Issue 86, 2017, Issue in Progress

Blue-white screening as a new readout for deoxyribozyme activity in bacterial cells

Abstract

The 10–23 deoxyribozyme is considered as sequence-specific “molecular scissors” for RNA molecules. Extensive investigations have been reported for this deoxyribozyme in vitro and in eukaryotic host cells. However, few investigations are reported in the literature on the activity of this deoxyribozyme inside bacterial cells. The available reports focus on the cleavage of target mRNAs that encode for proteins which are responsible for the viability of bacterial colonies. Hence, the growth of bacterial cells was blocked and the main readouts in such studies were colony counts or optical density at 600 nm. In the current study, blue-white screening was utilized as a novel readout for analysis of the activity of the 10–23 deoxyribozymes in viable bacterial cells. Two deoxyribozymes were designed to target the α-peptide fragment from β-galactosidase (lacZ) mRNA at two different positions, i.e. 5′ untranslated region and translated region. Control experiments were performed utilizing DNA oligos that lacked the catalytic core. The 3′–3 inverted thymidine modified deoxyribozymes were compared with unmodified ones to analyze the effect of such modification in prokaryotic cells. The activity of the designed deoxyribozymes caused a significant retardation in the formation of the blue-color in colonies with deoxyribozymes. Miller assay confirmed the blue-white screening results. This report showed a proof of concept for application of blue-white screening as a readout system for the activity of the 10–23 deoxyribozyme that is a model RNA-cleaving deoxyribozyme. The result of this report can promote future investigations on the activity of deoxyribozymes in prokaryotic cells.

Graphical abstract: Blue-white screening as a new readout for deoxyribozyme activity in bacterial cells

Supplementary files

Article information

Article type
Paper
Submitted
31 Aug 2017
Accepted
23 Nov 2017
First published
01 Dec 2017
This article is Open Access
Creative Commons BY license

RSC Adv., 2017,7, 54835-54843

Blue-white screening as a new readout for deoxyribozyme activity in bacterial cells

S. Sadeghi, N. Ahmadi, A. Esmaeili and F. Javadi-Zarnaghi, RSC Adv., 2017, 7, 54835 DOI: 10.1039/C7RA09679H

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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