This work describes advances in nanopore sequencing technology as they apply to RNA. RNAs in the cellular environment have a wide range of functions and structures, by nature being much more dynamic in their activities than their chemical counterpart, DNA. The diversity of activities and functions that RNAs assume in the cell is reflected in the diverse research interests of those who study RNA. Any effort to develop nanopore-based direct RNA sequencing applications requires accounting for this diversity.
Given the spectrum of interests in RNA biology, much of this dissertation attempts to address a fundamental challenge in directly analyzing RNAs by nanopore - how to deliver and read diverse classes of RNA at single-nucleotide resolution using a nanopore sequencer? The introductory first chapter describes background on nanopores as sequencing sensors and samples of some biology surrounding this diverse class of molecules. The state of the art and challenges in RNA sequencing in general are discussed.
The second chapter of this work demonstrates a method specifically preparing tRNA for nanopore sequencing. The primary challenge in this effort is to be able to consistently load a single tRNA end and process the tRNA strand through the pore in a linear order. A molecular adapter composed of double-stranded DNA ligated to the tRNA termini facilitates this process. The adapter specifically targets tRNA by hybridizing to the universally conserved CCA tail of tRNA. It also provides a binding site for ϕ29 DNA polymerase, which acts a molecular brake on RNA under non-catalytic conditions. These two features allow for discrimination between two tRNA species from E. coli. This work demonstrates that it is possible to use a nanopore to analyze individual tRNAs as linear strands, which is a necessary prerequisite for nanopore-based tRNA sequencing.
The third chapter of this work describes applying the Oxford Nanopore MinION sequencer to directly sequence 16S ribosomal RNA (16S rRNA). Demonstration of direct RNA sequencing of poly-adenylated RNA by Oxford Nanopore in August of 2016 was a critical development towards direct sequencing of RNA in general. The work described in Chapter 3 again involves designing an adapter, but this time specific for prokaryotic 16S rRNA. This adapter can be used with the existing Oxford Nanopore direct RNA sequencing kit. Sequencing of 16S rRNA from E. coli and other three other microbial species is demonstrated. The data and confirmatory experiments show that it is possible to directly detect modified ribonucleotides in nanopore-based sequencing data, which has long been a promised benefit of direct RNA sequencing methods. The 16S rRNA adapter is designed to hybridize to conserved 3′-end sequence of E. coli 16S rRNA. It could be generalized to all prokaryotic 16S rRNA, which would be desirable for rapid identification of prokaryotic microbes in a clinical or environmental setting.
The appendices cover unpublished work on helicase proteins to control RNA strand movement through a nanopore. The appendices also contain protocols for preparing different classes of RNA for nanopore sequencing and preparation of the helicase fragment of the SV40 Large T-antigen.