binding to a specific nucleic-acid sequence and consequent binding followed by cleavage. The CRISPR-Cas system has recently emerged as a revolutionary and widely employed gene editing tool. For use in diagnostic assays, we first need to benchmark the specificity of the ribonucleoprotein complexes (RNPs) – something we can do using nanopores and DNA nanotechnology. We can use the combination of nanopores and DNA nanotechnology to investigate the utility of the RNPs not only for differentiating single nucleotide changes in double-stranded DNA, but also in single stranded RNA, through the construction of RNA-DNA hybrid nanostructures. The ability to detect single nucleotide changes is important in sensing for both small non-coding RNAs and ribosomal RNA. The technique can also be expanded to investigate proteins beyond Cas. For example, we can study interactions of, alpha-synuclein oligomers, a biomarker for Parkinson’s Disease, with various small-molecule drugs. DNA nanostructures allow for highly multiplexed evaluation of the efficacy of various drugs. The combination of nanopore sensing and DNA nanostructures enables single-molecule protein sensing. This can greatly impact not only our fundamental understanding of proteins and their behaviour but can influence areas of biotechnology where protein sensing is essential to both the diagnosis of diseases as well as the evaluation of novel therapeutic approaches.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Sandler, Sarah
- Advisors dc:contributor.advisor
-
- Keyser, Ulrich
- Knowles, Tuomas
Subjects
dc:subject × 6Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
- 0000-0001-9689-8684
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/368210