Accurate diagnosis can make a life-changing difference to therapeutic options and outcomes, and that is highly dependent on the progress of biomarker discovery as well as the precision of analytical tools available. Nucleic acid has been a popular molecular tool for biosensing applications. This is in particular due to the unique Watson-Crick base pairs that allow single-base programming of the nucleic acid reactions by modulating the sequence information. The biophysics and quantitative characterization, for example the reaction Gibbs free energy underlying the nucleic acid hybridization reactions, enable rational design of the nucleic acid machineries to finely tune the analytical performance. With rapidly falling synthesis cost and versatile chemical moieties that can be func...[ Read more ]

Accurate diagnosis can make a life-changing difference to therapeutic options and outcomes, and that is highly dependent on the progress of biomarker discovery as well as the precision of analytical tools available. Nucleic acid has been a popular molecular tool for biosensing applications. This is in particular due to the unique Watson-Crick base pairs that allow single-base programming of the nucleic acid reactions by modulating the sequence information. The biophysics and quantitative characterization, for example the reaction Gibbs free energy underlying the nucleic acid hybridization reactions, enable rational design of the nucleic acid machineries to finely tune the analytical performance. With rapidly falling synthesis cost and versatile chemical moieties that can be functionalized on synthetic nucleic acids, frontier nucleic acid sensing strategies can be developed to address critical biosensing challenges.

In this thesis, we hinge on the thermodynamics, kinetics and functional properties of synthetic nucleic acids to 1) develop a nucleic acid recycling circuit that kinetically discriminates single base mutants through cyclic toehold exchange reactions. This work overcomes the deteriorating specificity amid the signal amplification process in conventional analyte decoupled amplification reactions; 2) create in a first homogeneous single-molecule immunoassay which translates the transient antibody-antigen binding event to amplifiable nucleic acid signals based on multiple nucleic acid extension reactions. The proximity required several times for generating a complete reporter sequence immensely suppresses the background signal suffered in commercialized immunoassays and achieves ultrasensitivity using exceedingly small sample volume; and lastly 3) depict the phase –the exact nucleotide content on a strand by developing a homogeneous polymerase assisted hybridization-based approach where the polymerase is programmed to cause a change in fluorescence signal only if the studied alleles are in cis-conformation. The simplicity of the approach is promising to substitute costly next generation sequencing, indirect statistical computation or assays that require tedious single molecule isolation techniques for phasing at multiple allelic sites.