Development of a Modular Biosensor Platform for Direct, Sequence-Specific DNA Detection

Abstract

Point of care (POC) diagnostic tests are useful as they provide fast time-to-results and are easy to use, minimising time to diagnosis and helping combat the spread of infectious diseases. Consequently, POC tests are increasingly in demand, however no existing POC test for DNA analytes is comparable in accuracy and sensitivity to lab-based methods. This thesis, comprised of six chapters, describes the development of a new biosensor for sequence-specific DNA detection by coupling a DNA-binding domain to a bioluminescence resonance energy transfer (BRET) signalling component.

Chapter 1 highlights the need for POC diagnostic tests for genetic sequence detection, and the suitability of synthetic biology technologies to meet this demand. Biosensors are introduced as ideal candidates for POC testing devices, and the properties of DNA as a target analyte are discussed. Additionally, the utility of amplifying specific DNA sequences to improve detection thresholds is emphasised.

Chapter 2 introduces the materials and methods used throughout the thesis. Reagent sources, equipment, and methods relevant to all chapters are presented. Methods and experimental design specific for individual chapters are described in each experimental chapter.

Chapter 3 details the development and application of biosensors for sequence-specific DNA detection. Two DNA-binding proteins were used to produce several unique biosensor designs, which were tested to identify the best candidate. The chosen biosensor's performance was assessed, including measuring output under application-relevant conditions. Overall, a proof of principle bipartite biosensor was developed by combining the DNA-binding specificity of zinc finger proteins (ZFPs) with the ratiometric signal from BRET signal transduction. Importantly, this meant the biosensor was produced in a polypeptidic fashion, obviating the need for expensive synthesis steps using harsh chemicals. To modify the biosensor specificity to a new, clinically relevant, analyte was engineered to bind a DNA sequence specific to Mycobacterium tuberculosis, the causative agent of tuberculosis (TB).

Chapter 4 introduces isothermal DNA amplification (IA) to improve the biosensor limit of detection without compromising its applicability to POC. The gold standard for DNA amplification is polymerase chain reaction, however this method is not suitable for application at POC. Instead, IA was used due to its amplification of nucleic acids without requiring expensive equipment and reagents, or skilled technicians. Novel primer sets for two different IA technologies, loop-mediated IA and recombinase polymerase amplification, were developed to amplify the biosensor's DNA targets. Following amplification of target DNA, the crude reaction product was successfully detected with the biosensor.

Chapter 5 details the development of a screen to find DNA-binding proteins for specific DNA sequences. The screen facilitated rapid selection of the best-performing biosensors and removed the need for a modular assembly approach to ZFP programming, further speeding up the biosensor development process. Inclusion of cell-free protein synthesis (CFPS) in the screen reduced the time required for protein expression from a week to 3 hours. Furthermore, the crude CFPS product was compatible with the biosensor, obviating the need for protein purification. The screen was used to test different ZFPs for the development of new biosensors, and to create an alternate version of the TB biosensor described in Chapter 3. In the future, the screen could take DNA templates of biosensor designs and return a compatibility result for a DNA target within ~3 hours.

Chapter 6 concludes, sets the findings in the context of the literature and provides future directions. Show more