Nucleic acid analysis using deoxyribozyme technology

Columbia University Health Sciences, New York NY

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Abstract

[unreadable] 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. [unreadable] [unreadable] [unreadable]