AbstractsBiology & Animal Science

The impact of after-school childcare arrangements on the developmental outcomes of low-income children and the labor conditions of their working mothers

by Benjamin M Brandsen

Institution: University of Illinois – Urbana-Champaign
Year: 2016
Keywords: deoxyribozyme; ribozyme; DNA enzyme; DNA catalyst; protease
Posted: 02/05/2017
Record ID: 2133735
Full text PDF: http://hdl.handle.net/2142/90458


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,… Advisors/Committee Members: Silverman, Scott K (advisor), Silverman, Scott K (Committee Chair), Mitchell, Douglas A (committee member), Moore, Jeffrey S (committee member), Morrissey, James H (committee member).