Recognition of precursor microRNAs by the Dicer cofactor TRBP

MicroRNAs (miRNAs) are small non-coding RNAs that enable post-transcriptional gene regulation through RNA interference (RNAi). The biogenesis of miRNAs involves distinct enzymatic processing steps, each of which requires a specific set of proteins. The processing of precursor miRNAs (pre-miRNAs) is performed by the endoribonuclease Dicer. Dicer enzymes are conserved in all organisms that utilise RNAi and they always associate with a double-stranded (ds) RNA-binding protein. In mammals, Dicer associates with TAR element binding-protein (TRBP) but the precise role of TRBP in miRNA biogenesis remains unclear. Research over the last 15 years has suggested that TRBP aids Dicer substrate recognition, facilitates diffusion of Dicer along dsRNA substrates, influences the length of Dicer products, and regulates Dicer activity or stability. Human TRBP is a 366-residue protein that comprises three dsRNA-binding domains (dsRBDs) separated by long unstructured linkers. The two N-terminal Type-A dsRBDs bind dsRNA while the C-terminal Type-B domain mediates protein-protein interactions. The goal of this thesis is to explore the structure-function relationship and RNA-binding properties of TRBP. DsRBDs recognise dsRNA via three interaction surfaces: Regions 1-3. Previous research has highlighted the contribution of Region 2 and suggested that the sequence and molecular dynamics of this loop may tune the dsRNA-binding affinity of the domain. Here, nuclear magnetic resonance (NMR) spectroscopy was used to evaluate the structure and ps-ns backbone dynamics of the two Type-A dsRBDs of TRBP, with particular focus on RNA-binding region 2. Analysis of 15N NMR relaxation parameters and residue composition of this region revealed differences between the two dsRBDs. A series of single and double mutant variants were made in single and tandem dsRBD constructs, and their dsRNA-binding properties were tested using several pre-miRNAs. A previously reported difference in binding affinity between dsRBD-1 and 2 was shown to be dependent on pH, which was also shown to affect the foldedness of dsRBD-1. Single-site mutations were shown to alter the dynamics of Region 2 and to affect RNA-binding affinity. These analyses suggested that Region 2 may be required for sensing the presence of non-Watson-Crick features in a pre-miRNA. The implications of these results on Dicer activity are unclear, but the characterisation of binding properties and backbone dynamics reported here provide further insight into pre-miRNA recognition by TRBP and proteins with multiple dsRBD more generally

SARS-CoV-2 aptasensors based on electrochemical impedance spectroscopy and low-cost gold electrode substrates

SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter

A SARS-CoV-2 aptasensor based on electrochemical impedance spectroscopy and low-cost gold electrode substrates.

SARS-CoV-2 diagnostic practices broadly involve either qPCR based nucleic amplification or lateral flow assays (LFAs). qPCR based techniques suffer from the disadvantage of requiring thermal cycling (difficult to implement for low-cost field use) leading to limitation on sample to answer time, the potential to amplify viral RNA sequences after a person is no longer infectious and being reagent intense. LFA performance is restricted by qualitative or semi-quantitative readouts, limits on sensitivity and poor reproducibility. Electrochemical biosensors, and particularly glucose test strips, present an appealing platform for development of biosensing solutions for SARS-CoV-2 as they can be multiplexed and implemented at very low cost at point of use with high sensitivity and quantitative digital readout. This work reports the successful raising of an Opti-mer sequence for the spike protein of SARS-CoV-2 and then development of an impedimetric biosensor which utilises thin film gold sensors on low-cost laminate substrates from home blood glucose monitoring. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times of 15 minutes. The biosensor developed here is compatible with mass manufacture, is sensitive and low-cost CE marked readout instruments already exist. These findings pave the way to a low cost and mass manufacturable test with the potential to overcome the limitations associated with current technologies