In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca2+ and Mg2+
Loading...
Date
2016-05-27
Authors
Zhou, Wenhu
Zhang, Yupei
Ding, Jinsong
Liu, Juewen
Journal Title
Journal ISSN
Volume Title
Publisher
American Chemical Society
Abstract
RNA-cleaving DNAzymes have been attempted as in vivo analytical probes and gene silencing reagents over the past two decades. Despite progress already achieved, concerns still exist regarding the activity of DNAzymes in biological fluids. An example is the low activity of the 10-23 DNAzyme in intracellular Mg2+ concentrations. To obtain DNAzymes that work optimally in biological samples, we herein report the first DNAzyme in vitro selection in undiluted human blood serum. The selection starts with a large DNA library containing 50 random nucleotides, and sequences that can be cleaved in serum were isolated and amplified. After deep sequencing analysis, 80% of the final library are a variant of the 8-17 DNAzyme (named 17EV1). The main difference between 17E and 17EV1 is a single mutation at the N-12 position of the catalytic core. 17EV1 is similar to 6-fold faster in serum than 17E, since 17EV1 is preferentially activated by Ca2+ and serum is rich in Ca2+ over Mg2+. On the other hand, 17E has a similar activity with Ca2+ or Mg2+. With this observation, a method for measuring the Ca2+/Mg2+ ratio was developed by combining the 17E and 17EV1 DNAzymes. This study demonstrates the feasibility of selecting DNAzymes in biological fluids and will facilitate the application of DNAzymes in bioanalytical chemistry and gene therapy.
Description
This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Sensors, © 2016 American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Zhou, W., Zhang, Y., Ding, J., & Liu, J. (2016). In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca2+ and Mg2+. Acs Sensors, 1(5), 600–606. https://doi.org/10.1021/acssensors.5b00306
Keywords
8-17 Deoxyribozyme, Living Cells, DNA Enzyme, Metal-Ions, Lead, Biosensor, Cleavage, Probes, Gene, Nanoparticles