Nucleic acid analysis using deoxyribozyme technology

描述（由申请人提供）：本项目的目标是开发一种用于核酸分析的新技术，该技术允许在温和条件下检测核酸，具有非凡的特异性和高灵敏度，无需PCR扩增。这种新方法是基于使用由两条DNA链组成的二元脱氧核酶。在不存在核酸分析物的情况下，链解离，并且脱氧核酶无活性。添加特定的DNA/RNA分析物导致两条脱氧核酶DNA链与分析物的相邻位置杂交并形成脱氧核酶催化核心。由于探针的每条DNA链与相对短的分析物片段（8-10个核苷酸）结合，因此单个错配的碱基对基本上使每个杂交体不稳定，从而使探针具有非凡的选择性。活性二元脱氧核酶触发自催化级联反应，其能够指数放大催化作用，从而显著增强阳性信号。该级联的催化活性将使用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收益：（i）建立一个新的概念，通过将探针分为两个片段，提高核酸识别的选择性，（ii）前所未有的高选择性：该方法将允许可靠的区别在任何位置的16-20个核苷酸的DNA分析物的单碱基取代。(iii)高灵敏度：潜在地，单个核酸分子可以在没有PCR扩增的情况下被检测到，（iv）温和的反应条件：该方法将在接近生理条件的缓冲液中和在室温下工作，因此潜在地适用于活细胞，（v）相对较低的成本。目标1。高特异性识别核酸的双元脱氧核酶的结构优化。目标2。使用交叉催化切割脱氧核酶原的级联放大二元脱氧核酶的催化活性。目标3。脱氧核酶催化活性的光学检测。

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.