In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca2+ and Mg2+

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• 作者：Wenhu Zhou ; Yupei Zhang ; Jinsong Ding ; Juewen Liu
• 刊名：ACS Sensor
• 出版年：2016
• 出版时间：May 27, 2016
• 年：2016
• 卷：1
• 期：5
• 页码：600-606
• 全文大小：580K
• ISSN：2379-3694

文摘

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 N₁₂ position of the catalytic core. 17EV1 is ∼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.