Collaborative Research: L-RNA Based Reactive Oxygen Species Detection and Response Systems

非技术综述：放射治疗(放射治疗)用于治疗癌症已有100多年的历史，新的临床创新使非常集中的辐射束能够杀死恶性细胞。这些光束是由高能轻粒子组成的，进入人体后，水和氧气发生反应，分裂成许多不同的活性氧物种(ROS)。然后，这些RO继续破坏细胞内的遗传物质，最终导致细胞死亡。在癌症治疗中，放射治疗本身通常不是完全有效的。有时它会错过癌细胞。有时它没有足够的氧气来产生治疗性的ROS。事实上，在后续预约之前，几乎不可能判断它是否有效。由于辐射是由如此高能的粒子组成，与其直接相互作用的分子并不是很多；然而，众所周知，辐射会杀死细胞，因为遗传物质非常容易与这些RO发生化学反应。因此，由遗传物质制成的分子探测器应该能够对辐射损伤做出反应是有道理的。这项提议的重点是设计新的基于人造生物材料的传感器，这些传感器几乎与你体内的遗传物质“看起来”一模一样，只是细胞不能像正常的RNA或DNA那样阅读它，而且它不会被体内的酶降解，但辐射以完全相同的方式影响它。这些新的探针将被设计成瞄准线粒体或“细胞的发动机”，以确定辐射如何对细胞造成损害。最后，这项工作建议制造一种新型的纳米材料，当它受到辐射时可以发光。有一天，这种材料可能会实时告诉医生放射治疗是否有效。技术综述：活性氧(ROS)的过度产生会损害遗传物质，导致细胞死亡。ROS过度产生的原因是多方面的，从线粒体功能障碍到辐射暴露。羟基自由基--反应性·OH--不能通过酶反应消除，它的威力独一无二，足以诱导RNA和DNA单链断裂。了解·OH的化学对于在细胞和组织水平上解决许多领域的根本挑战是至关重要的。存在几个·OH敏感的荧光探针，但它们缺乏检测细胞内个位数μM浓度的灵敏度和/或无法区分·OH和其他ROS。因此，由于缺乏足够的工具，检测或利用·OH--最有害的ROS之一--受到阻碍。这项建议的目标是优化新的L-核糖核酸低聚物，作为一种双正交工具，可以进行·OH“触发”的链断裂。研究小组将展示这些优化的L核糖核酸可以被合成地定制成目标细胞细胞器，以报告·OH浓度。最后，在ROS的存在下，L-RNA链的断裂将被触发为纳米配方，以制造具有高度选择性的“智能”材料。中心假设是，L核糖核酸将在生理相关的·OH浓度下发生单链切割，无毒，不会在体外或体内被酶破坏。在这项概念验证研究的结论中，预期的结果将是在体外验证L核糖核酸作为一种长寿命和靶向的传感器。这里奠定的基础将进一步促进开发能够在体内和体外工作的新的“双正交”DNA和RNA结构。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Non-Technical Summary: Radiation therapy (radiotherapy) has been used to treat cancer for more than 100 years and new clinical innovations have allowed for very focused beams of radiation to kill malignant cells. These beams are made of high energy light particles that enter the body and cause water and oxygen to react and split into many different reactive oxygen species (ROS). These ROS then go on to destroy the genetic material inside a cell, which ultimately leads to the cell’s death. In cancer treatment, radiotherapy is usually not completely effective on its own. Sometimes it misses cancerous cells. Sometimes there’s not enough oxygen for it to create the therapeutic ROS. In fact, it is almost impossible to tell if it is working until a follow up appointment. Because radiation is composed of such high energy particles, not very many molecules directly interact with it; however, it is known that radiation kills cells because genetic material is so susceptible to chemical reactions with these ROS. It makes sense then that a molecular probe made from genetic material should be able to respond to radiation damage. This proposal focuses on designing new artificial biomaterial-based sensors that “look” almost exactly like the genetic material in your body except cells can’t read it like normal RNA or DNA and it won’t be degraded by enzymes in the body yet radiation affects it the exact same way. These new probes will be designed to target the mitochondria or the “powerhouse of the cell” to identify how radiation causes damage to cells. Finally, this work proposes to make a new type of nanomaterial that can glow when it is hit by radiation. One day, this sort of material might be able to tell doctors in real time if radiotherapy is working.Technical Summary: The overproduction of reactive oxygen species (ROS) damages genetic material, causing cell death. The causes of ROS overproduction are various and range from mitochondrial dysfunction to radiation exposure. Hydroxy radicals—reactive •OH—cannot be eliminated by enzymatic reactions and are uniquely powerful enough to induce single strand breaks in RNA and DNA. Understanding the chemistry of •OH is essential to address fundamental challenges in a number of arenas at the cellular and tissue levels. Several •OH sensitive fluorescent probes exist but they lack the sensitivity to detect the single digit μM concentrations in cells and/or cannot distinguish between •OH and other ROS. Thus, detecting or exploiting •OH—one of the most pernicious ROS—is hamstrung by a lack of adequate tools. The objective in this proposal is to optimize new L-RNA oligomers as a biorthogonal tool that can undergo strand scission “triggered” by •OH. The research team will show these optimized L-RNA can be synthetically tailored to target cellular organelles to report on •OH concentrations. Finally, L-RNA strand scission will be triggered as a nano-formulation in the presence of ROS to make a highly selective “smart” material. The central hypothesis is that L-RNA will undergo single strand cleavage at physiologically relevant •OH concentrations, is non-toxic, and will not be enzymatically destroyed in vitro or in vivo. At the conclusion of this proof-of-concept study, the expected outcome will be the validation of L-RNA as a long-lived and targetable sensor in vitro. The groundwork laid here will be further enabling in the development of new “biorthogonal” DNA and RNA architectures that can work in vivo and in vitro.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.