RPG Student List

 

Tsz Wing FAN

Doctor Candidate in Chemical and Biomolecular Engineering

Supervisor: Prof. I Ming HSING

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Research Topic

Nucleic Acid Based Circuits for Specific and Sensitive Molecular Diagnostics

Abstract

Target recycling has frequently been employed as a strategy to achieve high sensitivity in nucleic acid assays, yet this approach, in most reported literatures, involves the use of enzymes, limiting performance fidelity under steady or variant operating conditions. More importantly, spontaneity of the reactions will further intensify leakage while reducing specificity during the amplification process. Meanwhile, a long assay time is often required in particular for the detection of analyte at low concentrations.

In this thesis, an enzyme-free, highly specific target recycling approach with minimal leakage based on toehold mediated strand displacement reactions of a partially double stranded substrate was explored. Unlike traditional approaches, the double stranded substrate was designed with mismatches or insertions which imposed a kinetic barrier to the reactions to mitigate unfavorable interactions and impart higher detection specificity. With the iterative recycling process, differential interaction between on and off-targets to that of Sensing Probes accumulated for prolonged measurement. This allows sustainable or even increased specificity against excess mutants (i.e. 100X) with time.

To shorten the assay time, a simple nucleic acid circuitry with two partial duplexes were also devised which triggered an autonomous and alternative polymerization reaction similar to the hybridization chain reaction(HCR). Since Gibbs free energies of the reactions were set close to 0, high discrimination between target and spurious counterparts was achieved. In addition, the short time for the reactions to reach equilibrium, for example, around 20 minutes, provides this assay a short period of detection.

Applications of these approaches to real clinical sample detection were also explored in the study.

Journal Publication


Fan, Tsz Wing; Yu, Henson L. Lee; Hsing, I-Ming, "Conditional Displacement Hybridization Assay for Multiple SNP Phasing", (2017)
 
Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming, "Integrating DNA Strand Displacement Circuitry to the Nonlinear Hybridization Chain Reaction", (2017)
 
Fan, Tsz Wing; Hsing, I-Ming, "Kinetically Modulated Specificity Against Single-base Mutants in Nucleic Acid Recycling Circuitry Using the Destabilization Motif", (2017)
 
Xuan, Feng; Fan, Tsz Wing; Hsing, I-Ming, "Peptide Nucleic Acid Mediated Dendritic Growth of Nucleic Acid Self-Assembly for Amplified Homogeneous Electrochemical Nucleic Acid Assay", (2015)