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Title:In Vitro Selection of Kinase Deoxyribozymes
Author(s):Lammer, Nickolaus C.
Contributor(s):Silverman, Scott K.
Subject(s):deoxyribozymes
DNA enzymes
kinases
phosphorylation
in vitro selection
Abstract:Kinases, enzymes that catalyze phosphorylation, are found throughout biology as major components in cellular regulation and maintenance and also in the laboratory as tools. While natural protein enzymes can be used to achieve amino acid side chain or DNA phosphorylation, there is still room for improvement. Deoxyribozymes may fill this gap as versatile, biopolymer catalysts. These kinase deoxyribozymes could be more easily and generally applicable in the case of protein phosphorylation, or fill a hole in the protein enzyme toolkit in the case of DNA 3′-phosphorylation. Here, I describe the use of in vitro selection to identify serine kinase deoxyribozymes as well as an ongoing effort to select for improved DNA 3′-phosphorylating deoxyribozymes. In vitro selection subjects a pool of random DNA sequences to desired reaction conditions and the catalytic sequences are separated by a capture step. The remaining pool is then amplified by PCR and the process is iterated until the desired catalytic activity dominates the pool. During the capture step of the serine kinase deoxyribozyme selections, the 8VP1 deoxyribozyme was used, which attaches a 5′-triphosphorylated RNA oligonucleotide to the phosphorylated serine residue present in a peptide substrate ligated to the DNA pool. After 15 rounds of in vitro selection, no deoxyribozymes capable of serine phosphorylation were identified. A side reaction during the 8VP1 reaction step was observed for one of the selections that caused sequences with undesired catalytic activity to be carried forward. To avoid this issue in future studies, work is being done to employ Phos-tag-based PAGE, which uses Phos-tag to separate phosphorylated peptides rather than 8VP1. Preliminary experiments using Phos-tag acrylamide gels showed promising separation with tyrosine-containing peptides and synthesis of Phos-tag is underway. Modified nucleotides that contain a uracil attached to a functional group, such as a benzyl or amino group, will also be used. The modification may give the DNA more functionality and improve the chances of identifying serine kinase deoxyribozymes. These nucleotides contain a uracil with a functional group attached, such as a benzyl or amino group. The selections for DNA 3′-phosphorylating deoxyribozymes that are faster or more sequence-general than the previously identified 3′Kin1 deoxyribozyme are ongoing.
Issue Date:2017
Genre:Dissertation / Thesis
Type:Text
URI:http://hdl.handle.net/2142/96047
Rights Information:Copyright 2017 Nickolaus C. Lammer
Date Available in IDEALS:2017-05-15


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