Defining the substrate scope of DNAzyme catalysis for reductive amination with aliphatic amines

Yang, Shukun

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https://hdl.handle.net/2142/120360 Copy

Description

Title

Defining the substrate scope of DNAzyme catalysis for reductive amination with aliphatic amines

Author(s)

Yang, Shukun

Issue Date

2023-04-13

Director of Research (if dissertation) or Advisor (if thesis)

Silverman, Scott K.

Doctoral Committee Chair(s)

Silverman, Scott K.

Committee Member(s)

• Fratti, Rutilio A.
• Lu, Yi
• Procko, Erik

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Artificial Enzymes
• DNAzymes
• In Vitro Selection
• Reductive Amination
• Substrate Scope
• Lysine Alkylation
• Kinetic studies

Abstract

Proteins and RNA are naturally occurring enzymes characterized by their complex secondary and tertiary structures, which facilitate substrate binding and catalysis. Although DNA is typically double-stranded and non-catalytic, its chemical structure closely resembles RNA, suggesting that single-stranded DNA may also form intricate structures. Indeed, various artificial DNAzymes have been discovered in laboratories through in vitro selection. The identification of new artificial enzymes tends to favor nucleic acids over proteins for several reasons. The most important reason is that nucleic acids can be directly amplified by polymerase chain reaction (PCR), while proteins lack a comparable amplification method. In addition, the potential sequence variety is smaller for nucleic acids (4^n, where n represents the biopolymer's length) than for proteins (20^n), allowing selection experiments for nucleic acid enzymes to explore a larger fraction of the total sequence space. Moreover, a significant portion of nucleic acid sequences can easily form secondary or tertiary structures, while many random protein sequences fail to fold properly and subsequently aggregate. Among nucleic acid polymers, DNA presents additional practical advantages over RNA. DNA can be directly amplified by polymerases, whereas RNA necessitates an extra reverse transcription step. Furthermore, DNA is easier to synthesize, more stable and cost-effective than RNA. Amines can be alkylated using various reactions, such as reductive amination of aldehydes. In this study, we sought DNAzymes as catalytic DNA sequences that promote reductive amination with aliphatic amines, including DNA-anchored peptide substrates that have lysine residues. By in vitro selection starting with either N40 or N20 random DNA pools, we identified many DNAzymes that catalyze reductive amination between the DNA oligonucleotide-anchored aliphatic amino group of DNA-C3-NH2 (C3 = short three-carbon tether) and a DNA-anchored benzaldehyde group in the presence of NaCNBH3 as reducing agent. At pH 5.2, 6.0, 7.5, or 9.0 in the presence of various divalent metal ion cofactors including Mg2+, Mn2+, Zn2+ and Ni2+, the DNAzymes have kobs up to 0.12 h^(−1) and up to 130-fold rate enhancement relative to the DNA-splinted but uncatalyzed background reaction. However, analogous selection experiments did not lead to any DNAzymes that function with DNA-HEG-NH2 [HEG = long hexa(ethylene glycol) tether], or with short- and long-tethered DNA-AAAKAA and DNA-HEG-AAAKAA lysine-containing hexapeptide substrates (A = alanine, K = lysine). Including a variety of other amino acids in place of the neighboring alanines also did not lead to DNAzymes. These findings establish a practical limit on the substrate scope of DNAzyme catalysis for N-alkylation of aliphatic amines by reductive amination. The lack of DNAzymes for reductive amination with any substrate more structurally complex than DNA-C3-NH2 is likely related to the challenge in binding and spatially organizing those other substrates. Because other reactions such as aliphatic amine N-acylation are feasible for DNAzymes with DNA-anchored peptides, our findings show that the ability to identify DNAzymes depends strongly on both the investigated reaction and the composition of the substrate.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Shukun Yang

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