Ultrasensitive DNAzyme‐Based Ca²⁺ Detection Boosted by Ethanol and a Solvent‐Compatible Scaffold for Aptazyme Design

Abstract

Functional DNA includes aptamers and DNAzymes, and metal ions are often importantf or achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA-based biosensors. In this work, the effect of ethanol on the activity of the EtNa DNAzyme was studied for Ca 2 + detection. With 30 %e thanol, the sensor has ad etection limit of 1.4 mm Ca 2 + ,w hich is a1 6-fold improvement relative to that in water.T his EtNa DNAzyme is unique because other tested DNAzymes are all inhibited by 50 %e thanol. Finally,b yu sing the EtNa DNAzyme as as caffold, the adenosinem onophosphate (AMP) aptamer was inserted to construct an aptazyme, which allowed the measurement of AMP in ethanol. In summary,t his study has reported the most sensitiveD NA-based sensorf or Ca 2 + ,a nd its sensitivity and selectivity can approach those of proteins or small-molecule ligands. Thisw ork also provides aw ay to measurea ptamer binding in organic solvents.In the past three decades, the functionofDNA has significantly expanded. In particular,D NA has emerged as ap owerful platform for biosensor development. [1][2][3][4][5] In addition to complementaryn ucleic acids, DNA can also detect variousm etal ions, small molecules, proteins, and even cells by using DNA aptamers and DNAzymes. The majority of detections, to date, have been performed in aqueous buffers, and an interesting question is whether it is possible to use DNA sensors in organic solvents. We reason such studies have at least the following implications and applications.F irst, it might broaden the range of analytes that can be detected, and enable such sensors in more research areas. In addition, for certain analytes,t he sensitivity might be even higher in solvents than that in water due to lower dielectric constants of organic solvents, or differences in other physicalproperties. Finally,itm ight allow new physical insights into DNA.DNA can maintain its base pairing in many water-miscible solvents up to ac ertain solvent fraction. [6,7] The most extensively studied solvent for DNA is ethanol. TheD NA duplex is first destabilizedw ith increasing ethanolc oncentration (e.g., lowered DNA melting temperature, T m ). [8] At ac riticale thanol concentration, which is af unctiono ft he type and concentration of cation in the system,D NA starts to aggregate. With 70-80 %e thanol, aB -to-A form transition occurs that is attributable to extensive DNA dehydration.M ost previouss tudies focusedo nt he stabilitya nd hybridization kinetics of DNA in organic solvents. [9][10][11][12][13] Since 1994, research on DNA catalysis has been explored. [14] DNAzymes are DNA-based catalysts, and they often require metal ions as cofactors for activity. [15][16][17][18] All known DNAzymes were obtained by ac ombinatorial biology technique called in vitro selection and D...

An RNA‐Cleaving Catalytic DNA Accelerated by Freezing

“…We chose a DNAzyme that required a very high metal concentration for catalysis so that we could better observe the effect of freezing. The EtNa DNAzyme is highly specific for Na + among the monovalent metals, but it has almost no activity in water when the Na + concentration is below 100 m m ; cleavage occurs efficiently with 1 m Na + . Its activity can be significantly enhanced by adding an organic solvent, such as ethanol, attributed to stronger electrostatic interactions between Na + and DNA in solutions with a lower dielectric constant .…”

“…Its activity can be significantly enhanced by adding an organic solvent, such as ethanol, attributed to stronger electrostatic interactions between Na + and DNA in solutions with a lower dielectric constant . EtNa also works with a divalent metal, Ca 2+ , allowing for a systematic comparison of the effects of metal ions . Therefore, we selected EtNa for this study.…”

Nucleic Acid Catalysis under Potential Prebiotic Conditions

“…Interestingly, EtNa activity drastically decreases at ethanol concentrations beyond 72 % (v/v) ethanol, where the B‐form helix of DNA is converted into the A‐form that (in contrast to ribozymes) seems to be incompatible with the formation of the active DNAzyme conformation. Given that EtNa shows cooperative binding of and activation by Ca 2+ (in contrast to Mg 2+ ) it can also be used as an ultrasensitive biosensor capable of detecting Ca 2+ levels down to 1.4 μ m Ca 2+ . Eutectic freezing can also activate EtNa, while other DNAzymes that depend on divalent or trivalent metals are inhibited under these conditions .…”

“…Given that EtNa shows cooperative binding of anda ctivation by Ca 2 + (in contrastt oM g 2 + ) [202] it can also be used as an ultrasensitive biosensor capable of de-tectingC a 2 + levels down to 1.4 mm Ca 2 + . [203] Eutectic freezing can also activate EtNa, while other DNAzymes that depend on divalent or trivalent metals are inhibited under these conditions. [204] This again highlights the interchangeability of freezing, organic dehydration or evaporation to achieve activation of metal-independent nucleic acid catalysts.…”

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

“…Although the detailed structure and enzymatic mechanism of the 17E DNAzyme have not been elucidated, biophysical studies have shown the importance of Mg 2+ binding and non-canonical hydrogen bonding in the catalytic core for catalysis [53][54][55]. It is noted that an RNA-cleaving DNAzyme exhibiting high activity in the presence of high ethanol concentration have been reported [27,28]. The active complex of this enzyme might be stable enough to overcome the destabilization effect of ethanol, unlike the case of 17E(S).…”