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. ? ? ?

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Exploratory/Developmental Grants (R21)
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Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Ozenberger, Bradley
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University of Central Florida
Schools of Arts and Sciences
United States
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Bengtson, Hillary N; Kolpashchikov, Dmitry M (2014) A differential fluorescent receptor for nucleic acid analysis. Chembiochem 15:228-31
Gerasimova, Yulia V; Cornett, Evan M; Edwards, Emily et al. (2013) Deoxyribozyme cascade for visual detection of bacterial RNA. Chembiochem 14:2087-90
Gerasimova, Yulia V; Kolpashchikov, Dmitry M (2013) Folding of 16S rRNA in a signal-producing structure for the detection of bacteria. Angew Chem Int Ed Engl 52:10586-8
Cornett, Evan M; O'Steen, Martin R; Kolpashchikov, Dmitry M (2013) Operating Cooperatively (OC) sensor for highly specific recognition of nucleic acids. PLoS One 8:e55919
Cornett, Evan M; Gerasimova, Yulia V; Kolpashchikov, Dmitry M (2013) Two-component covalent inhibitor. Bioorg Med Chem 21:1988-91
Gerasimova, Yulia V; Kolpashchikov, Dmitry M (2013) Detection of bacterial 16S rRNA using a molecular beacon-based X sensor. Biosens Bioelectron 41:386-90
Labib, Mahmoud; Ghobadloo, Shahrokh M; Khan, Nasrin et al. (2013) Four-way junction formation promoting ultrasensitive electrochemical detection of microRNA. Anal Chem 85:9422-7
Cornett, Evan M; Campbell, Eleanor A; Gulenay, George et al. (2012) Molecular logic gates for DNA analysis: detection of rifampin resistance in M. tuberculosis DNA. Angew Chem Int Ed Engl 51:9075-7
Gerasimova, Yulia V; Kolpashchikov, Dmitry M (2012) Connectable DNA logic gates: OR and XOR logics. Chem Asian J 7:534-40
Kolpashchikov, Dmitry M (2012) An elegant biosensor molecular beacon probe: challenges and recent solutions. Scientifica (Cairo) 2012:928783

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