DNAzymes for amine and peptide lysine acylation

Yao, Tianjiong

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https://hdl.handle.net/2142/113964 Copy

Description

Title

DNAzymes for amine and peptide lysine acylation

Author(s)

Yao, Tianjiong

Issue Date

2021-11-23

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

Silverman, Scott

Doctoral Committee Chair(s)

Silverman, Scott

Committee Member(s)

• Lu, Yi
• Procko, Erik
• van der Donk, Wilfred

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• DNAzyme
• in vitro selection

Abstract

The typical enzymatic biopolymers evolved by nature are proteins and RNA. These biopolymers can fold into complex secondary and tertiary structures capable of performing catalysis. In contrast, no natural DNAzymes (deoxyribozymes) have been identified. Given the structural similarities between DNA and RNA, theoretically DNA is also capable of forming complex structures thus functioning as enzymes. Engineered DNAzymes are identified and developed in laboratories by in vitro selection, and there are several reasons to develop artificial DNAzymes to act as comparable enzymes to proteins and RNA. First, the fundamental base for selection is that candidate pool needs to be amplified after each selection round, otherwise selection cannot be done. Oligonucleotides can be directly amplified by natural polymerases, whereas proteins cannot be amplified directly by any means. Second, oligonucleotide synthesis does not involve many practical challenges associated with protein expression and purification. Third, random oligonucleotide sequences can readily fold into secondary, then tertiary structures, whereas most random peptide sequences will misfold and aggregate. Last but not least, the total number of possible sequences is much smaller for oligonucleotides (4n, where n is the length of the biopolymer) than for proteins (20n). Therefore, by combining last two reasons, selections for oligonucleotide enzymes can cover a larger meaningful fraction of total sequence space than for proteins. Furthermore, between the two types of oligonucleotides, DNA has several practical advantages over RNA: DNA is cheaper, more stable, and easier to synthesize. The field of DNAzymes research is relatively unexplored, thus it is more likely to discover novel DNAzymes that perform chemical reactions for which protein enzymes are limited. My research focus is on lysine acylation, a common post-translational modification (PTM) important in gene expression and regulation, control of protein function, and primary metabolism. Besides the most well-studied acylation type, acetylation, many other acylation types such as malonylation, succinylation, and glutarylation have also been discovered yet are poorly understood. Engineering of natural acetyltransferases to alter their parent substrates to other longer chain acyl donors is an exciting prospect, but this process usually leads to relaxed substrate selectivity, rather than a true alteration in substrate specificity. Because DNAzymes are identified from pools of random DNA sequences, no inherent peptide sequence biases must be overcome during the selection process, and thus the prospect of truly selective artificial acyltransferases is reasonable. Previous efforts by our lab identified DNAzymes that can catalyze lysine modification by using 5’-phosphorimidazolide (5’-Imp), resulting in the formation of a Lys-phosphoramidite bond. Then, the efforts to investigate DNAzymes (with canonical nucleotides) that catalyze the lysine acylation by using thioesters as electrophile were performed, however no DNAzymes were identified. Directly following the last effort seeking DNAzymes with canonical nucleotides, Chapter 2 describes in vitro selection efforts seeking lysine acylation that employed chemically modified nucleotides to expand the functionality of DNA and facilitate catalysis. The in vitro selection in Chapter 2 also used thioesters as the acyl donor electrophile; meanwhile two amino substrates 5’-DNA-C3-NH2 and 5’-DNA-HEG-AAAKAA were used as the nucleophiles. Ten rounds of in vitro selection were performed, but no DNAzymes were identified. We concluded that a thioester is insufficiently reactive as an electrophile to allow the identification of amine-acylating DNAzymes, and a more reactive acyl donor is required. Therefore, aryl esters were considered as more appropriate acyl donor electrophiles than thioesters. Chapter 3 describes the in vitro selection efforts using highly reactive aryl ester (DMT ester and TFP ester) as acyl donor electrophiles. Acyl donor oligonucleotides were activated in situ from their 5’-carboxylic acid (5’-CO2H) precursors using two common amide-forming coupling reagents, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) or the combination of the water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 2,3,5,6-tetrafluorophenol (TFP), resulting in the formation of DMT ester and TFP ester in the selection process. The purpose of selection in this stage is to identify DNAzymes that catalyze the amine acylation using DMT or TFP ester as electrophiles and two amino substrates as nucleophiles. The random DNA pool sequences were composed of canonical nucleotides, and same two amino substrates 5’-DNA-C3-NH2 and 5’-DNA-HEG-AAAKAA in Chapter 2 were again used as the nucleophiles. After four rounds of selection, several DNA sequences were identified by cloning and sequencing. However, the emergent DNA sequences had no rate enhancement above the uncatalyzed, splinted background reaction under the same incubation conditions. We concluded that each individual DNA sequence likely adopts a combination of secondary and tertiary structure that merely recapitulates a complementary splint. Apparently, rate enhancement beyond the splinting effect cannot be achieved because the DMT or TFP ester electrophile is too reactive. The lessons learned by the two previous efforts are that neither too low nor too high reactive electrophiles can be considered as appropriate acyl donors; then, the attention was turned to acyl donors with intermediate reactivity. Chapter 4 describes the in vitro selection efforts using intermediate reactive aryl ester acyl donors which are phenyl ester and 4-fluorophenyl ester. The random DNA pool sequences were still composed of canonical nucleotides, and two amino substrates 5’-DNA-C3-NH2 and 5’-DNA-HEG-AAAKAA were used as the nucleophile as well. After seven/eight or eleven rounds respectively for the two amino substrates, several DNA sequences emerged from each selection and were identified by cloning and sequencing. These DNA sequences were assayed and have much higher rate enhancement above the uncatalyzed, splinted background reaction under the same incubation conditions, the highest rate enhancement is about 103 fold. So, DNAzymes for amine or peptide lysine acylation have been identified by using intermediate aryl ester acyl donor electrophiles. These identified DNAzymes were also assayed for activity by using free peptide as the nucleophile; unfortunately, no free peptide activity was observed. Future efforts will seek DNAzymes that can catalyze lysine acylation of modified free peptide such as azido-peptide by adopting appropriate electrophiles. While DNAzymes that transfer the big acyl oligonucleotide onto the amino group or peptide lysine have been identified, efforts towards investigating DNAzymes that can transfer a small acyl group onto amino group have also been performed. Chapter 5 describes the efforts seeking DNAzymes that transfer a glutaryl group to the amine group or peptide lysine. Nine rounds of in vitro selection were performed, but no DNAzymes were identified. Interestingly, when assaying DNAzymes discovered from the lysine acylation selections, it turned out that three DNAzymes, among these previously identified lysine acylation DNAzymes, also have the ability to transfer the glutaryl group to the amine group. This result could develop strategy that, when aiming for DNAzymes that catalyze a difficult substrate, we can first select with an easier counterpart molecule with same reaction mechanism as the target substrate. The resulting DNAzymes could also have the ability to catalyze the target substrate, then we can improve the identified DNAzymes using several methods such as reselection.

Graduation Semester

2021-12

Type of Resource

Thesis

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