Selection and characterization of DNA aptamers for estradiol and ethynylestradiol for aptasensor development

Akki, Spurti Umesh

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Description

Title

Selection and characterization of DNA aptamers for estradiol and ethynylestradiol for aptasensor development

Author(s)

Akki, Spurti Umesh

Issue Date

2018-07-10

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

Werth, Charles J.

Doctoral Committee Chair(s)

Guest, Jeremy S.

Committee Member(s)

• Silverman, Scott K.
• Cusick, Roland

Department of Study

Civil & Environmental Eng

Discipline

Environ Engr in Civil Engr

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• aptamer
• aptasensor
• estradiol
• ethynylestradiol
• DNA
• SELEX
• immobilization
• electrochemical sensor

Abstract

Small organic contaminants have been widely detected in the surface and ground waters of this nation. A sub-class of these contaminants called endocrine disrupting compounds (EDCs) are known to have adverse effects on aquatic and human health. Among the EDCs, natural hormone 17β-estradiol (E2) and synthetic hormone 17α-ethynylestradiol (EE) possess high estrogenic potency and hence are contaminants of interest. Conventional methods to detect these compounds are expensive, time consuming and need implementation by an expert. By contrast, antibody-based assays are relatively inexpensive and commercially available but suffer from poor selectivity. A promising alternative makes use of DNA aptamers as molecular recognition elements. In order to evaluate the potential of DNA aptamers and aptasensors to detect small organics in natural waters, the following objectives were pursued: (1) critically review DNA aptamers and aptasensors developed for small organic molecules and assess their use for monitoring environmentally relevant organics, (2) select and characterize DNA aptamers that bind to E2 and EE and, (3) study the effect of immobilization on the binding affinity of the selected E2 and EE aptamers. A review of ~80 aptamers and ~200 aptasensors for small organics was conducted to identify factors that affect binding affinity of the aptamer and limits of detection (LODs) of the aptasensor. Based on regression analyses, aptamer binding affinities are found to have a weak relationship with hydrophobicity of the target and length of the aptamer (p-values<0.05). Independent t-tests comparing aptasensor LODs suggest that the electrochemical platform is significantly more sensitive than colorimetric and fluorescence-based platforms. The inherent binding affinity of the aptamer was found to have a significant effect on the LOD of the aptasensor. While some fabricated aptasensors are sufficiently sensitive to detect contaminants at environmentally relevant concentrations, they are often associated with complex fabrication steps, and/or interference from structurally similar analogs. As a result, aptasensor commercialization faces many challenges including reusability, reproducibility and robustness. In vitro selections were conducted with different selection pressures to isolate sensitive and selective DNA aptamers for E2 and EE. An equilibrium-filtration assay was used to determine dissociation constants (Kd) of the aptamer towards its parent target and its analogues. The E2 aptamers, E2Apt1 and E2Apt2 were found to have Kd values of 0.6 µM. They bound to analogue estrone (E1) with a similar affinity but were at least 74-fold more selective over EE. The EE aptamers Kd values are 0.5-1 µM. While one EE aptamer (EEApt1) was 53-fold more selective for EE over E2 and E1, the second EE aptamer (EEApt2) bound to all three EDCs (E1, E2 and EE) with similar affinities. The aptamers maintained their binding affinities in natural waters samples (tap water and lake water). DMS probing of the structure of the DNA aptamer revealed that the binding regions were mostly located in the single-stranded loop regions of the aptamer. Aptasensors typically employ immobilized aptamers though the aptamers are selected and characterized while free or unattached in solution. The Kd values of immobilized selective aptamers were evaluated using magnetic microbeads surface for attachment. E2Apt1 immobilized at either end (5′ or 3′) and E2Apt2 immobilized at the 3′ end retain their binding affinity. The binding affinity is inversely correlated to the average linear distance of the binding pocket from the immobilized end. This result suggests that unwanted interactions between the aptamer and other moieties are more likely when the binding pocket is further away from the surface. Binding curve of E2Apt2 immobilized at the 5′ end indicates potential dimerization at high loadings of aptamer on the beads due to increased proximity between aptamer strands. EEApt1 loses its binding affinity upon immobilization potentially due to disruption in its tertiary structure upon attachment to the surface. Despite no loss in binding affinity upon immobilization, E2Apt1 (5′) shows no significant change in electrochemical current on binding to E2 when incorporated into an electrochemical sensor. This result implies an insufficient conformational change of the aptamer on binding to the target. Overall, this work identifies the first aptamers for EE and selective aptamers for E2, while also highlighting the issues with development of aptamers and their eventual incorporation into aptasensors to detect small organics. Two major concerns are (1) immobilizing aptamers in sensor platforms while selections of aptamers are conducted with free/unattached aptamers, resulting in loss of binding affinity and (2) insufficient conformational change of the aptamer on binding to small molecule targets, resulting in a lack of change in the sensor signal. The findings from this dissertation support additional research directions regarding employing free aptamers in sensors and/or conducting new selections for aptamers using a DNA pool that is attached to a surface.

Graduation Semester

2018-08

Type of Resource

text

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Copyright 2018 Spurti Umesh Akki

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