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Light-activated binary nucleotide reagent for inactivation of DNA polymerase

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Date Issued:
2012
Abstract/Description:
This work explores a binary reagent approach to increase the specificity of covalent inhibitors. In this approach, two ligand analogs equipped with inert pre-reactive groups specifically bind a target biopolymer. The binding event brings the pre-reactive groups in proximity with each other. The two groups react, generating active chemical intermediates that covalently modify and inactivate the target. In the present study we compare the new approach with the traditional single-component reagent strategy using DNA polymerase from bacteriophage T4 as a model target biopolymer. We report the design and synthesis of two analogs of deoxythymidine triphosphate, a natural DNA polymerase substrate. Together, the analogs function as a binary nucleotide reagent which is activated by light with wavelengths 365 nm and longer. However, the active analog functions as a traditional single component reagent when activated by light with wavelengths at 300 nm and longer. The traditional single-component reagent efficiently inactivated DNA polymerase. However, in the presence of non-target protein the inactivation efficiency is greatly diminished. Under the same conditions, the binary nucleotide reagent also inactivated DNA polymerase, and the inactivation efficiency is not affected by the presence of the non-target protein. Our results validate that a binary approach can be employed to design highly specific covalent inhibitors. The binary reagent strategy might be useful as a research tool for investigation of ligand-protein interactions in complex biological systems and for drug design.
Title: Light-activated binary nucleotide reagent for inactivation of DNA polymerase.
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Name(s): Cornett, Evan, Author
, , Committee Chair
, Committee Member
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2012
Publisher: University of Central Florida
Language(s): English
Abstract/Description: This work explores a binary reagent approach to increase the specificity of covalent inhibitors. In this approach, two ligand analogs equipped with inert pre-reactive groups specifically bind a target biopolymer. The binding event brings the pre-reactive groups in proximity with each other. The two groups react, generating active chemical intermediates that covalently modify and inactivate the target. In the present study we compare the new approach with the traditional single-component reagent strategy using DNA polymerase from bacteriophage T4 as a model target biopolymer. We report the design and synthesis of two analogs of deoxythymidine triphosphate, a natural DNA polymerase substrate. Together, the analogs function as a binary nucleotide reagent which is activated by light with wavelengths 365 nm and longer. However, the active analog functions as a traditional single component reagent when activated by light with wavelengths at 300 nm and longer. The traditional single-component reagent efficiently inactivated DNA polymerase. However, in the presence of non-target protein the inactivation efficiency is greatly diminished. Under the same conditions, the binary nucleotide reagent also inactivated DNA polymerase, and the inactivation efficiency is not affected by the presence of the non-target protein. Our results validate that a binary approach can be employed to design highly specific covalent inhibitors. The binary reagent strategy might be useful as a research tool for investigation of ligand-protein interactions in complex biological systems and for drug design.
Identifier: CFE0004366 (IID), ucf:49429 (fedora)
Note(s): 2012-08-01
M.S.
Medicine, Molecular Biology and Microbiology
Masters
This record was generated from author submitted information.
Subject(s): drug design -- inhibitors -- covalent inhibitors -- biochemistry
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFE0004366
Restrictions on Access: campus 2013-08-15
Host Institution: UCF

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