title: Hemin/G-quadruplex-catalyzed aerobic oxidation of thiols to disulfides: application of the process for the development of sensors and aptasensors and for probing acetylcholine esterase activity.
abstract: This study describes the novel hemin/G-quadruplex DNAzyme-catalyzed aerobic oxidation of thiols to disulfides and the respective mechanism. The mechanism of the reaction involves the DNAzyme-catalyzed oxidation of thiols to disulfides and the thiol-mediated autocatalytic generation of H2O2 from oxygen. The coupling of a concomitant H2O2-mediated hemin/G-quadruplex-catalyzed oxidation of Amplex Red to the fluorescent resorufin as a transduction module provides a fluorescent signal for probing the catalyzed oxidation of the thiol to disulfides and for probing sensing processes that yield the hemin/G-quadruplex as a functional label. Accordingly, a versatile sensing method for analyzing thiols (L-cysteine, glutathione) using the H2O2-mediated DNAzyme-catalyzed oxidation of Amplex Red to the resorufin was developed. Also, the L-cysteine and Amplex Red system was implemented as an auxiliary fluorescent transduction module for probing recognition events that form the catalytic hemin/G-quadruplex structures. This is exemplified with the development of thrombin aptasensor. The thrombin/thrombin binding aptamer recognition complex binds hemin, and the resulting catalytic complex activates the auxiliary transduction module, involving the aerobic oxidation of l-cysteine and the concomitant formation of the fluorescent resorufin. Finally, the hemin/G-quadruplex DNAzyme/Amplex Red system was used to follow the activity of acetylcholine esterase, AChE, and to probe its inhibition. The AChE-catalyzed hydrolysis of acetylthiocholine to the thiol-functionalized thiocholine enabled the probing of the enzymatic activity of AChE through the hemin/G-quadruplex-catalyzed aerobic oxidation of thiocholine to the respective disulfide and the concomitant generation of the fluorescent resorufin product.
authors.affiliation: The Institute of Chemistry, The Minerva Center for Biohybrid Complex Systems, The Hebrew University of Jerusalem , Jerusalem 91904, Israel.
authors.forename: Eyal
authors.initials: E
authors.lastname: Golub
authors.forename: Ronit
authors.initials: R
authors.lastname: Freeman
authors.forename: Itamar
authors.initials: I
authors.lastname: Willner
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism*; D000332:Aerobiosis; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D055162:Biocatalysis; D015374:Biosensing Techniques / Q000379:methods*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D021881:DNA, Catalytic / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D054856:G-Quadruplexes; D006427:Hemin / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013917:Thrombin / Q000032:analysis
chemical_list: D052157:Aptamers, Nucleotide; D002800:Cholinesterase Inhibitors; D021881:DNA, Catalytic; D004220:Disulfides; D013438:Sulfhydryl Compounds; D006427:Hemin; D006861:Hydrogen Peroxide; D000110:Acetylcholinesterase; D013917:Thrombin