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Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection

Shaoru Wang,† Boshi Fu,† Jiaqi Wang,† Yuelin Long,† Xiaoe Zhang,† Shuang Peng,† Pu Guo,† Tian Tian,*,† and Xiang Zhou*,†,‡

 

†College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, State Key Laboratory of Virology, Wuhan University, Wuhan, Hubei 430072, P. R. of China


‡State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China

 


ABSTRACT: G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or Gquadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.


 

 

 

Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection
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