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Title:In Vitro Selection of Phosphoserine and Phosphothreonine Lyase DNA Enzymes
Author(s):Wylder, Adam C.
Contributor(s):Silverman, Scott K.
Abstract:In nature, the canonical role of DNA is the storing and transferring of genetic information, while proteins and RNA can act as catalysts, or enzymes. Despite this delegation by nature, artificial single-stranded DNA catalysts, or deoxyribozymes, can be identified by in vitro selection. The genotype is an intrinsic property of nucleic acid catalysts; any sequence exhibiting the desired phenotype can be amplified. This is a notable advantage of nucleic acid catalysts over protein catalysts, as protein catalysts, independent from their genotypic information, cannot be amplified. Compared to RNA catalysts, DNA catalysts are more stable, cheaper to synthesize, directly amplifiable by polymerases, and exhibit similar catalytic proficiency. Therefore, the identification of deoxyribozymes through in vitro selection is a tractable endeavor to solve modern chemical and biological problems. One such biological problem is a lack of chemoselective catalysts for the formation of post-translational modifications on proteins and peptides. This problem can be rectified by the de novo approach of in vitro selection, which allows the identification of novel enzymes to catalyze specific chemical reactions, including the modification of peptide and protein substrates. One post-translational modification of interest is the catalytic β-elimination of phosphate from phosphoserine (pSer) to form dehydroalanine (Dha), a non-proteogenic, electrophilic, α,β-dehydroamino acid. An enzyme that catalyzes this conversion of pSer to Dha is known as a pSer lyase. The actions of pSer lyases are of vital importance to several organisms, as Dha is an intermediate in the synthesis of many classes of cyclic, bioactive peptides, including lantibiotics, thiopeptides, and microcystins. Selections performed in the Silverman lab identified two pSer lyase deoxyribozymes, which expanded the known catalytic scope of DNA. The practical applications of one pSer lyase deoxyribozyme, DhaDz1, were demonstrated through its use in the chemoenzymatic synthesis of a cyclic cystathionine-containing peptide, a more stable analogue of the biologically active compstatin peptide. While DhaDz1 could act on several model peptide substrates, its activity was not general enough to function on many of the tested pSer-containing peptides, demonstrating the deoxyribozyme’s limited scope of potential substrates. In vitro selection experiments were thus designed to identify pSer lyase deoxyribozymes which could act on a more diverse set of substrates. This selection strategy alternates which chemically distinct peptide is presented to the oligonucleotide sequences, thereby applying pressure for the DNA enzymes to accommodate diverse substrates. Additionally, this alternating selection strategy is being incorporated in selections which aim to identify phosphothreonine (pThr) lyase deoxyribozymes, which convert pThr to dehydrobutyrine (Dhb) through a reaction analogous to that of pSer lyases. Enzymes identified through these selections are expected to have practical applications in both synthesis and phosphoproteomics studies.
Issue Date:2017
Genre:Dissertation / Thesis
Rights Information:Copyright 2017 Adam C. Wylder
Date Available in IDEALS:2017-05-15

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