Expanding Oligonucleotide Properties through Chemical Modification: from Gene Editing and Abasic Detection to Self-Replication

• Author / Creator

  Park, Hansol

• DNA stores the genetic information of human beings and all living organisms. Research on oligonucleotides can lead us to find the answer to the origin of life. Self-replication of nucleic acids in the absence of enzymes such as polymerase or ligase represents an important step in the origin of life. Yet, product inhibition remains a major challenge despite efforts to develop strategies for the nonenzymatic self-replication of nucleic acids. We propose that studying a successful example of enzymatic DNA self-replication based on a simple ligation chain reaction could shed light on critical steps in self-replication. Previously our group showed an isothermal self-replication system called lesion-induced DNA amplification (LIDA). It was proposed that the destabilizing abasic group played an important role in reducing product inhibition. This thesis focuses on determining all of the factors that allow LIDA to overcome product inhibition. Thermodynamic studies were performed to measure the binding affinity of DNA complexes in the absence of enzymes, and a kinetic model was used to determine the corresponding values in the presence of T4 DNA ligase. This study suggests that T4 ligase helps overcome product inhibition by reducing the binding affinity difference between the product duplex and the intermediate complex, implying that catalysts or enzyme-like substances that stabilize the intermediate DNA complex might be a route to nonenzymatic replication. Unnatural nucleobases have been designed to extend the role of natural bases in templated synthesis and molecular recognition. Some laboratory-designed bases that imitate the hydrogen bonding of the natural base pairs often contain pyrimidines or purine derivatives. In contrast, other unnatural base pairs rely on the geometric fit and packing force. Prior work had shown a hydrophobic pyrene nucleotide forms a strong base pair with an abasic site. As abasic lesions are the most common types of DNA damage, pyrene nucleotides are a promising candidate to detect abasic sites using their high stability. Remarkable selectivity was observed in the ligation of 5’-phosphate-pyrene nucleotides across from an abasic lesion in a templated ligation reaction suggesting that pyrene-terminated probes could be used in abasic detection when followed by amplification of the ligated product. This result also suggested that other DNA adducts could be detected by the incorporation of DNA glycosylase in a prior step, which removes specific DNA adducts to form an abasic lesion, broadening pyrene nucleotides' application in DNA lesion or adduct detection.  The CRISPR-Cas9 system is a powerful gene-editing tool that allows researchers to alter DNA sequences and modify gene function, the identification of which was just recognized with the 2020 Nobel Prize in Chemistry. The CRISPR-Cas9 system requires Cas9 protein, crRNA, the 20 nucleotides (nt) programmable target sequence, and tracrRNA, which binds the Cas9 nuclease and complexes the crRNA through partial hybridization. The crRNA and tracrRNA sequences can also be fused into one strand resulting in a single guide RNA (sgRNA). For some applications targeting multiple genes is required to study complex biopathways and avoid the difficulty in deconvoluting overlapping and redundant roles of proteins. Yet, the synthesis of sgRNA in solid-phase synthesis and the modification of sgRNA remain challenging. Here I describe a strategy to synthesize multiple fragments that together constitute the sgRNA and connect these pieces with bioconjugation: specifically, by copper-catalyzed azide-alkyne cycloaddition (CuAAC), the most common “click” reaction. This modular bioconjugation method enables multi-color labeling with different fluorophores as well as different chemical modifications. This modular synthesis was successfully used to edit two genes in cells combined with fluorescence-activated cell sorting.    Comprehensive studies on oligonucleotides serve to further the knowledge and understanding of the oligonucleotides and their applications. As oligonucleotide plays a key role in biotechnologies, the observation and methodologies within this thesis may prove insightful for understanding the self-replication of the oligonucleotide, developing probes for DNA damage detections, and enabling the preparation of highly modified single guide RNA for CRISPR-Cas9-based biotechnologies. 
  

• Subjects / Keywords

  • Oligonucleotide
  • chemical modification
  • gene editing
  • abasic detection
  • self-replication

• Graduation date

  Spring 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-gf72-gf46

• 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.