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Publication

RNA-cleaving DNAzymes: from characterization at the single-molecule level to multiplex applications

Book - Dissertation

The development of biosensors is a widely multidisciplinary challenge. A good biosensor is able to measure the target of interest with high specificity, sensitivity and reliability, but is also cheap, fast and easy to operate. Moreover, the power to detect a panel of biomarkers simultaneously - called multiplexing - is a real advantage in medical diagnostics. In view of these requirements, RNA-cleaving DNA enzymes (RCDs) are attractive candidates as biorecognition elements thanks to their high specificity, and as signal generation elements on account of their capacity to amplify the signal through the cleavage of multiple substrate sequences. In this context, the aim of this dissertation was to study the potential of RCDs as signal generation elements, both from the application and fundamental point of view with the ultimate purpose of developing more performant biosensors. In the first part of this work, RCDs were used in combination with aptamers (aptazymes) to demonstrate their potential for the simultaneous detection of proteins and NAs in the same solution with great specificity and nanomolar limits of detection. The development of this multiplex bioassay led to the discovery of a methodology to rationally design new aptazymes, which was based on comparing the secondary structure of the RCD and the aptamer within the aptazyme to their individual sequences. The second part of this dissertation was focused on a more fundamental study of RCDs with the ultimate aim of unravelling information to aid in the design of a novel generation of RCD-based bioassays. Two methodologies were presented for the study and characterization of the cleavage reactions catalysed by RCDs. These novel methods facilitated the understanding of these sequences with the ultimate goal of further expanding the applications of RCD-based biosensors. In a first approach the RCDs were studied in bulk using a mathematical model that was developed and optimized to estimate the rate constant of each reaction step. The resulting model was used to accurately estimate the time-course of reactions for a wide range of substrate concentrations, temperatures and cofactor concentrations. Moreover, based upon a sensitivity analysis, the cleavage and association steps were determined to be the main rate-limiting steps of the reaction. In a second approach the performance of individual RCDs and the diversity among them was studied at the single-molecule level using Förster Resonance Energy Transfer. The analysis of individual RCDs under different reaction conditions allowed for the discrimination between cleavage and non-cleavage events. The quantification of such events was used to determine the amount of active DNAzyme molecules. The results showed that regardless of the experimental conditions, at least 25% of the RCDs on the surface were inactive. Based on the cleavage events, the substrate turnover of each RCD was calculated, resulting in an average of 2.5 cleavages per minute. Nevertheless, the distribution of values indicated a large variability among DNAzymes. Although many challenges still remain, we believe that the results reported in this thesis will serve as a good foundation for further research in this interesting field.
Publication year:2023
Accessibility:Open