Methods and applications of using DNA-cleaving DNAzymes to cleave double-stranded DNA

DNAzymes are DNA molecules that can catalyze specific reactions. Like protein-based enzymes, DNAzymes have high efficiency and specificity, yet they also own additional advantages such as more feasible customizability and lower cost. Particularly, DNAzymes that catalyze the cleavage of another DNA molecule, called DNA-cleaving DNAzymes, may specifically recognize and cleave user-defined DNA sequences with high efficiency. However, there are only limited applications of DNA-cleaving DNAzymes, because they could only recognize and cleave single-stranded DNA (ssDNA), whereas most DNA targets of interest (such as eukaryotic genomic DNA) are double-stranded DNA (dsDNA). To overcome this limitation, methods that can expand the substrate scope of DNA-cleaving DNAzymes from ssDNA to dsDNA are investigated. Here, a system called peptide nucleic acid (PNA)-assisted dsDNA nicking by DNAzymes (PANDA) is proven to be the first example of using DNA-cleaving DNAzymes to cleave dsDNA. PANDA may recognize almost any user-defined target sequences and cleave the target in a sequence-specific manner. The application of PANDA to mimic restriction enzymes is proven by using PANDA systems in molecular cloning. The potential and barriers of using PANDA systems in genome editing are investigated and discussed. Then, another system called ssDNA-assisted dsDNA nicking by DNAzymes (DANDA) is described, which proves that PANDA is not the only method to expand the substrate scope. The unique feature of DANDA to selectively cleave negatively supercoiled dsDNA rather than relaxed dsDNA has inspired a novel DNA detection method that has specificity on the target superhelicity. These results have proved that using DNA-cleaving DNAzymes to cleave dsDNA is feasible and has huge potential for further applications.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Lyu, Mingkuan

Contributors dc:contributor

• Lu, Yi
• Chan, Jefferson
• Silverman, Scott K.
• Zhao, Huimin

• Dnazyme
• Peptide Nucleic Acid
• Genetic Engineering
• Gene Editing
• Cccdna

Statement dc:rights

• Copyright 2022 Mingkuan Lyu

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/121185

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F121185