Identification of DNA aptazymes for small-molecule sensors

描述（由申请人提供）：本区域申请中的拟议研究旨在利用DNA的结合和催化能力来开发小分子传感器的新方法。从药品到制造化学品的许多小分子都存在于环境中，并可能影响人类健康。农药是一类对人类构成重大危害的小分子污染物。目前的检测方法依赖于质谱和液相色谱等技术，能够检测浓度非常低的污染物，但需要大量的样品制备，必须在实验室中完成。由于产生小分子污染物的抗体所需的努力以及抗体对反应条件的敏感性，开发用于污染物的ELISA测定是具有挑战性的。用简单的设备现场检测这些污染物的能力，而不是需要在实验室中运输和处理样本，将允许对潜在的有害情况进行快速评估。适体是以其以高亲和力和选择性结合靶标的能力为特征的核酸序列。脱氧核酶或DNA酶是催化DNA序列，其可以与适体配对以形成适体酶。DNA的稳定性使得适体酶成为传感器的有吸引力的组分。虽然适体酶已经被开发用于检测金属离子的存在，并且在原理验证研究中，一些小分子的存在，但是我们打算开发用于检测农药的新的适体酶系统。依赖于催化活性而不是RNA裂解的污染物的DNA传感器的开发将代表便携式传感器的新方向，将探索DNA催化的新领域，并可以提供关于小分子如何与核酸相互作用的信息。本提案中讨论的DNA适体酶的成功鉴定最终可以为个人提供评估其个人健康风险的新工具，并很容易应用于其他小分子。该提案的三个具体目标是：（1）鉴定依赖农药活性的DNA适体酶;（2）鉴定农药特异性DNA适体酶;（3）开发DNA适体酶的信号传导方法。 公共卫生相关性：这个区域应用的目标是利用DNA的结合和催化能力来开发新的环境污染物小分子传感器。这些传感器的成功开发将为个人提供新的工具来监测他们的个人健康风险，而无需等待实验室测试结果。

DESCRIPTION (provided by applicant): The proposed research in this AREA application seeks to harness the binding and catalytic abilities of DNA to develop new approaches for small-molecule sensors. Many small molecules ranging from pharmaceuticals to manufacturing chemicals are present in the environment and potentially impact human health. Pesticides are one class of small-molecule pollutants that represent a significant hazard to humans. Current testing methods relying on techniques including mass spectrometry and liquid chromatography are able to detect pollutants at very low concentrations, but require extensive sample preparation that must be done in a lab. Developing ELISA assays for pollutants is challenging because of the efforts required to generate antibodies for small- molecule pollutants and the sensitivity of antibodies to reaction conditions. The ability to detect these pollutants on-site wih simple equipment rather than requiring samples be transported and processed in a lab would allow for rapid assessment of potentially harmful situations. Aptamers are nucleic acid sequences characterized by their ability to bind a target with high affinity and selectivity. Deoxyribozymes or DNAzymes are catalytic DNA sequences that can be paired with aptamers to form aptazymes. The stability of DNA makes aptazymes attractive components for sensors. While aptazymes have been developed to detect the presence of metal ions and, in proof-of-principle studies, some small molecules, we intend to develop new aptazyme systems for the detection of pesticides. The development of DNA sensors for pollutants that rely on catalytic activities other than RNA cleavage would represent new directions for portable sensors, would explore new areas of DNA catalysis, and could provide information about how small molecules interact with nucleic acids. The successful identification of the DNA aptazymes discussed in this proposal could eventually provide individuals with new tools for assessing their personal health risks and would be easily applied to other small molecules. The three specific aims of the proposal are to (1) identify DNA aptazymes dependent on pesticides for activity; (2) characterize pesticide-specific DNA aptazymes; and (3) develop signaling approaches for DNA aptazymes. PUBLIC HEALTH RELEVANCE: The goal of this AREA application is to use the binding and catalytic abilities of DNA to develop new small- molecule sensors for environmental pollutants. The successful development of these sensors will provide individuals with new tools for monitoring their personal health risks without needing to wait on lab testing results.