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Title:DNA enzymes for peptide-nucleic acid conjugation and for lysine methylation
Author(s):Chu, Chih-Chi
Director of Research:Silverman, Scott K
Doctoral Committee Chair(s):Silverman, Scott K
Doctoral Committee Member(s):Hergenrother, Paul J; Mitchell, Douglas A; Zimmerman, Steven C
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Nucleic acids
DNAzymes
Deoxyribozymes
Deoxyribonucleic acid (DNA) enzymes
Site-specific
Protein modification
Peptide modification
Peptide-nucleic acid conjugation
Lysine methylation
Recruiting
Histidine tag
TrisNTA
Modified nucleotides
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
Click chemistry
Abstract:Proteins and RNA are known to be enzymes in nature. These biopolymers have complex secondary and tertiary structures that can enable substrate binding and catalysis. DNA is primarily double-stranded and is not known to be catalytic in nature. However, given the similarities in chemical structure between DNA and RNA, it is reasonable to think that singlestranded DNA can also form complex structures. In fact, artificial DNA enzymes have been identified in laboratories by in vitro selection. The identification of new enzymes favors the use of nucleic acids over proteins for several reasons. First, nucleic acids can be amplified by natural enzymes whereas proteins cannot be amplified in any way. Second, the number of possible sequences is smaller for nucleic acids (4n, where n is the length of the biopolymer) than for proteins (20n). Therefore, selection experiments for identifying nucleic acid enzymes will cover a larger fraction of total sequence space. Furthermore, of the sequence space that is covered, a large portion of nucleic acid sequences can fold into secondary or tertiary structures, whereas most random sequences of proteins often will not fold and thus will aggregate. Between the two nucleic acid polymers, DNA offers additional advantages over RNA because DNA can be directly amplified by polymerases whereas RNA requires an extra reverse transcription step. DNA is also cheaper and more stable compared to RNA. An approach to site-specifically modify peptide and protein side chains will be valuable to study natural post translation modification of proteins. That approach will also be useful for artificial modification of proteins to generate homogeneous protein conjugates. The idea of using DNA enzymes for site-specific protein modification is very attractive. With a suitable in vitro selection strategy, peptide-sequence-selective DNA enzymes can be identified to modify peptide and protein side chains. The formation of covalent linkages between protein side chains and nucleic acids plays an essential role in many biological processes. Deoxyribozymes have been previously identified to join tyrosine-containing peptides to RNA by reaction of the tyrosine (Tyr) hydroxyl group with the a-phosphate of a 5′-triphosphorylated RNA oligonucleotide (5′-pppRNA). The selection approach for identifying those DNA enzymes required the peptide substrate to be either embedded in or tethered to a DNA oligonucleotide. Consequently, the resulting deoxyribozymes either cannot function with untethered peptide substrates, or require high peptide concentration for catalysis. In Chapter 2, a new in vitro selection approach for DNA-catalyzed peptide nucleic acid conjugation is described. This new selection approach uses untethered, free peptide substrates containing an azido group at the N-terminus. Capture of the catalytically active sequences by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with a 3'-alkyne-modified DNA oligonucleotide provides a substantial mass difference to allow PAGE separation of the catalytically active DNA. Selection experiments led to several deoxyribozymes that catalyze peptide-RNA conjugation of untethered peptides. Separate selection experiments were performed using a 5′-phosphorimidazolated DNA oligonucleotide (5′-ImpDNA) as the conjugation partner. Imp is a more generalizable electrophile relative to ppp because Imp can be easily placed at either terminus (5′ or 3′) of both RNA and DNA. The selection experiments resulted in numerous DNA enzymes that catalyze peptide DNA conjugation. In addition, imposition of selection pressure via reduced peptide concentration led to a deoxyribozyme with a preparatively useful peptide Km value. A key challenge for DNA-catalyzed peptide and protein modification is to achieve catalysis using low peptide and protein concentrations that enable practical utility. For deoxyribozymes that require high peptide concentration for catalysis, recruiting strategies were sought to direct DNA enzymes to their substrates. Chapter 3 describes the development of recruiting strategies for DNA-catalyzed reactions. The use of a histidine tag successfully recruited a peptide–RNA-conjugating deoxyribozyme to its peptide substrate and enhanced the conjugation yield and rate at low peptide concentrations. The histidine recruiting strategy also allowed a Tyr kinase deoxyribozyme to achieve, for the first time, DNA catalyzed Tyr phosphorylation of a discrete, untethered peptide substrate. However, the recruiting effect was not observed for protein substrates, likely because the tested deoxyribozymes either cannot access the target peptide segments or cannot function when these segments are presented in a structured protein context. Chapter 4 describes the efforts toward DNA-catalyzed lysine (Lys) methylation. Lys methylation is an important protein post-translational modification. Developing DNA enzymes to site-specifically methylate Lys side chains on protein surfaces is valuable for biochemical studies. SAM analogs with terminal alkyne containing chains were used in selection experiments as the alkyl donors. The terminal alkyne enables CuAAC reaction with a 5′-azido modified DNA to capture the catalytically active DNA sequences. An initial set of selection experiments did not lead to deoxyribozymes. Further efforts have focused on incorporating modified nucleotides with protein-like functional groups to facilitate catalysis.
Issue Date:2017-07-26
Type:Text
URI:http://hdl.handle.net/2142/99165
Rights Information:Copyright 2017 Chih-Chi Chu
Date Available in IDEALS:2018-03-13
Date Deposited:2017-12


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