Radiofrequency Remote Control of Enzyme-Nanocluster Conjugates

描述（由申请人提供）：通过自然发生的机制（如磷酸化）调节酶是生命的基础。酶的小分子控制是许多药物作用的基础。酶活性的整体热控制使许多实验室和工业过程，如PCR和阿斯巴甜合成。我们的总体目标是建立一种全新的调节酶的方法。该方法利用金属纳米团簇（如Au102(SR)44）在射频照射下产生的纳米局域热来热影响纳米团簇/酶偶联物的活性。所选择的射频与混合物的其他组分的相互作用最小，类似于用于Wi-Fi的射频。因此，这种纳米局部热酶控制不会改变溶液温度，也不应该影响没有直接共轭到纳米团簇上的酶的活性。我们建议通过4个具体目标来实现这一目标。Aim 1测试了我们可以在酶具有最小可测量活性的实验温度下“激活”嗜热酶/纳米簇偶联物（如热溶酶/Au102(SR)44）的假设。目标2测试假设，我们可以可逆地“失活”酶，大概是通过可逆展开。对于这一假设，我们首先测试了纳米簇偶联物与溶菌酶、RNAse A和b -半乳糖苷酶等教科书酶的结合。在目标1和目标2中，我们测试了纳米簇偶联酶的位置影响偶联物活性的子假设。这种远程控制酶学的全面实施需要定量了解纳米团簇如何在射频中加热。这样的理解将允许精确预测纳米簇温度，这是了解酶的局部温度的先决条件。目前，有三种被提出的纳米簇加热机制。它们是感应机制、磁机制和电泳机制。所有机制对所施加射频场的频率有不同的响应。目的3是测量少量定义良好的纳米团簇和纳米颗粒的频率响应。目的4是综合改变纳米颗粒的性质，从而改变其在不同机制下的热耗散。目标3和目标4都结合了利用现有机制的理论建模，认识到可能需要结合机制或发展新的理论机制来理解热耗散。这种机制的理解不仅将开辟一个远程酶控制的新领域，而且还可能使其他基于纳米颗粒的高温方法成为可能，如无创高温癌症治疗和远程控制分子生物物理学。

DESCRIPTION (provided by applicant): Regulation of enzymes by naturally occurring mechanisms such as phosphorylation is fundamental to life. Small molecule control of enzymes underlies the action of many pharmaceutical drugs. Bulk thermal control of enzymatic activity enables many laboratory and industrial processes such as PCR and aspartame synthesis. Our overall goal is to establish an entirely new method for regulating enzymes. This method uses the nano- localized heat generated by metal nanoclusters such as Au102(SR)44 under radiofrequency irradiation to thermally influence the activity of a nanocluster/enzyme conjugate. The RF is chosen to interact minimally with other components of the mixture, analogous to the RF used for Wi-Fi. Thus, this nano-local thermal enzyme control does not modify the solution temperature, nor should it influence the activity of enzymes that are not directly conjugated to nanoclusters. We propose to accomplish this goal in a set of 4 specific aims. Aim 1 tests the hypothesis that we can 'activate' thermophilic enzyme/nanocluster conjugates such as thermolysin/Au102(SR)44 at assay temperatures in which the enzyme has minimal measurable activity. Aim 2 tests the hypothesis that we can reversibly 'deactivate' enzymes, presumably by reversible unfolding. For this hypothesis we begin by testing nanocluster conjugates to textbook enzymes such as lysozyme, RNAse A and B-galactosidase. In both Aims 1 and 2 we test the sub-hypothesis that the site of nanocluster conjugation on the enzyme influences the activity of the conjugate. A full implementation of this remote-control enzymology requires quantitative understanding of how nanoclusters heat in radiofrequencies. Such an understanding will allow accurate prediction of nanocluster temperature, which is prerequisite for understanding how locally hot an enzyme is. Currently, there are three proposed mechanisms for nanocluster heating. These are an inductive mechanism, a magnetic mechanism, and an electrophoretic mechanism. All mechanisms have different responses to the frequency of the applied radiofrequency filed. Aim 3 is to measure the frequency response of a small number of well-defined nanoclusters and nanoparticles. Aim 4 is to synthetically change the properties of nanoparticles in a manner that will change their thermal dissipation under different mechanisms. Both aims 3 and 4 incorporate theoretical modeling using existing mechanisms, recognizing the possible need for combining mechanisms or developing a novel theoretical mechanism for understanding thermal dissipation. Such mechanistic understanding will not only enable a new field of remote enzyme control but may also enable other nanoparticle based hyperthermal methods such as noninvasive hyperthermal cancer therapy and a remote control molecular biophysics.