Ligand-Dependent Exponential Amplification of RNA

描述（由申请人提供）：生物医学科学的最新技术进步使得以前所未有的详细程度对生物系统的组成部分进行盘点成为可能。这不仅适用于基因和基因调控元件，也适用于存在于生物体中的蛋白质和小分子。了解这些成分在健康和疾病方面的功能相互作用的关键是对其在生物样品中的浓度进行定量测量。对于基因及其转录产物，这很容易通过定量聚合酶链反应（qPCR）等技术来实现，这些技术已经彻底改变了分子生物学和临床诊断。然而，对于蛋白质和小分子，分析方法更加专业化，并且不像qPCR那样受益于指数信号扩增。在之前的项目期间，开发了一种新的蛋白质和小分子定量检测方法，该方法具有高特异性，并且由于指数信号放大而具有高灵敏度。该方法依赖于一种新型的自我复制核酸酶，这种酶在恒温下进行指数扩增，并且可以根据对目标配体的识别来进行扩增。拟议的研究将扩大该系统的通用性，使其可以应用于广泛的目标，包括疾病相关蛋白质、药物和代谢物。实时荧光分析将开发，使高通量，多重分析使用标准仪器。自我复制酶将通过位点特异性化学修饰和定向进化的结合进行优化，使其在生物样品中稳定并具有高催化效率。将配体识别元件连接到复制酶的方法将被推广并应用于与生物传感和临床诊断相关的各种蛋白质。拟议的研究还将研究一种潜在的更深远的方法，利用复制酶以自我维持的方式进化的独特能力，使它们能够自我配置以识别目标分子。这类似于免疫系统对抗原识别的成熟，但将完全发生在试管中合成遗传系统的背景下。这些努力将为开发一类新的智能材料提供机会，以帮助跟上新生物靶点的快速发现速度。

DESCRIPTION (provided by applicant): Recent technological advances in the biomedical sciences have made it possible to take inventory of the components of biological systems at an unprecedented level of detail. This applies not only to genes and genetic regulatory elements, but also to the proteins and small molecules that exist within living organisms. Critical to understanding the functional interplay of these components, in both health and disease, is the quantitative measurement of their concentration in biological samples. For genes and their transcription products this is readily accomplished through techniques such as the quantitative polymerase chain reaction (qPCR), which have revolutionized molecular biology and clinical diagnostics. For proteins and small molecules, however, the analytical methods are more specialized, and do not benefit from exponential signal amplification as occurs with qPCR. During the previous project period, a new approach was developed for the quantitative detection of proteins and small molecules that has high specificity and achieves high sensitivity due to exponential signal amplification. The approach relies on a novel class of self-replicating nucleic acid enzymes that undergo exponential amplification at constant temperature, and can be made to do so contingent upon recognition of a target ligand. The proposed research will expand the generality of the system so that it can be applied to a broad range of targets, including disease-related proteins, drugs, and metabolites. A real-time fluorescent assay will be developed that enables high-throughput, multiplex analysis using standard instrumentation. The self-replicating enzymes will be optimized using a combination of site-specific chemical modification and directed evolution so that they are stable in biological samples and operate with high catalytic efficiency. The method for linking the ligand-recognition element to the replicating enzymes will be generalized and applied to a variety of proteins of relevance to biosensing and clinical diagnostics. The proposed research also will investigate a potentially more far-reaching approach that takes advantage of the unique ability of the replicating enzymes to evolve in a self-sustained manner, enabling them to configure themselves to recognize a target molecule. This is analogous to the maturation of antigen recognition by the immune system, but would occur entirely in the test tube within the context of a synthetic genetic system. These efforts will provide the opportunity to develop a new class of smart materials to help keep pace with the rapid rate of discovery of novel biological targets.