DNAzyme Walkers for the Detection of Enzyme Activity
Creators: Tao, Jeffrey Jiaen
Publisher: name: University of Alberta Library
Year: 2024

<p>DNAzyme walkers represent a dynamic class of DNA nanotechnology with immense potential to enhance diagnostics and drug delivery. These locomotive nanomachines consist of a mobile DNA strand that navigates a designated track of DNA, offering potential applications in biosensing, imaging, and cargo delivery. The integration of gold nanoparticles (AuNPs) with DNA nanotechnology has propelled DNAzyme walker technology forward in three significant ways: 1) enabling the assembly of densely packed spherical DNA tracks, 2) providing strong fluorescence quenching properties for biosensor development, and 3) facilitating the transport of DNA components into intracellular environments of living cells. Previous research on DNAzyme walkers has primarily focused on detecting and imaging nucleic acids, such as microRNA. Investigations into enzyme-triggered DNAzyme walkers are still in their early stages. The primary objective of my research was to develop DNAzyme walkers that respond to specific enzymes within living cancer cells, thereby broadening the scope of targets for DNAzyme walker technology. Furthermore, the ability to target enzyme activity would enable the DNAzyme walker to respond to active cancerous processes such as therapeutic resistance, instead of merely measuring the presence of nucleic acid biomarkers. My thesis research aimed to develop DNAzyme walkers for the detection of six enzymes that are overexpressed in most advanced cancers and implicated in the DNA damage response.</p>

<p>I developed a DNAzyme walker that responded to apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme involved in base excision repair (BER) that processes abasic sites. I designed a blocker DNA with an abasic site to inhibit DNAzyme activity, ensuring that only APE1 activity would cleave and remove the blocker DNA and initiate DNAzyme activity. For APE1 detection, I achieved limits of detection (LODs) of 0.16 pM APE1 in buffer and an amount of APE1 in diluted lysate equivalent to a single HeLa cell. After incubation with HeLa cells, the DNAzyme walker successfully signalled the presence and enzyme activity of APE1 within live cells, which was imaged using confocal microscopy.</p>

<p>By designing the blocker DNA to contain additional lesions, I developed DNAzyme walkers that could respond to four DNA glycosylases: uracil DNA glycosylase (UDG), single-strand-selective monofunctional uracil DNA glycosylase (SMUG1), formamidopyrimidine DNA glycosylase (Fpg), and alkyl adenine DNA glycosylase (AAG). These DNA glycosylases precede APE1 in the BER pathway. I achieved LODs of 3.2 pM for UDG, 3.0 pM for SMUG1, 31 pM for Fpg, and 78 pM for AAG. The DNAzyme walker detected UDG and SMUG1 in diluted A549 cell lysate, with an LOD of 48 cells in 100 μL. Using UDG inhibitor, I determined that UDG was predominantly active over SMUG1 in A549 cell lysate. About 501 nM of UDG was estimated to reside within each A549 cell.</p>

<p>In addition to detecting hydrolytic enzymes, I attempted to measure the activity of telomerase, a reverse transcriptase. I designed the DNAzyme walker to leverage the telomeric extension activity of telomerase, which would facilitate toehold-mediated strand displacement (TSMD) of the blocker DNA to activate the DNAzyme. I discuss challenges in the detection of telomerase activity in cancer cells and lysates using the DNAzyme walker and other DNA-based techniques,</p>

<p>Throughout my thesis, I demonstrate the adaptability of the DNAzyme walker for detecting enzyme activity within a living cell. A well-designed DNAzyme walker can be a sensitive and specific analytical device that operates in ambient temperature, carrying promising potential for point-of-care detection of a variety of target molecules.</p>