Aptamer Sensors for Drugs of Abuse and Medical Biomarkers: Design, Engineering and Application in Complex Samples

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors

The development of capillary electrophoresis assays to study enzyme inhibition

New methods for studying enzymes and enzyme inhibition using capillary electrophoresis (CE) were developed and applied. Because CE is a separation technique, spectral interference is minimized by separation of substrates, products, inhibitors, and sample matrix components. Two types of CE enzyme assays were explored in this dissertation research. The first assay was based on optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence detection (LIF). This approach involves periodic photobleaching of fluorescent substrates and products. The initial goal of the study was to develop an assay for adenosine deaminase (ADA). A fluorescent ADA substrate was synthesized; however, the substrate was unusually photostable and could not be photobleached. In Chapter 2, a comparative study is presented that quantified the photostability of the fluorescent ADA substrate relative to other fluorophores that have been used for OGVCE in order to obtain a better understanding of the ideal properties of a dye for OGVCE assays. The development of an off-column CE assay for measuring acetyl coenzyme A carboxylase holoenzyme (holo-ACC) activity and inhibition is presented in Chapter 3. The two reactions catalyzed by the holo-ACC components, biotin carboxylase (BC) and carboxyltransferase (CT), were simultaneously monitored in the assay, which required successful separation of two substrates and two products using micellar electrokinetic chromatography (MEKC). Additionally, a previously reported off-column CE assay for only the CT component of ACC was optimized, and an off-column CE assay for only the BC component of ACC was developed. Finally, the CE-based enzyme assays developed in Chapter 3 were used for screening of botanical extracts against holo-ACC, BC and CT (Chapter 4). Compounds known to be present in the botanical extracts that inhibited holo-ACC were selected based on a detailed literature search and tested for inhibition of holo-ACC, BC and CT. Synthetic compounds selected based on ligand homology studies were also tested for inhibition of holo-ACC. These CE assays were simple, off-column methods, where spectral interference and other limitations of previous methods were greatly reduced. The development of methods that can directly monitor reactants and products for multiple enzyme-catalyzed reactions will have impact beyond the enzymes presented in this dissertation

Fluorescent sensors using DNA-functionalized graphene oxide

The final publication is available at Springer via http://dx.doi.org/10.1007/s00216-014-7888-3In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNA-based biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence-quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.University of Waterloo || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation || Foundation for Shenghua Scholar || National Natural Science Foundation of China || Grant No. 81301258, 21301195 Postdoctoral Science Foundation of Central South University and Hunan province ||Grant No. 124896 China Postdoctoral Science Foundation || Grant No. 2013M540644 Hunan Provincial Natural Science Foundation of China || Grant No. 13JJ4029 Specialized Research Fund for the Doctoral Program of Higher Education of China || Grant No. 2013016212007

Target profiling of PARP inhibitors and necroptosis inhibitors using photoaffinity labelling

Target profiling of a small molecule therapeutic is essential to fully understand how that compound works in the clinic. Photoaffinity labelling (PAL) has become a widely utilised strategy for in-cell target identification campaigns for reversible, small molecule drugs. After an overview of target profiling and PAL, this Thesis discusses the application of PAL to two classes of molecules with incomplete target profiles. The Thesis focusses initially on the generation of the first photo-activatable probe for inhibitors of the PARP family of enzymes, PARPYnD, based on a novel anti-cancer PARP1/2/6 inhibitor AZ0108 with unexplained off-target toxicity. The design, synthesis and validation of the probe is discussed, along with the application of PARPYnD to PAL studies. Herein, simultaneous live-cell target engagement of PARP1/2 is shown for the first time by a photo-activatable probe, and this labelling is used to quantify live-cell engagement of these PARPs by known PARP inhibitors in competitive PAL experiments. For AZ0108 and clinical PARP inhibitor olaparib, novel off-targets are identified, demonstrating the power of PAL to capture weaker, secondary binders. Finally, PARPYnD fails to label PARP6 in live cells, but is able to label recombinant PARP6, highlighting a biomolecular disparity that raises questions about the proposed mechanism of action of AZ0108. PAL is then applied to a novel series of inhibitors of necroptosis, an inflammatory form of cell death, with an unknown mechanism of action. Design and synthesis of cell-active photo-activatable probe 7PQYnD1 is presented, along with the development of a bespoke live-cell necroptosis assay to evaluate necroptosis inhibitors in-house. 7PQYnD1 is then applied to the PAL workflow and five bona fide target proteins are identified through proteomics. Preliminary functional analysis of these hits is then undertaken to begin to identify the target interaction(s) responsible for the anti-necroptosis phenotype of these compounds.Open Acces

Novel fluorescent cell-based sensors for detection of viral pathogens

Diseases caused by viruses are responsible for millions of deaths worldwide. However, viruses are used for development of virus-based biopharmaceuticals (VBBs), like vaccines and viral vectors to treat/prevent diseases. These applications require reliable methods for virus and viral vectors quantification. Nevertheless, current titration techniques fail to provide fast, reliable method with high-throughput. This thesis aimed at developing genetically encoded fluorescent cell-based sensors for virus detection - VISENSORS. These are activated upon recognition by viral proteases (VP) of specific cleavable sequences, allowing detection of label-free virus and viral vectors. Three strategies were developed based on split-Green Fluorescent Protein (GFP) fluorescence inhibition caused by inducing a structural distortion (SD): coiled-coil, embedded and cyclized split-GFP VISENSORS. After VP proteolysis, SD is relieved, and fluorescence is restored. Different backbones were optimized per strategy, and their performance evaluated under VP activity for detection of Adenovirus and Human Immunodeficiency Virus type one (HIV-1) by transient screenings and latter, stably expressing VISENSORS in mammalian cells and analysing its response to viral infection. The coiled coil strategy for Adenovirus-VISENSOR showed the lowest performance being only tested in transient. The cyclized and embedded strategies were tested upon adenovirus infection, the first exhibited a lower Signal/Noise ratio (S/N) (1.6) possibly caused by sensor instability, the latter showed promising performance - S/N of 2.0 - with room for enhancement through improving SD. HIV-1-VISENSORS were successfully established, where embedded and cyclized strategies proved similar S/N performances in transient. This work contributes for the optimization of Adenovirus and HIV-1 label-free sensors, by analysing the impact of SD strategies and VP cleavable sequences, showing these have high impact in sensor performance. Embedded strategy showed potential although further improvements to reduce the Noise are needed VISENSORS can be adapted to different viruses for detection and quantification of viruses and VBBs

Molecular evidence of adenosine deaminase linking adenosine A2A receptor and CD26 proteins

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits

Point of care diagnostics for tuberculosis

The goals of the End TB strategy, which aims to achieve a 90% reduction in tuberculosis (TB) incidence and a 95% reduction in TB mortality by 2035, will not be achieved without new tools to fight TB. These include improved point of care (POC) diagnostic tests that are meant to be delivered at the most decentralised levels of care where the patients make the initial contact with the health system, as well as within the community. These tests should be able to be performed on an easily accessible sample and provide results in a timely manner, allowing a quick treatment turnaround time of a few minutes or hours (in a single clinical encounter), hence avoiding patient loss-to-follow-up. There have been exciting developments in recent years, including the WHO endorsement of Xpert MTB/RIF, Xpert MTB/RIF Ultra, loop-mediated isothermal amplification (TB-LAMP) and lateral flow lipoarabinomannan (LAM). However, these tests have limitations that must be overcome before they can be optimally applied at the POC. Furthermore, worrying short- to medium-term gaps exist in the POC diagnostic test development pipeline. Thus, not only is better implementation of existing tools and algorithms needed, but new research is required to develop new POC tests that allow the TB community to truly make an impact and find the ‘‘missed TB cases’’

DNA stabilized fluorescent metal nanoclusters for biosensor development

The final publication is available at Elsevier via https://doi.org/10.1016/j.trac.2013.12.014." © 2014. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Fluorescent silver, gold and copper nanoclusters (NCs) have emerged for biosensor development. Compared to semiconductor quantum dots, there is less concern about the toxicity of metal NCs, which can be more easily conjugated to biopolymers. These NCs need a stabilizing ligand. Many polymers, proteins and nucleic acids stabilize NCs, and many DNA sequences produce highly-fluorescent NCs. Coupling these DNA stabilizers with other sequences, such as aptamers, has generated a large number of biosensors. We summarize the synthesis of DNA and nucleotide-templated NCs; and, we discuss their chemical interactions. We briefly review properties of NCs, such as fluorescence quantum yield, emission wavelength and lifetime, structure and photostability. We categorize sensor-design strategies using these NCs into: (1) fluorescence de-quenching; (2) generation of templating DNA sequences to produce NCs; (3) change of nearby environment; and, (4) reacting with heavy metal ions or other quenchers. Finally, we discuss future trends.University of Waterloo || Canadian Foundation for Innovation || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |