Abstract:
Nucleic acid sequence detection is very important for many applications such as the diagnosis of genetic diseases. The development of new, simple yet effective methods is still in great demand. Among several options, colorimetric detection is a technique that does not require complicated instruments and allows the detection by naked eyes. In this work, a new colorimetric DNA detection system was developed using the combination of a pyrrolidinyl peptide nucleic acid (acpcPNA) probe and a G-quadruplex/hemin DNAzyme. The G-quadruplex is formed from G-rich DNA sequences, and in the presence of hemin, a DNAzyme is formed which effectively catalyzes the oxidation of the colorless 3,3',5,5'-tetramethylbenzidine (TMB) substrate into the blue oxidized TMB in the presence of H2O2. The G-quadruplex formation is inhibited by the partial hybridization with the acpcPNA probe via the Watson and Crick base-pairing. Accordingly, the acpcPNA suppresses the DNAzyme activity and also the blue color formation. A strand displacement occurs in the presence of the target DNA, which has a sequence complementary to the G-quadruplex DNA probe but does not directly overlap with the G-quadruplex-forming region. This results in the displacement of the acpcPNA strand leading to the re-formation of the G-quadruplex structure as well as the restoration of the DNAzyme activities. The G-quadruplex disruption and formation were confirmed by CD spectroscopy, gel electrophoresis, and UV-visible spectrophotometry. The principle was successfully applied for colorimetric DNA detection in a sequence-specific fashion with a linearity range from 0.25 µM to 0.025 µM and an LOD of 7 nM. Thus, this developed platform has the potential to be applied for diagnostic applications as well as other problems. To improve the sensitivity further, the target-mediated partial digestion of the G-quadruplex DNA probe by Exonuclease III was proposed to amplify the signal by target recycling. However, the target recycling was not successful due to the competitive binding of the acpcPNA probe to the partially digested G-quadruplex DNA probe.
