Development of nucleic acid-based tools for pathogen diagnostics and real-time RNA imaging using droplet microfluidics

Abstract

Nucleic acid is a heritable genetic information system that is indispensable in the continuation of life. Both DNA and RNA can not only propagate information with high stability and accuracy but also can easily adapt through evolution. This makes them a distinctive biomolecule as they can be used both as an information hub and also as functional biopolymers through evolution-mediated ligands and enzymes. Therefore, they are an excellent tool for the detection of any organism comprising genetic material which can be effectively used for infective pathogen diagnosis. It will enable us to also further detect potential mutations and antibiotic resistances in bacteria and viruses which are essential during pandemics and epidemics. Furthermore, their ligand-based functional capacity also makes them an emerging tool for live cellular RNA imaging to visualize and understand the roles of biologically significant RNAs as they are discovered. One such ligand is a fluorogenic RNA aptamer that is a single-stranded oligonucleotide that exhibits strong fluorescence upon target-binding and showcase great potential in real-time RNA visualization. Therefore, in this thesis, droplet-based microfluidic platforms were used as a system here to facilitate the use of nucleic acid as a tool for pathogen detection as well as to screen potential fluorogenic RNA aptamers with robust functionality for real time RNA imaging. The first chapter elaborates on microfluidics, specifically droplet-based microfluidics and their biological applications and how it has been used in the directed evolution of fluorogenic RNA aptamers and their applications. It also discusses how traditional nucleic acid amplification testing have been adopting a digital droplet amplification technique over the years because of its high sensitivity, specificity and potentials of point-of-care diagnosis. It also discusses the current lacking still present in the technologies and how they can be combatted. The first sections of chapters two and three discuss about how fluorescence-activated droplet sorting (FADS) was used to screen for new RNA aptamers that bind a modified green fluorogenic dye that has shown to have superior thermal and photo stability in vivo compared to other existing green fluorophores. Five rounds of selections were conducted and new RNA aptamers were discovered that were compared to existing aptamers screened against other dyes. The last sections of the chapters discuss on developing an integrated on-chip nucleic acid amplification platform that can conduct both digital droplet polymerase chain reaction and loop-mediated isothermal amplification using an Arduino-powered heating system. Preliminary results of the SARS-CoV-2 viral RNA amplification were demonstrated, mostly comprising qualitative fluorescence images of droplets encapsulating the amplified RNA.