University of Illinois Urbana-Champaign

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

Lyu, Mingkuan

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

Description

Title
Methods and applications of using DNA-cleaving DNAzymes to cleave double-stranded DNA
Author(s)
Lyu, Mingkuan
Issue Date
2022-11-29
Director of Research (if dissertation) or Advisor (if thesis)
Lu, Yi
Doctoral Committee Chair(s)
Lu, Yi
Committee Member(s)
  • Chan, Jefferson
  • Silverman, Scott K.
  • Zhao, Huimin
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • DNAzyme
  • peptide nucleic acid
  • genetic engineering
  • gene editing
  • cccDNA
Abstract
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.
Graduation Semester
2022-12
Type of Resource
Thesis
Copyright and License Information
Copyright 2022 Mingkuan Lyu

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