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

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 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.

Theranostic DNAzymes

“…A key advantage of in vitro selection is that DNAzymes can be isolated under physiological conditions or even using biological fluids directly to ensure their activity for the intended applications 17. On the other hand, extreme conditions such as high temperature or low pH can be used for other tailored applications 18, 19.…”

“…We reported that the 8-17 is even faster than the 10-23 for cleaving chimeric substrates in the presence of Mg 2+ 25. The 8-17 motif has been repeated selected by several labs using Zn 2+ 26, Mg 2+ 27, 28, Ca 2+ 17, 28, Cd 2+ 29, and other conditions with the best activity observed with Pb 2+ 30. Its recurrence has been attributed to the small size, high tolerance to mutations, and high activity 27.…”

“…Therefore, their concentration monitoring is important in medicine. We recently studied the stability of a DNAzyme in undiluted serum and also performed an in vitro selection in serum 17, 101. We found that DNAzymes can work stably in serum with minimal degradation and a few mutants of the 8-17 DNAzyme showed substantial activity due to the presence of Ca 2+ and Mg 2+ .…”

Replacing Mg2+ by Fe2+ for RNA‐Cleaving DNAzymes

“…The 17E DNAzyme is an extensively studied model enzyme, and it is active in the presence of many divalent metal ions. For this reason, together with its small size and tolerance to mutations, 17E has recurred in many in vitro selections from different labs under different selection conditions . The highest activity is achieved with Pb 2+ , followed by Zn 2+ , Cd 2+ , Mn 2+ , and Co 2+ , whereas a high metal concentration is needed for Mg 2+ and Ca 2+ .…”

DNAzyme sensors for detection of metal ions in the environment and imaging them in living cells

“…In vitro selection starts with approximately 10 15 different DNA sequences and, based on the selection conditions, can produce DNAzymes which are highly specific for metal ion cofactors in a range of concentrations. Thus far, DNAzymes have been selected that are specific for Pb 2+ [6]-[8], Zn 2+ [9,10], Mg 2+ [8,11], Cu 2+ [12], Ca 2+ [13], UO2 2+ [14], Na + [15], Hg 2+ [16], Cd 2+ [17], Cr 3+ [18], Ln 3+ [19-21], Ce 3+ [22], and Ag + [23]. …”