THESIS
2022
1 online resource (xvii, 133 pages) : illustrations (chiefly color)
Abstract
Detection of small molecules is increasingly needed in environmental monitoring, food safety,
medical research, among other industries. Allosteric transcription factors (aTFs) and aptamers
have emerged as potential biorecognition elements to expand the biosensing toolkit and
overcome limitations of rapid antibody-based detection. Rational design of signal transduction
and amplification methods, and better understanding of bioreceptor-analyte interactions are
paramount to build simple, efficient, affordable and reliable biosensing platforms.
This dissertation showcases several unexplored and unaddressed factors that aid the
development of rationally designed aTF- and aptamer-based biosensors. First, we introduce a
novel endonuclease-mediated strand displacement circuit that is the first...[
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Detection of small molecules is increasingly needed in environmental monitoring, food safety,
medical research, among other industries. Allosteric transcription factors (aTFs) and aptamers
have emerged as potential biorecognition elements to expand the biosensing toolkit and
overcome limitations of rapid antibody-based detection. Rational design of signal transduction
and amplification methods, and better understanding of bioreceptor-analyte interactions are
paramount to build simple, efficient, affordable and reliable biosensing platforms.
This dissertation showcases several unexplored and unaddressed factors that aid the
development of rationally designed aTF- and aptamer-based biosensors. First, we introduce a
novel endonuclease-mediated strand displacement circuit that is the first report of small
molecule-aTFs interaction to actuate DNA toehold-mediated strand displacement reactions,
which had been typically triggered by nucleic acid inputs only. This simple yet efficient
mechanism was used to build tunable and modular biosensors for detecting nanomolar
concentration of antibiotics in water samples in 20 minutes. Second, we introduce a
computational pipeline that addresses fundamental issues in predicting aptamer-ligand
interactions using modelling tools. This approach shows that using metastable representative
structures extracted from multiple independent simulations can more accurately predict
aptamer-small molecule interactions compared to conventional in silico approaches that assume
that the bound and unbound aptamer conformations are the same. Third, we show how these
predictions can support the design of aptasensors using an aflatoxin aptamer as an example.
Through non-equilibrium competitive binding assays, we show that experimental results are in
agreement with the hypothesized conformational selection binding pathway drawn from the computational simulations. Finally, we demonstrate the modularity of the signal amplifier
previously developed by incorporating it into a sensitive and rapid aflatoxin optical aptasensor.
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