A High-Resolution High-Efficiency Force Spectroscopy for Measuring Drug-DNA Interactions

分子-分子键是生物学中分子识别和药物靶向的基础。然而，这种类型的相互作用仍然很难量化，主要是因为缺乏适当的技术来提供可靠的和有特征的参数。这项提议将开发一种新型的力光谱，它使用精确控制的超声波来测量结合力，以区分不同的分子键。数万个分子键将被同时研究并根据它们不同的结合力进行解析。这项被称为超声力诱导剩余磁化光谱(UsFIRMS)的技术将成为研究分子相互作用的一种新的科学工具，具有高灵敏度、前所未有的分辨率和高探测效率。通过克服超声波和磁探测器集成所涉及的工程挑战，所产生的仪器将能够准确、可靠和高效地识别不同的分子相互作用。这项新技术的应用范围将集中在药物分子与DNA双链的结合上，以精确揭示它们的结合力和热力学。结果将定量地鉴定结合的选择性。缺乏选择性一直是DNA靶向药物的一个重大挫折。因此，通过新技术获得的结果具有很高的科学价值。除了工程上的进步和科学价值，这项计划中的研究活动将为来自广泛背景的学生提供极好的教育机会，包括研究生、本科生和高中生。特别是，将积极招募少数族裔本科生参与并最终领导部分项目。这项提议旨在建立一种新的usFIRMS技术，该技术利用精确衰减的超声产生的声辐射力来选择性地解离标记有磁性粒子的非共价键。磁信号由原子磁强计监测，原子磁强计是磁检测最灵敏的设备。我们提出的红外光谱技术将为表征非共价键开辟一个新的领域，而非共价键在生物化学和生物学中却鲜为人知。在技术方面，与现有技术相比，这项技术具有三个独特的能力。首先，它具有高的力分辨率，可以清楚地解析分子键，结合力差小于两个皮牛顿差，比现有技术高一个数量级。其次，这是第一次使用超声波来区分分子键。通过将超声波组件与原子磁强计相结合，该仪器实现了高效和自动化的生化分析，这是任何其他形式的机械力都无法实现的。第三，宽带力光谱的概念将在一次采集中解析不同的键，而不是耗时的力扫描。这一革命性的进步将带来高吞吐量的应用。在基础科学方面，将红外光谱技术应用于药物与DNA的相互作用，将通过建立一个新的差异结合力的物理化学参数来定量鉴定药物与DNA的结合选择性。它将成为药物优化的新平台。由于完全分解了不同的分子键以消除测量中的潜在干扰，因此还可以精确地确定结合常数、自由能和热焓。此外，红外光谱技术以其高分辨率、高效率、高适用性的独特优势，将在生物研究中得到广泛的应用。

The molecule-molecule bonds are the basis of molecular recognition and drug targeting in biology. However, this type of interactions remains difficult to quantify, mainly because of the lack of a suitable technique to provide a reliable and characteristic parameter. This proposal will develop a novel force spectroscopy that uses precisely controlled ultrasound to measure the binding forces to distinguish different molecular bonds. Tens of thousands of molecular bonds will be studied simultaneously and resolved based on their different binding forces. The technique, termed as ultrasound force-induced remnant magnetization spectroscopy (usFIRMS), will be a new scientific tool for studying molecular interactions, with high sensitivity, unprecedented resolution, and high detection efficiency. By overcoming the engineering challenges involved in the integration of ultrasound and the magnetic detector, the resulting instrument will be able to identify different molecular interactions precisely, reliably, and efficiently. The application scope of the new technique will focus on drug molecules binding with DNA duplexes to precisely reveal their binding forces and thermodynamics. The results will quantitatively identify the binding selectivity. Lack of selectivity has been a major setback for DNA-targeting drugs. Results obtained by the new technique are thus of high scientific merit. In addition to the engineering advancements and scientific values, the research activities in this proposal will provide excellent educational opportunities for students from a broad background, ranging from graduate students, undergraduates, and high school students. In particular, minority undergraduate students will be actively recruited for participation and ultimately leading a subset of the projects. This proposal aims at establishing the novel usFIRMS technique that uses acoustic radiation force generated by precisely attenuated ultrasound to selectively dissociate noncovalent bonds labeled with magnetically particles. The magnetic signal is monitored by an atomic magnetometer, which is the most sensitive device for magnetic detection. The proposed usFIRMS technique will open up a new field for characterizing noncovalent bonding, which is poorly understood but widely encountered in biochemistry and biology. On the technological front, this technique has three unique capabilities comparing with existing techniques. First, it possesses high force resolution that can clearly resolve molecular bonds with less than two pico-newton difference in the binding forces, which is one order of magnitude better than that of existing techniques. Second, it represents the first time that ultrasound is used for distinguishing molecular bonds. By integrating ultrasound components with an atomic magnetometer, the instrument allows for efficient and automated biochemical analysis that cannot be obtained with any other forms of mechanical forces. Third, the concept of broadband force spectroscopy will resolve different bonds in a single acquisition, instead of the time-consuming force sweeping. This revolutionary advancement will lead to high-throughput applications. On the fundamental science front, the application of usFIRMS in drug-DNA interactions will quantitatively identify the binding selectivity by establishing a new physicochemical parameter of differential binding force. It will serve as a new platform for drug optimization. The binding constants, free energy, and enthalpy can also be precisely determined because different molecular bonds are completely resolved to remove potential interference in measurements. Furthermore, the usFIRMS technique will find broad applications in biological research because of its unique combination of high resolution, high efficiency, and high applicability.