Nucleic acid analysis using deoxyribozyme technology

使用脱氧核酶技术进行核酸分析

• 批准号: 7870705
• 负责人: Dmitry Mikhaylovich Kolpashchikov
• 金额: $ 7.88万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7870705
• 关键词: Agriculture; Base Pair Mismatch; Binding; Buffers; Catalysis; Catalytic DNA; Catalytic Domain; Cells; Cleaved cell; Color; Condition; DNA; DNA Sequence; Dependence; Detection; Development; Discrimination; Energy Transfer; Ensure; Equipment; Eye; Fluorescence; Genes; Goals; Gold; Growth; Hybrids; Imagery; Life; Methods; Modern Medicine; Nucleic Acids; Nucleotides; Optics; Physiological; Polymerase Chain Reaction; Positioning Attribute; Principal Investigator; Reaction; Science; Science of Microbial Genetics; Signal Transduction; Single base substitution; Solutions; Specificity; Structure; Techniques; Technology; Temperature; TimeLine; Work; base; clinically significant; concept; cost; design; improved; nanoparticle; novel strategies; nucleic acid detection; programs; response; tau Proteins; trait; viral detection

DESCRIPTION (provided by applicant): The goal of this project is to develop a new technique for nucleic acid analysis, which allows the detection of nucleic acids under mild conditions with extraordinary specificity and high sensitivity without PCR amplification. The novel approach is based on the use of a binary deoxyribozyme that consists of two DNA strands. In the absence of a nucleic acid analyte the strands are dissociated, and the deoxyribozyme is inactive. Addition of a specific DNA/RNA analyte results in the hybridization of the two deoxyribozyme DNA strands to the adjacent positions of the analyte and formation of the deoxyribozyme catalytic core. Since each DNA strand of the probe is bound to a relatively short analyte fragment (8-10 nucleotides), a single mismatched base pair substantially destabilizes each of the hybrids, enabling an extraordinary selectivity of the probe. The active binary deoxyribozyme triggers an autocatalytic cascade that is capable of an exponential amplification of catalysis that dramatically enhances the positive signal. The catalytic activity of the cascade will be detected using fluorescent resonance energy transfer or optically using gold nanoparticles. The method will contribute to the following major gains: (i) The establishment of a new concept for improving selectivity of nucleic acid recognition by dividing the probe into two fragments, (ii) Unprecedented high selectivity: the method will allow reliable discrimination of a single base substitution at any position of the 16-20 nucleotide DNA analyte. (iii) High sensitivity: potentially a single nucleic acid molecule can be detected without PCR amplification, (iv) Mild reaction conditions: the method will work in buffers close to physiological conditions and at room temperature, thus being potentially applicable in living cells, (v) Relatively lower costs. AIM # 1. Structural optimization of the binary deoxyribozyme for highly specific recognition of nucleic acid. AIM # 2. Amplification of the catalytic activity of the binary deoxyribozyme using a cascade of cross-catalytic cleaving deoxyribozymogens. AIM # 3. Optical detection of deoxyribozyme catalytic activity.

描述(申请人提供)：该项目的目标是开发一种新的核酸分析技术，允许在温和的条件下检测核酸，具有非凡的特异性和高灵敏度，而不需要PCR扩增。这一新方法是基于使用由两条DNA链组成的二元脱氧核酶。在没有核酸分析物的情况下，链被解离，脱氧核酶不活跃。加入特定的DNA/RNA分析物会导致两条脱氧核酶DNA链杂交到分析物的相邻位置，并形成脱氧核酶催化核心。由于探针的每条DNA链都与相对较短的分析物片段(8-10个核苷酸)结合，单个不匹配的碱基对大大破坏了每个杂交体的稳定，使探针具有非凡的选择性。活性的二元脱氧核酶触发了一个自我催化级联反应，能够以指数级联的方式放大催化作用，从而显著增强正面信号。级联的催化活性将通过荧光共振能量转移或使用金纳米颗粒进行光学检测。该方法将有助于以下主要成果：(I)通过将探针分成两个片段来提高核酸识别选择性的新概念的建立，(Ii)前所未有的高选择性：该方法将允许可靠地区分16-20个核苷酸DNA分析物的任何位置的单个碱基取代。(Iii)高灵敏度：潜在地可以在不进行PCR扩增的情况下检测到单个核酸分子，(Iv)温和的反应条件：该方法将在接近生理条件的缓冲液中工作，并在室温下工作，因此可能适用于活细胞，(V)相对较低的成本。目的#1.对脱氧核酶进行结构优化，以实现对核酸的高度特异性识别。目的#2.用交叉催化裂解脱氧核酶的级联反应扩增二元脱氧核酶的催化活性。目的#3.光学检测脱氧核酶催化活性。