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Title:DNA enzymes for tyrosine PEGylation and azido-adenylylation of peptide and protein substrates
Author(s):Wang, Puzhou
Director of Research:Silverman, Scott K.
Doctoral Committee Chair(s):Silverman, Scott K.
Doctoral Committee Member(s):Moore, Jeffrey S.; Sarlah, David; van der Donk, Wilfred A.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):DNA enzyme
deoxyribozyme
peptide
protein
PEGylation
azido-adenylylation
Abstract:Proteins and RNA are used as enzymes in nature, while DNA is used for the storage and transfer of genetic information. Proteins and RNA are biopolymers that can fold into complex secondary and tertiary structures to enable substrate binding and catalysis. Given the structural similarity to RNA, single-stranded DNA should also be able to function as enzymes. DNA enzymes, or deoxyribozymes, have not been found in nature, but in vitro selection has led to the identification of deoxyribozymes for a variety of reactions. De novo enzyme identification 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, within the sequence space evaluated, a large portion of nucleic acid sequences will fold into secondary and tertiary structures, whereas most random protein sequences are unlikely to fold into high-order structures. Considering nucleic acid enzymes, DNA has 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. Post-translational modifications (PTMs) are essential for protein functions. The ability to site-specifically modify peptides and proteins will enable better understanding and applications of these biomolecules. The idea of using DNA enzymes for peptide and protein modification is very attractive, especially considering that DNA enzymes with site selectivity can be de novo identified without the requirement of a known enzyme as the starting point. PEGylation is an important artificial PTM for therapeutic peptides and proteins. PEGylation improves the pharmacokinetic properties of biopharmaceuticals by increasing circulation half time, reducing immunogenicity, increasing solubility, and suppressing aggregation. Peptide and protein PEGylation is most commonly achieved by solely chemical means. However, these chemical strategies generally lack site selectivity among different target sites in the substrates. Some chemical strategies also suffer from off-target reactivity, i.e., poor chemoselectivity. Enzymatic approaches for PEGylation have also been developed, yet their application is limited by the substrate specificities and the sequence selectivities of the natural enzymes used. In Chapter 2, DNA enzymes were identified for PEGylation of tyrosine in a DNA-tethered peptide substrate using a 5′-phosphorimidazolide-activated oligonucleotide-PEG conjugate (Imp-oligo-PEG5k) as the PEG donor. Two different approaches are described for the identification of DNA enzymes that are functional with untethered peptide substrates. The first approach is to increase the length of the tether between the peptide substrate and the DNA anchor for mimicking a peptide free in solution. The selection experiment using this approach did not lead to deoxyribozymes, and further analysis of other deoxyribozymes with untethered peptide reactivities suggests that the long tethers may interfere with catalysis. Thus, the first approach was discontinued. The second approach is to alternate the position of the tether between the peptide substrate and the DNA anchor. The rationale is that DNA enzymes are expected to perform catalysis without the requirement of any tether if the enzymes are identified from selection experiments with alternating tether positions. Ongoing efforts include selection experiments using the second approach and mixed-sequence peptide substrates to identify deoxyribozymes with untethered peptide reactivity. In Chapter 3, a two-step strategy is described for DNA-catalyzed peptide modification. In this strategy, a DNA enzyme first catalyzes the transfer of the 2′-azido-2′-deoxyadenosine 5′ monophosphoryl group (2′-Az-dAMP) from the analogous 5′-triphosphate (2′-Az-dATP) onto the tyrosine hydroxyl group (azido-adenylylation). Second, a particular modification of interest is attached to the azido group by copper-catalyzed azide-alkyne cycloaddition (CuAAC) using an alkyne-functionalized reagent. Eleven deoxyribozymes with azido-adenylylation activity are described in Chapter 3. One of the DNA enzymes is selective for the YPR sequence motif and is able to discriminate between tyrosine residues within a single peptide on the basis of sequence context. Another deoxyribozyme is peptide sequence-general, functions with free peptides, and allows their subsequent CuAAC labeling with moieties such as PEG and fluorescein. The use of azido-adenylylation deoxyribozymes is a versatile method for the synthesis of site-specifically modified peptides and proteins, since the azide group installed by the DNA enzyme can be used for any particular modification as long as the corresponding alkyne derivative is available. One of our long-term goals is DNA-catalyzed site-specific modification of protein substrates. In Chapter 4, two proteins, human annexin V and human TNF-related apoptosis-inducing ligand (TRAIL) 114–281, and a 36-mer peptide pancreatic polypeptide (PP) with an additional C-terminal cysteine were used as the substrates to evaluate two different approaches for identifying deoxyribozymes. The first approach is to directly use protein substrates during in vitro selection experiments. This approach requires the surviving deoxyribozymes to simultaneously adopt functions of both binding to the protein substrates and catalyzing the modification. However, the selection experiments using this approach did not lead to deoxyribozymes. The second approach is to decouple the binding and catalytic functions required for DNA-catalyzed protein modification. In this modular approach, the binding function is assigned to the predefined aptamer domain, which is placed adjacent to the initially random enzyme domain. The sequence of the enzyme domain will be subsequently identified through in vitro selection in the presence of the aptamer domain. Ongoing efforts are focused on the identification of DNA aptamers that bind to annexin V, TRAIL, and PP, with benzyl, naphthyl, and indolyl modifications. Once the DNA aptamers are identified, they will be used as the binding modules in selection experiments to identify DNA enzymes for protein modification.
Issue Date:2018-07-03
Type:Text
URI:http://hdl.handle.net/2142/101524
Rights Information:Copyright 2018 Puzhou Wang
Date Available in IDEALS:2018-09-27
Date Deposited:2018-08


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