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难以量化，主要是因为缺乏适当的技术来提供可靠的特征参数。本提案将发展一种新的力谱，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。数以万计的分子键将同时被研究，并根据它们不同的结合力来解决。该技术被称为超声力致残余磁化光谱（usFIRMS），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检测效率。通过克服集成超声波和磁探测器所涉及的工程挑战，所产生的仪器将能够精确、可靠和有效地识别不同的分子相互作用。新技术的应用范围将集中在药物分子与DNA双链的结合上，以精确地揭示它们的结合力和热力学。结果将定量地确定结合选择性。缺乏选择性一直是dna靶向药物的主要挫折。新技术所得结果具有很高的科学价值。除了工程进步和科学价值外，本提案中的研究活动将为来自广泛背景的学生提供良好的教育机会，包括研究生，本科生和高中生。特别是，少数族裔本科生将被积极招募参与并最终领导一部分项目。本提案旨在建立新的usFIRMS技术，该技术利用精确衰减超声产生的声辐射力选择性地解离带有磁性粒子标记的非共价键。磁信号由原子磁强计监测，原子磁强计是最灵敏的磁探测装置。提出的usFIRMS技术将为表征非共价键开辟一个新的领域，非共价键在生物化学和生物学中知之甚少，但却广泛遇到。在技术方面，与现有技术相比，该技术具有三个独特的功能。首先，它具有很高的力分辨率，可以清晰地分辨出作用力差小于2皮牛顿的分子键，比现有技术提高了一个数量级。其次，它代表了超声波第一次被用于区分分子键。通过将超声元件与原子磁强计集成，该仪器可以进行任何其他形式的机械力无法获得的高效自动化生化分析。第三，宽带力谱的概念将在一次采集中解决不同的键，而不是耗时的力扫描。这一革命性的进步将带来高通量的应用。在基础科学方面，usFIRMS在药物- dna相互作用中的应用将通过建立一种新的差动结合力的物理化学参数来定量地确定结合选择性。它将为药物优化提供一个新的平台。结合常数，自由能，和焓也可以精确地确定，因为不同的分子键被完全分解，以消除测量中的潜在干扰。此外，usFIRMS技术由于其独特的高分辨率、高效率和高适用性的结合，将在生物学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.