In vitro selection and characterization of mono-, di-, and trivalent metal-dependent DNAzymes and their sensing applications

by Seyed Fakhreddin Torabi

Institution: University of Illinois – Urbana-Champaign
Department: 0318
Degree: PhD
Year: 2015
Keywords: In vitro selection
Record ID: 2060587
Full text PDF: http://hdl.handle.net/2142/73057


Since the discovery of nucleic acid enzymes and aptamers that can perform functional roles other than storing genetic information; a new paradigm in nucleic acid chemistry has been opened. In 1990 the first artificially isolated functional nucleic acid was discovered through an in vitro process called SELEX, Systematic Evolution of Ligands by Exponential Enrichment. Aptamers are known as in vitro isolated functional nucleic acids that can bind their target ligands with high affinity and selectivity. Catalytic DNA or DNAzymes, another class of functional nucleic acids, were first isolated from pools of random DNA in 1994 using the in vitro selection procedure to catalyze the cleavage of a phosphodiester bond. Since the discovery of the first aptamer and DNAzyme, many more functional nucleic acids have been isolated and engineered to perform various functions, including binding to a wide variety of different ligands and catalysis of many different chemical reactions. Due to the superior properties of functional nucleic acids, they have found particular interest in environmental sensing and monitoring, biomedical diagnostics and therapy. Iron is a critical component of oxygen transportation and electron transfer, and is tightly associated with functions of many enzymes. Deficiency in iron leads to anemia, which is especially detrimental to pregnant women. Knowing the concentration of both Fe(II) and Fe(III) will be beneficial for clinical diagnoses. A DNAzyme pair selective for Fe(II) and Fe(III) is of particular interest, because of their interconversion in an biological environment. Also comparison of the DNAzymes selective for each would provide a fundamental understanding about DNAzymes??? abilities to distinguish between different oxidation states of the same metal ion. In-vitro selection experiments for Fe(II) and Fe(III)-dependent RNA-cleaving DNAzymes were carried out. In-vitro selection of Fe(II)-dependent DNAzyme selection was carried out in presence/absence of reduced glutathione in oxygen free condition. For each condition the effect of counter selection was investigated using mixture of divalent metal ions including Pb2+, Mn2+, Cd2+, Zn2+, and Co2+. Different pools were isolated that require Fe(II) for activity. Cis-acting DNAzymes were tested in presence of Fe(II) to find most active and selective Fe(II)-dependent DNAzymes. A number of DNAzymes converted into trans-cleaving DNAzymes through systematic truncation studies. Fe(II)-dependent DNAzymes including H5, the most active DNAzyme, were characterized. It was shown that the H5 DNAzyme selectivity reacts with Fe(II) in a mixture containing both Fe(II) and Fe(III). We are in the process of designing a fluorescent sensor for Fe(II) based on the H5 DNAzyme. This sensor will be tested for intracellular imaging of Fe(II) in living cells. To obtain Fe(III)-dependent DNAzymes, different selection protocols and selection conditions were investigated. In order to select active Fe(III)-DNAzymes, various attempts were remained fruitless, including efforts of…