Appropriately ordered guanosine bases in DNA chains form G-quartets stabilized by interbase hydrogen bonds. These Gquartets are further stacked, in the presence of ions, into a Gquadruplex structure. [1] Different organic molecules are known to interact with G-quadruplex structures. [2] Specifically, hemin was found to bind to a G-quadruplex, and the resulting complex revealed horseradish peroxidise mimicking activities. [3] For example, the hemin/G-quadruplex catalyzes the H 2 O 2 -mediated oxidation of the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate dianion) (ABTS 2ร ) to the colored product ABTSC ร [3] or leads to the generation of chemiluminescence [4] in the presence of H 2 O 2 /luminol. Also, the hemin/ G-quadruplex structure linked to electrodes was reported to act as an electrocatalyst for the reduction of H 2 O 2 , [5] and the hemin/G-quadruplex associated with semiconductor quantum dots was found to quench the luminescence of the quantum dots. [6] These properties enabled the use of the hemin/Gquadruplex as a versatile label for numerous sensors including enzyme-, [7] DNA-, [8] and aptamer-based [9] sensors. In the present study, we demonstrate that the hemin/G-quadruplex acts not only as a horseradish peroxidase mimicking DNAzyme, but, also, as an NADH oxidase and NADH peroxidase mimicking DNAzyme. The regeneration of the nicotinamide adenine dinucleotide, NAD + , cofactor attracted substantial interest for biotechnological applications, and NAD + -dependent enzymes have been widely used for chemical transformations. [10] Our results pave the way to use the hemin/Gquadruplex as a biocatalyst for the regeneration of the NAD + cofactor and to apply the DNAzyme as a catalyst for enzymedriven transformations.
NADH oxidase catalyzes the oxidation of NADH by O 2 with the concomitant formation of H 2 O 2 . [11] The present system consists of reduced nicotinamide adenine dinucleotide (NADH), hemin/G-quadruplex (1), and Amplex Red (2), as a fluorescent reporter dye (Scheme 1 A). Under aerobic conditions, NADH is oxidized to NAD + (see below), while 2 is oxidized to resorufin (3), which generates fluorescence at l max = 581 nm. Figure 1 A, curve (a), shows the time-dependent increase in the fluorescence generated by the system. Control experiments reveal minor background fluorescence generated under an inert argon atmosphere (curve (e)), or Scheme 1. A) Frame I: Hemin/G-quadruplex-catalyzed oxidation of NADH by O 2 to NAD + and H 2 O 2 , respectively, and the concomitant oxidation of Amplex Red (2) to resorufin (3) by H 2 O 2 . Frame II: Coupled DNAzyme-alcohol dehydrogenase biocatalytic system for the formation of NADH and the concomitant regeneration of the NAD + cofactor by the hemin/G-quadruplex. B) Suggested mechanism for the DNAzyme-catalyzed oxidation of NADH.