Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Marit Nilsen-Hamilton

Abstract

Detection and quantification of molecules has an enormous importance and potential in the field of scientific development and basic discovery research. Advances in colorimetric techniques, fluorescent labeled probes and biosensor based technologies have resulted in the specific detection and precise quantification of analytes. The effective development of a particular detection method needs optimization in terms of overall speed, efficiency, and accuracy of the sensing system. Current biosensor approaches often rely on cumbersome procedural steps and reagent requirements. Along with the complexity of the assay, the size of the analytical instrument is a deterrent factor for on-site applications. To overcome such disadvantages, novel approaches that challenge traditional methods need to be developed. Today, technological advancements have enhanced the ability of researchers to change the properties of molecules. An in vitro selection and amplification technique, called SELEX, has allowed for the discovery of specific oligonucleotide sequences referred to as 'aptamers'. Synthetic oligonucleotide aptamers display a high specificity and affinity binding to different classes of target molecules. Aptamers can discriminate between closely related targets with subtle structural differences. Currently, considerable efforts are being applied to miniaturize the nucleic-acid based sensor systems and assays for analytical and diagnostic screenings based on fluorescence approaches, calorimetric detections and other biosensing methodologies. The efficacy of aptamer based biosensors has been shown on a number of biosensing platforms and these sensors can provide revolutionary sensitivity for forensic detection and identification of controlled substances. Most of the aptamers used in sensor technologies are modified post selection for their use in analytical, diagnostic, forensic and therapeutic applications. Here we detail the specificity profile of cocaine aptamer including a range of metabolites such as benzoylecgonine (BZE), ecgonine methyl ester (EME), ecgonine ethyle ester (EEE), ecgonine (ECG), cocaethylene (COE), norcocaine (NOR) and anhydroecgonine methyl ester (AME). We tested different variants of cocaine aptamer and determined that the specificity of the aptamers is wider than previously reported. The cocaine aptamer binds to cocaine COC, NOR and COE. Extremely weak affinity was observed for BZE. All variants of cocaine aptamer tested showed uniform specificity but different affinities. We determined the motifs of the cocaine that are important for aptamer recognition. We hypothesized that if the cocaine aptamer binds selectively to cocaine and some of its metabolites then the functional groups that are common to these ligands should indicate the determinants of aptamer recognition. The affinity of the aptamers was tested against cocaine and its natural metabolites and against the unnatural cocaine molecules such as cocaine-tertiary-N-biotin (GK69), cocaine-tertiary-N-Boc (GK68), amethyl cocaine (GK71), dithiomethyl cocaine (GK94) and dihydroxy cocaine (GK66). It was determined if the aptamer recognition of cocaine dependent on the functional groups of cocaine such as benzoyl ester, methyl ester and N-methyl group and also upon the underivitized part of the cocaine molecule. The information of the functional groups important for aptamer recognition will be helpful towards the design of aptamer based analytical methods where the knowledge of steric aspects, such as the geometric shape, and interaction with the functional groups, plays an important role. For diagnostic and analytical assays, such information will help in predicting the ligands that have the potential to cross react, depending upon their structural similarity with cocaine. Further, we report a novel method for the fluorescence based detection of cocaine and cocaine metabolites using a 2-aminopurine (2AP) modified cocaine aptamer. Fluorescence quenching of the 2AP upon cocaine binding was used to detect cocaine and its metabolites. 2AP is a structural analogue of adenine in which the amino group is located at the C2 position instead of the C6 position. 2AP is an important molecule owing to its fluorescence properties and has been used as a reporter in structural studies of oligonucleotides. This molecule is reported to form structurally and thermodynamically similar Watson Crick base pairs with thymine as adenosine. We tested several 2AP substitutions of the cocaine aptamer that provided different fluorescence quenching and affinity profiles. Substitution of 2AP for A at the 23rd position was best suited for the fluorescence detection of cocaine and metabolites in homogeneous samples including 10% urine. Interestingly, 2AP substitution at the 6th position increased the affinity of the cocaine aptamer. The increase in affinity of the aptamer upon 2AP substitution could be due to the perturbation of the aptamer structure by 2AP or to direct interaction of 2AP with the cocaine. The cocaine aptamer has been reported to undergo large structural changes upon cocaine binding. However, in our studies we found relatively small 2AP fluorescence changes in the regions of the aptamer reported to undergo large changes. While the cocaine aptamer can be used for the detection of cocaine and metabolites in homogeneous solutions, the forensic application of this aptamer, especially the detection of cocaine in urine is limited due to its specificity profiles and its low binding affinity.

Copyright Owner

Ashish Sachan

Language

en

File Format

application/pdf

File Size

142 pages

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