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

Torabi, Seyed Fakhreddin

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Description

Title

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

Author(s)

Torabi, Seyed Fakhreddin

Issue Date

2015-01-21

Director of Research (if dissertation) or Advisor (if thesis)

Lu, Yi

Doctoral Committee Chair(s)

Lu, Yi

Committee Member(s)

• Martinis, Susan A.
• Silverman, Scott K.
• Tajkhorshid, Emad

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• In vitro selection
• functional DNA
• DNAzyme
• iron
• sodium
• mercury
• biosensor

Abstract

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 two previous Lu lab members. The choice of negative selection condition, Fe(III)-stabilizing agent and other selection parameters were shown to be vital to the success of Fe(III)-dependent DNAzyme selection. Finally, several classes of Fe(III)-dependent DNAzymes were selected in presence of 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris) as a stabilizing agent for Fe(III) at pH 5.5. Activity of several clones from selected pools were examined in presence of Fe(III) to find the most active sequence. A number of active DNAzymes were converted into trans-cleaving DNAzymes including B12 and D13, the most active clones. Buffer, ligand, and pH requirements of both DNAzymes were investigated. It was shown that Fe(III)-dependent DNAzymes are highly selective and are able to react with Fe(III) without any interference from Fe(II). A turn-on fluorescent sensor for Fe(III) is designed based on the B12 DNAzyme sequence. We would like to test our fluorescent sensor capability in imaging Fe(III) in endosome. Sodium ion detection is of particular interest. Infants suffer from diabetes insipidus (DI) are prone to wide sodium fluctuations, due to a high fluid diet and uncertainties in the assessment of hydration status. These individuals require daily assessment of serum and urine sodium. Monitoring sodium level is important in several other health conditions such as syndrome of inappropriate antidiuretic hormone secretion, hepatorenal syndrome, hypertension and different types of hyponatremia and hypernatremia. Two parallel in vitro selections were carried out to expand capability of DNAzymes in cleaving a ribonucleotide phosphodiester bond independent of any divalent or trivalent metal ion and, specifically selective for the sodium ion. Having efficient sodium specific DNAzyme can be used to design fast and simple point-of-care sodium sensors. A column-based strategy was used to isolate sodium-specific DNAzymes. NaA43 DNAzyme was isolated, truncated into a trans-acting DNAzyme, and biochemically characterized. Interestingly NaA43 is at least 10,000-fold selective for sodium over other monovalent ions such as lithium. A turn-on fluorescent sensor was designed based on NaA43 and tested in imaging sodium inside living cells. As a proof of concept we demonstrated that our sodium DNAzyme-based sensor can be used for cellular studies. We are designing a ratiometric fluorescent sensor based on NaA43 that can be used for quantitative assessment of sodium inside living cells. Additionally, a biosensor based on personal glucose meter and NaA43 was developed. It was shown that our NaA43-based sensor can be used to detect sodium in human serum with high accuracy (0.99). Mercury is a highly toxic and widely distributed pollutant in the environment. It is known that mercury ion is able to bind to a class of functional DNA containing thymine-thymine mismatch. To develop a platform for detection of mercury ion, ability of functional DNA–linked gold nanoparticles was demonstrated for a fast and simple detection and quantification of Hg2+ ions in aqueous solution, with high sensitivity and selectivity over competing metal ions. DNA functionalized gold nanoparticles were aggregated using a functional DNA containing several thymine-thymine mismatches. Mercury ions induce the folding of linker DNA strand by thymine–Hg2+–thymine formation which disassemble aggregated gold nanoparticles rapidly and cause color change from purple to red. Such a system has been converted into dipstick tests using lateral flow devices to make it even more practical for on-site detection.

Graduation Semester

2014-12

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Copyright 2014 Seyed Fakhreddin Torabi

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