Enzymatic reaction modulated DNA assembly on graphitic carbon nitride nanosheets for sensitive fluorescence detection of acetylcholinesterase activity and inhibition.

A novel fluorescent strategy has been developed by using an enzymatic reaction modulated DNA assembly on graphitic carbon nitride nanosheets (CNNS) for the detection of acetylcholinesterase (AChE) activity and its inhibitors. The two-dimensional and ultrathin-layer CNNS-material was successfully synthesized through a chemical oxidation and ultrasound exfoliation method. Because of its excellent adsorption selectivity to ssDNA over dsDNA and superior quenching ability toward the fluorophore labels, CNNS were employed to construct a sensitive fluorescence sensing platform for the detection of AChE activity and inhibition. The detection was based on enzymatic reaction modulated DNA assembly on CNNS, which involved the specific AChE-catalyzed reaction-mediated DNA/Hg2+ conformational change and subsequent signal transduction and amplification via hybridization chain reaction (HCR). Under the excitation at 485 nm, the fluorescence signal from 500 to 650 nm (λmax = 518 nm) of the developed sensing system was gradually increased with increasing concentration of AChE. The quantitative determination range of AChE is from 0.02 to 1 mU/mL and the detection limit was 0.006 mU/mL. The developed strategy was successfully applied to the assay of AChE in human serum samples, and can also be used to effectively screen AChE inhibitors, showing great promise providing a robust and effective platform for AChE-related diagnosis, drug screening, and therapy.

G-Quadruplex-Induced Liquid-Liquid Phase Separation in Biomimetic Protocells.

Biomolecular condensates comprised of specific proteins and nucleic acids are now recognized as one of the key organizing mechanisms in eukaryotic cells. However, the specific roles played by the nucleic acid secondary structure and sequence in biomolecular phase separation are still not clear. Here, utilizing giant membrane vesicles (GMVs) as a protocell model, we found that single-stranded DNA (ssDNA) with a parallel G-quadruplex structure could functionally cooperate with a G-quadruplex-binding protein to form speckle-like puncta inside the GMVs. The clustering behavior is dependent on the structural diversity of G-quadruplexes, and the reversible clustering behavior implicated a new pathway in dynamically regulating the formation of biomolecular condensates. This finding represents a potential link between G-quadruplex-binding proteins and the resulting G-quadruplex-mediated biomolecular phase separation, which would gain insight into a wide range of biological processes associated with nucleic acid-modulated phase separation inside living cells.