Isothermal and Homogeneous Detection of Nucleic Acids and Proteins Using the Cleavage Activities of CRISPR-Cas and DNAzyme

• Author / Creator

  Cao, Yiren

• Nucleic acids and proteins play essential roles in biological systems; and the detection of these molecules can be applied to the diagnoses of diseases. Extensive detection of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral RNA has contributed to the containment of the recent coronavirus disease of 2019 (COVID-19) pandemic. The gold standards for the detection of nucleic acids and proteins are the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Although PCR and ELISA are sensitive and accurate, they are not ideal for point-of-care (POC) analysis because PCR requires thermal cycling and ELISA protocols include time-consuming washing steps. Isothermal amplification techniques and homogeneous binding assays are promising alternatives for POC applications. The former can be performed under readily achievable temperature and the latter in a single test tube.
  Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been integrated with the isothermal amplification techniques to improve the specificity of the detection of SARS-CoV-2 viral RNA to avoid false positive results. Composed of a guide RNA (gRNA) and a Cas protein, the CRISPR-Cas system can be activated only when the gRNA hybridizes with the specific amplification product of target gene. Most developed assays for SARS-CoV-2 rely on fluorescence detection and/or a lateral flow format, which requires an excitation light source and sophisticated equipment. Colorimetric assays, on the other hand, have not been
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  fully exploited for sensitive and rapid detection. Described in Chapter 2, a molecular transducer with hairpin structure was used to facilitate the trans-cleavage activity of CRISPR-Cas12a, resulting in the aggregation of gold nanoparticles (AuNPs) and color change. A simple centrifugation step for 10 seconds was sufficient to achieve a clear color change within 1 minute. The assay maintained the sensitivity of isothermal amplification and had a detection limit of 225 copies of the nucleocapsid (N) gene of the viral RNA.
  The cleavage activity of DNAzyme has also been incorporated with isothermal amplification techniques to construct DNA circuits with enhanced sensitivity or lower background. In a typical circuit, the amplification reaction and cleavage reaction form a positive feedback loop to amplify the target exponentially. However, the detection of different nucleic acid targets requires individually redesigned sequences of the circuit. Taking advantage of the multiple component nucleic acid enzyme (MNAzyme) technique derived from DNAzyme, I designed two subunits to recognize the target and initiate the DNA circuit (Chapter 3). The detection of a different target was readily achieved by changing only the complementary region of the subunits. To minimize background and maintain the cleavage activity of the MNAzyme, I investigated a series of blockers that were critical to the construction of circuit with positive feedback. I achieved a limit of detection at fM levels for two model targets.
  MNAzymes have been used for the detection not only of nucleic acids but also of proteins. MNAzymes with different secondary structures have been reported and the split
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  locations of MNAzymes used for proteins were the same as of MNAzymes used for nucleic acids. So far, there is no systematic study exploring how the secondary structure and split location affect the cleavage activity of MNAzymes in protein analysis. In Chapter 4, I systematically compared 14 split locations and two secondary structures to obtain MNAzymes with high cleavage activity. To achieve homogeneous detection of specific proteins, I combined the selected MNAzyme with binding-induced DNA assembly (BINDA) (Chapter 5). I designed two DNA motifs with short complementary regions. The two motifs assembled together to form the MNAzyme only in the presence of the protein target. For the detection of two protein targets, I achieved a sensitivity of pM level without polymerase-assisted amplification. The assay was performed in a single test tube and required only the mixing of reagents and reading of signals.
  I developed a colorimetric assay and a DNA circuit for isothermal detection of nucleic acid targets. I investigated the MNAzyme with the optimal split location and secondary structure for homogeneous detection of protein targets. My thesis research contributes to the development of POC tests of these biomolecules. By altering the recognition component (gRNA, sensor arm, aptamer, and antibody), I can potentially extend the developed assays to the detection of various targets.

• Subjects / Keywords

  • CRISPR/Cas
  • DNAzyme

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-dz6m-mz24

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.