Pulsed Field Surface Hybridization

Pi：Levky，Rastislav提案编号：1600584中心目标是推进DNA的生物传感，如人类基因组，以支持个性化医学、临床诊断和基础生命科学研究。基于合成DNA类似物，通过将分子行为的电场控制与优化的传感器设计相结合，将开发一种称为脉冲场杂交的技术，以消除误差并显著缩短分析时间。该项目还包括以设计说明分子性质的学习软件为中心的教育目标，以及组织可公开访问的科学讲座和演示。生物传感应用程序必须处理复杂的样本矩阵，其中需要将感兴趣的分析物与其他分子的背景区分开来。在核酸诊断中，背景干扰最常涉及与所需序列具有一定程度相似性的DNA或RNA分子的交叉杂交。交叉杂交会导致多种有害后果，包括阻断完全匹配的分析物的结合，显著减慢接近平衡的速度，从而使分析终点由动力学历史而不是路径无关的热力学定义，以及由于杂交物种的贡献而造成的精密度损失。对于包含更大基因组的富含序列的样本、来自多个来源的遗传物质的混合物，或者当感兴趣的序列是几个密切相关的序列之一时，这些担忧被放大。该项目提出了一种称为脉冲场杂交(PFH)的生物分析方法来解决这些挑战。通过使用被称为吗啉和调制电场的电荷中性DNA类似物，PFH寻求高效的、基于场的杂交操作，以显著缩短平衡时间，并通过分阶段操作实现对杂交产品纯度的“蒸馏”。该项目将深入了解表面场如何影响吗啉衍生传感器上杂交的热力学和动力学，以及分析物特性的影响，包括过量的核苷酸、未杂交的尾巴和各种位置的错配。这些基础研究将伴随着对临床情况下典型的复杂样本矩阵的应用，以展示一种功能齐全的实用方法。此外，项目参与者还将协助向公众开放的研究展览，参与组织纳米科学讲座，并合作开发学习软件，以提供对分子行为的有趣、信息丰富的观察。

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.