THESIS
2020
xv, 111 pages : illustrations (some color) ; 30 cm
Abstract
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...[
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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.
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