In vitro selection of deoxyribozymes for O-glycoside cleavage and for 3-nitrotyrosine modification

Yeh, Chih-Cheng

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https://hdl.handle.net/2142/105629 Copy

Description

Title

In vitro selection of deoxyribozymes for O-glycoside cleavage and for 3-nitrotyrosine modification

Author(s)

Yeh, Chih-Cheng

Issue Date

2019-07-01

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

Silverman, Scott K

Doctoral Committee Chair(s)

Silverman, Scott K

Committee Member(s)

• van der Donk, Wilfred A
• Hergenrother, Paul J
• Zimmerman, Steven C

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Deoxyribozymes
• Glycosidase
• Modified Nucleotides
• CuAAC
• Nitrotyrosine

Abstract

Proteins and RNA are biopolymers that form complex secondary and tertiary structures and perform catalysis in nature. Single-stranded DNA is structurally similar to RNA and is also capable of catalytic function. DNA enzymes, or deoxyribozymes, are not known to exist in nature, but they have been identified in lab by in vitro selection for a variety of chemical reactions. De novo identification of nucleic acid enzymes is possible because RNA and DNA can be amplified using the polymerase chain reaction. In contrast, there is no method to amplify proteins. Nucleic acids are uniquely suited for in vitro selection, because random sequences form secondary structures and do not aggregate, and because a relatively large fraction of sequence space can be explored when there are only four nucleic acid monomers. In vitro selection is a potent method to identify enzymes from scratch for reactions that do not have existing enzymes. For this purpose, DNA has additional advantages over RNA. DNA can be directly amplified without an extra reverse transcription step, and DNA is more stable and less expensive than RNA. The major disadvantage of nucleic acid enzymes compared to protein enzymes is the lack of side-chain functionality. Previous efforts in the Silverman lab attempted to mitigate this by installing protein-like functional groups on DNA using various modified dUTPs, which required a complicated synthetic preparation for each functional group. Chapter 2 describes the method of copper-catalyzed azide-alkyne cycloaddition (CuAAC) to install protein-like functional groups on alkyne-modified DNA. This modular method streamlines the preparative work and makes it easier to use a wide variety of functional groups for in vitro selection. Carbohydrates are a major class of biomolecules. The nonlinearity of carbohydrates leads to a much larger number of possible structures compared to proteins and nucleic acids of similar size. In addition, the non-templated biosynthesis of carbohydrates causes difficulty for both synthetic preparation and structural analysis. Glycosidases and glycosyltransferases are important components of the glycobiology toolbox that facilitate manipulation of complex carbohydrate structures. Chapter 3 describes the in vitro selection of deoxyribozymes for O-glycoside cleavage. N-glycosidase deoxyribozymes were reported nearly two decades ago, but O-glycosidase deoxyribozymes are not known, and ultimately were not identified successfully in the research described in this thesis. Chapter 3 also describes how the challenge to identify O-glycosidase deoxyribozymes was approached from multiple angles, such as by introducing noncanonical functional groups and by tuning sugar substrate reactivity. Among other functional groups, we provided the DNA sequences with boronic acid for diol binding, and carboxylate acid to enable mimicking of the dicarboxylate catalytic pathway of protein glycosidases. We also used reactive aryl glycoside substrates in addition to a disaccharide substrate. The in vitro selection experiments were unsuccessful, and no deoxyribozymes were identified. The balance of substrate reactivity is delicate for many in vitro selection experiments. A high background reaction rate leaves little room for rate enhancement, while a low background reaction rate may indicate a difficult reaction for which the fraction of catalytic sequences in a random population is too low to be identified by in vitro selection. We explored substrate reactivity in the in vitro selection of deoxyribozymes for 3-nitrotyrosine azido-adenylylation described in Chapter 4. Previous research had identified deoxyribozymes for tyrosine azido-adenylylation. The hydroxyl group of 3-nitrotyrosine is many orders of magnitude less reactive than that of tyrosine. In addition to potential biochemical significance of 3-nitrotyrosine modification and detection in the presence of tyrosine, we were interested to see if catalytic DNA sequences were able to overcome the difficulty of a less reactive substrate and emerge during in vitro selection. The in vitro selection experiments were unsuccessful, and no deoxyribozymes were identified.

Graduation Semester

2019-08

Type of Resource

text

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Copyright and License Information

Copyright 2019 Chih-Cheng Yeh

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