Novel Amplex Red Oxidases Based on Noncanonical DNA
Structures: Property Studies and Applications in MicroRNA Detection
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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 H<sub>2</sub>O<sub>2</sub> generation
from O<sub>2</sub> 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 G-quadruplex 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