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Ligand-Dependent Exponential Amplification of RNA

RNA 的配体依赖性指数扩增

基本信息

批准号：
8771441
负责人：
GERALD F JOYCE
金额：
$ 36.01万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8771441
关键词：
Acids Address Antigens Area Binding Biological Biological Assay Biomedical Research Biosensing Techniques Catalytic Domain Chemicals Clinical Complex Detection Diagnostic Disease Elements Ensure Environment Enzymes Equipment and supply inventories Evolution Fluorescence Resonance Energy Transfer Genes Genetic Genetic Transcription Health Hour Human Immune system In Vitro Life Ligand Binding Ligands Link Measurement Measures Methods Modification Molecular Molecular Biology Nucleic Acid Regulatory Sequences Nucleic Acids Organism Pharmaceutical Preparations Polymerase Chain Reaction Positioning Attribute Protein Binding Proteins Proteomics Protocols documentation RNA RNA amplification Reaction Reaction Time Research Resistance Ribonucleases Sampling Science Seeds Sensitivity and Specificity Serum Signal Transduction Site Specificity System Techniques Temperature Testing Therapeutic Time Tube amplification detection analytical method aptamer base biological systems directed evolution improved instrumentation meetings metabolomics novel novel strategies nuclease nucleotide analog replicator small molecule

项目摘要

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.

描述(由申请人提供)：生物医学科学的最新技术进步使以前所未有的详细程度清点生物系统的组件成为可能。这不仅适用于基因和遗传调控元件，也适用于活着的有机体中存在的蛋白质和小分子。要理解这些成分在健康和疾病中的功能相互作用，关键是对它们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式反应(QPCR)等技术实现，这些技术已经彻底改变了分子生物学和临床诊断学。然而，对于蛋白质和小分子，分析方法更加专业化，并不像qPCR那样受益于指数信号放大。在之前的项目期间，由于指数信号放大，开发了一种新的定量检测蛋白质和小分子的方法，该方法具有高特异性和高灵敏度。这种方法依赖于一种新型的自我复制的核酸酶，这种酶在恒温下经历指数放大，并且可以根据对目标配体的识别来实现这一点。拟议的研究将扩大该系统的通用性，以便它可以应用于广泛的靶标，包括与疾病相关的蛋白质、药物和代谢物。将开发一种实时荧光分析，使高通量、使用标准仪器的多重分析成为可能。这种自我复制的酶将通过结合定点特定的化学修饰和定向进化进行优化，以便它们在生物样品中稳定并具有高催化效率。将配基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。拟议的研究还将探索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，使它们能够配置自己识别目标分子。这类似于免疫系统识别抗原的成熟，但将完全发生在人工遗传系统的试管中。这些努力将为开发一类新的智能材料提供机会，以帮助跟上发现新生物靶标的快速速度。