Effective and timely management of genetic and infectious diseases highly depends on early, reliable, and accurate diagnosis of biomarkers, which in turn translates to a favorable prognosis of the disease. Nucleic acids are often the biomarkers of choice due to the ease of constructing DNA-based biosensors with high sensitivity and selectivity. With the available literature on the thermodynamics and kinetics of nucleic acid hybridization interactions and a plethora of well-characterized nucleic acid-acting enzymes, one can develop a nucleic acid biosensor with high degree of predictability and programmability.

This thesis showcases three applications of nucleic acid circuitries to address different challenges in nucleic acid diagnostics. The first project in chapter 2 focuses on...[ Read more ]

Effective and timely management of genetic and infectious diseases highly depends on early, reliable, and accurate diagnosis of biomarkers, which in turn translates to a favorable prognosis of the disease. Nucleic acids are often the biomarkers of choice due to the ease of constructing DNA-based biosensors with high sensitivity and selectivity. With the available literature on the thermodynamics and kinetics of nucleic acid hybridization interactions and a plethora of well-characterized nucleic acid-acting enzymes, one can develop a nucleic acid biosensor with high degree of predictability and programmability.

This thesis showcases three applications of nucleic acid circuitries to address different challenges in nucleic acid diagnostics. The first project in chapter 2 focuses on rapid signal amplification for point-of-care diagnostic applications. While sensitivity and selectivity are important parameters to consider in developing a biosensor, the speed of the detection process is also important especially in portable and home-use tests. We demonstrated that by employing a cascaded exonuclease III-aided target recycling strategy, we are able to detect picomolar quantities of DNA target within 15 minutes. The second project utilizes a two-step oligonucleotide probe-based hybridization assay in order to determine the haplotype phase of two SNPs, i.e., whether they are cis (in the same DNA strand) or trans (in different DNA strands). The first step is a toehold-mediated strand displacement reaction to interrogate the presence of the two SNPs, and the second reaction is a conditional magnetic separation step to differentiate cis and trans SNPs. Chapter 4 describes an underexplored function of Cas12a enzyme for detecting rare allele mutations. We first show how the three-nucleotide protospacer adjacent motif (PAM) sequence can be decoupled to the rest of the target sequence to universalize the applicable target, which previously was typically limited to PAM-containing targets; then, we characterized its double-stranded trans-cleavage activity which showed a much higher specificity than the ones used for signal amplification in most, if not all, of the currently available literature. As such, we show that our system can detect mutations in a genetically heterogeneous mixture where the desired target is a single base mutant diluted up to 10,000 times with the wild type sequence.

Lastly, this dissertation concludes with a summary on how these projects are able to expand the repertoire of DNA self-assembly reactions and add to the molecular toolbox in diagnosing genetic and infectious diseases. In particular, with the COVID19 pandemic affecting millions of lives globally, we offer a strategy and some preliminary results on how rational design of oligonucleotide probes can address the challenge of massively parallel testing, by identifying infected individuals in a pooled sample without the need for retesting.