DNAzymes for sequence-dependent peptide lysine acylation

Protein enzymes, ribozymes, and DNA enzymes (DNAzymes) are the three types of biopolymers that are known to exhibit catalytic activity. Of these biopolymers, protein enzymes are the most widely known, followed by ribozymes, and finally DNAzymes. However, because proteins cannot be amplified in vitro, new protein catalysts cannot be developed de novo. Instead, protein enzymes are developed using methods such as directed evolution, which rely on an existing enzyme as a starting point, limiting the types of reactions that can be targeted for catalysis. By contrast, RNA and DNA can be PCR-amplified, enabling these nucleic acids to undergo in vitro selection, starting with a random pool of single-stranded RNA or DNA sequences, to identify ribozymes or DNAzymes de novo. This ability to identify these nucleic acid catalysts from scratch provides significant flexibility in the types of reactions that can be pursued. Notably, DNA has several practical advantages over RNA, such as increased stability, ease of synthesis, and the ability to be directly amplified by PCR. Moreover, there is no evidence to suggest that ribozymes are more catalytically competent than DNAzymes. Therefore, the Silverman lab focuses on the identification of DNAzymes for various chemical reactions to explore the scope of DNAzyme catalysis. Beyond understanding the range of reactions that DNA can catalyze, developing DNAzymes holds potential for creating key chemical tools. For instance, DNAzymes can be identified for site-selective protein modification to complement existing bioconjugation methods. A well-reasoned approach to developing DNAzymes for protein modifications is a stepwise one. Sets of in vitro selection experiments can first be performed with peptides tethered to the DNAzyme, followed by selection experiments with untethered peptides, and finally with proteins. Each set of in vitro selection experiments can provide insights into the necessary aspects of selection design for identifying DNAzymes, such as pH conditions and the choice of electrophile. Lysine acylation is an important protein modification and, for example, is commonly used to generate antibody-drug conjugates (ADCs). Previous work in the Silverman lab has used aryliii ester functionalized oligonucleotides to identify DNAzymes that catalyze lysine acylation of a peptide that is tethered to an anchor oligonucleotide base-paired to the DNAzyme. Building on these findings, this thesis describes the identification and subsequent characterization of DNAzymes that acylate a lysine residue in an untethered peptide that is not connected to an anchor oligonucleotide. Chapter 2 describes the development of an in vitro selection strategy to identify DNAzymes for this reaction, detailing strategic elements such as tuning the reactivity of aryl ester acyl donors and designing the untethered peptide. This chapter also covers the progression of the in vitro selection experiments, which led to the identification of 11 unique DNAzymes. Chapter 3 describes the extensive assays conducted to characterize these 11 DNAzymes, investigating critical properties of the DNAzymes such as their kinetics, binding, and peptide sequence dependence. The activities of most of the DNAzymes were found to be dependent on both the position and sequence context of the lysine residue within the peptide. Furthermore, one of the DNAzymes was used in a preparative-scale synthesis of a peptide-oligonucleotide conjugate, demonstrating its practical utility. Finally, Chapter 4 describes plans for upcoming in vitro selection experiments with model proteins to develop DNAzymes that site-selectively acylate protein lysine residues. Nine model proteins have been identified for potential use in upcoming selection experiments. Success in this work would lead to future in vitro selection experiments featuring more pharmaceutically relevant proteins, such as antibodies, potentially opening avenues for novel DNAzyme applications while significantly expanding the scope of DNAzyme catalysis.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Das, Prakriti K.

Contributors dc:contributor

• Silverman, Scott K.
• Hegenrother, Paul J.
• Mirica, Liviu
• Chan, Jefferson

• Dnazymes
• Biocatalysis
• Nucleic Acids
• Peptides

Statement dc:rights

• Copyright 2025 Prakriti Das

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129901

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129901