Graduation Year
2024
Document Type
Campus Only Senior Thesis
Degree Name
Bachelor of Arts
Department
Biology
Reader 1
Aaron Leconte
Reader 2
Pete Chandrangsu
Abstract
DNA oligonucleotides are used for a plethora of biotechnological and biomedical applications such as molecular tagging, sequencing, and drug therapies, but are limited by susceptibility to nucleases. Xeno-Nucleic Acids (XNA) serve as a solution to improve nuclease stability but cannot be synthesized by naturally occurring DNA polymerases due to structural differences between DNA and XNA. 2’-Azide (2’Az) XNAs are of particular interest due to the capacity of the azido group to participate in the highly selective biorthogonal click chemistry reaction, allowing the conjugation of interesting molecules, dyes, or probes. While previous literature has demonstrated the incorporation of 2’-Az into DNA using laboratory-evolved XNA polymerases, there remains a gap in our knowledge regarding the ability of these polymerases to incorporate 2’-Az into an XNA polymer. Here, we evaluate whether SFM4-3, SFM4-6, and SFP1, 3 leading XNA polymerase mutants, can synthesize fully substituted mixed polymers containing a mixture of 2’-Az and 2’-fluoro (2’-F) modifications. All enzymes successfully synthesized a full-length 47 nucleotide 2’-Az/2’-F mixed XNA from a DNA template under mild synthesis conditions in 2 hours. To assess the accuracy of synthesis and better understand the impact of nucleotide concentration on accuracy, we employed a one-pot reverse transcription/amplification reaction to convert the XNA synthesis products back to DNA; the DNA was then analyzed using high-throughput DNA sequencing to quantify the error rate and error spectrum of the enzymes. We observed that polymerases had increased fidelity synthesizing 2’-Az/2’-F polymers relative to fully substituted 2’-F polymers. Additionally, SFP1 had the highest accuracy amongst these enzymes and lower nucleotide concentrations contributed to increased fidelity. This research serves to expand our understanding of XNA polymerases, synthesis of XNA with azide modifications, and identify promising mutants for XNA synthesis, thereby advancing the field of nucleic acid chemistry.
Recommended Citation
Liu, Serena C.; Leconte, Aaron M.; Venkatesh, Ananya; and Kang, Hailey E., "Assessing Fidelity of XNA Polymerase on 2’-Fluoro/2’-Azide Mixed XNA Synthesis" (2024). Scripps Senior Theses. 2320.
https://scholarship.claremont.edu/scripps_theses/2320
This thesis is restricted to the Claremont Colleges current faculty, students, and staff.
