Abstract
Nature is known to exploit both proteins and RNA as enzymes. No natural enzymes, however, have been discovered that are made of DNA. One can think of both RNA and proteins as large biopolymers with the potential to form complex secondary and tertiary structures capable of doing catalysis. Given the structural similarities between RNA and DNA, it is reasonable to think that DNA can also form these structures. If oligonucleotides can act as comparable catalysts to proteins, a number of practical reasons to favor their development exist. The number of possible sequences is much smaller for DNA and RNA than for proteins; a sequence n units long has 4n possible sequences of nucleotides and 20n possible sequences of amino acids. In addition, oligonucleotide synthesis lacks many of the practical challenges associated with protein expression and purification. DNA offers further advantages over RNA: DNA is cheaper, more stable, and easier to synthesize. If DNA can act as an efficient catalyst, the area of deoxyribozyme research is relatively unexplored, making the chances of discovering new deoxyribozymes likely. If deoxyribozymes can be identified that perform useful chemical reactions for which a catalyst has not yet been discovered, the impacts could be extensive. Protease enzymes, which catalyze cleavage of proteins, are essential enzymes for biotechnology. Engineering of natural proteases to change their site of cleavage is an exciting prospect, but this process usually leads to relaxed substrate selectivity, rather than a true change in enzyme specificity. Because DNA catalysts 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 proteases is reasonable. Chapter 2 describes our efforts to identify DNA catalysts which cleave peptide bonds. Previous efforts seeking DNA-catalyzed peptide cleavage resulted in DNA catalysts led to the identification of deoxyribozymes that cleave a DNA phosphodiester bond. In order to avoid identifying deoxyribozymes for phosphodiester cleavage in future efforts for peptide bond cleavage, an additional capture reaction was employed, and new in vitro selection experiments identified DNA enzymes for ester cleavage and aromatic amide cleavage. Unfortunately no enzymes for peptide cleavage were identified. Several additional efforts to identify DNA catalysts for peptide bond cleavage are described in chapter 3. Selection experiments were performed with stronger nucleophiles including amine, hydrazide, and thiol included in the in vitro selection step, with the hope that deoxyribozymes might be identified which can use one of these nucleophiles to enable the cleavage reaction. Selection experiments for DNA-catalyzed cleavage of N-acetylglycine, N-fluoroacetylglycine, and N-difluoroacetylglycine were performed, with the hope that new deoxyribozymes for cleavage of these activated substrates could facilitate our understanding of DNA-catalyzed amide cleavage. Finally, selection experiments for DNA catalysts that catalyze peptide self-cleavage using nucleophilic amine acids were performed, with the hope that deoxyribozymes which catalyze cleavage use an internal, tethered peptide nucleophile are more easily identified than those that catalyze hydrolysis. Unfortunately, each of these three selection strategies were unsuccessful. Additional selection experiments seeking amide cleavage using extremely long random regions were performed, based on the premise that much longer random regions are able to form the required complexes that shorter random region lengths cannot. Interestingly, deoxyribozymes identified from these selections do not catalyze amide cleavage, but instead they catalyze DNA cleavage by an oxidative mechanism similar to the natural product bleomycin. Previous efforts by our lab sought deoxyribozymes which catalyze the reaction between lysine and triphosphorylated RNA. Despite the strong nucleophilicity of primary amine functional groups, no DNA catalysts have been identified that can utilize it as a substrate. In one example, deoxyribozymes were identified which catalyze the reaction between phosphoramidate linkage and triphosphorylated RNA, even when a much stronger lysine nucleophile was available. In efforts described in chapter 4, a more reactive phosphorimidazolide substrate was employed to react with both DNA-C3-NH2 and DNA-HEG-CKA substrates. Deoxyribozymes that catalyze reaction between the amino group and phosphorimidazolide substrate were identified. In addition, a single deoxyribozyme was identified which utilizes the C4-NH2 of a cytosine nucleobase as a nucleophile, a finding which highlights the ongoing difficulty of identifying DNA catalysts for reactions involving amine-containing substrates. Chapter 5 describes in vitro selection efforts seeking lysine acylation, a common post-translational modification important in gene expression, control of protein function, and primary metabolism. The in vitro selection strategy employed relied on glutaric acid thioester acyl donors, which contain a carboxylic acid that could be used in a capture reaction to separate active sequences from inactive sequences. The substrates used during in vitro selections were DNA-C3-NH2 and DNA-HEG-AAAKAA. Additionally, N40 and N80 random region lengths and both oligonucleotide-anchored and free small molecule thioesters were employed. Thirteen rounds of in vitro selection were performed, but no DNA enzymes were identified. Future efforts seeking DNA-catalyzed acylation will use chemically-modified nucleotides to expand the functionality of DNA and facilitate catalysis. Efforts to identify deoxyribozymes which catalyze glycosylation are described in chapter 6. Protein glycosylation is a post-translational modification critically important in molecular and cellular recognition. Natural glycosylating protein enzymes, glycosyltransferases, use nucleoside diphosphate sugars (NDP sugars) as glycosyl donors in their catalyzed reactions. Attempts to identify glycosylating deoxyribozymes were hindered by the instability of NDP-sugars in the presence of certain divalent metal ions. Alternative glycosyl donors, O-aryl glycosides, were used to identify deoxyribozymes for 3⁰́ø-OH glycosylation of DNA. Attempts to identify DNA catalysts for protein glycosylation resulted in the DNA enzymes that catalyze self-glycosylation near the 5⁰́ø-terminus of the catalytic region. A new selection strategy that prevents the selection of self-glycosylating deoxyribozymes was designed. That strategy will be implemented in future selection experiments.