Pulsed Field Surface Hybridization

点击翻译按钮获取中文摘要

PI: Levicky, Rastislav Proposal Number: 1600584The central aim is to advance biosensing of DNA, such as the human genome, in support of personalized medicine, clinical diagnostics, and basic life science research. A technology termed pulsed-field hybridization will be developed by combining electric field control of molecular behavior with optimized sensor designs, based on synthetic DNA analogues, to eliminate errors and to significantly shorten analysis time. The project also encompasses educational goals centered on design of learning software that illustrates molecular properties and on organization of publicly accessible science lectures and demonstrations.Biosensing applications must handle complex sample matrices in which the analyte of interest needs to be distinguished against a background of other molecules. In nucleic acid diagnostics, background interference most often involves cross-hybridization from DNA or RNA molecules with a degree of similarity to the desired sequence. Cross-hybridization leads to multiple detrimental consequences including blocking binding of the perfectly matched analyte, markedly slowing down approach to equilibrium so that assay endpoints are defined by kinetic history rather than path-independent thermodynamics, and loss of precision due to contributions from the cross-hybridized species. These concerns become amplified for sequence-rich samples comprising larger genomes, mixtures of genetic material from multiple sources, or when the sequence of interest is one of several closely related sequences. This project proposes a bioanalytical method termed pulsed field hybridization (PFH) to solve these challenges. Through use of charge-neutral DNA analogues called morpholinos and modulated electric fields, PFH seeks highly efficient, field-based manipulation of hybridization to dramatically reduce equilibration timescales and to enable "distilling", through a stage-wise operation, of the purity of the hybridized product. The project will develop a thorough fundamental understanding of how surface fields influence thermodynamics and kinetics of hybridization on morpholino-derivatized sensors, and of the impact of analyte characteristics including excess nucleotides, unhybridized tails, and variously placed mismatches. These fundamental studies will be accompanied by application to complex sample matrices typical of clinical situations to demonstrate a fully functional, practical method. Project participants will in addition assist with research exhibitions open to the public, engage in organizing lectures on nanoscience, and collaborate on development of learning software to provide a fun, informative look into the behavior of molecules.