G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3′ end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3′ adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid–base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions.

DNA methylation catalyzed by methyltransferase (MTase) is a significant epigenetic process for modulating gene expression. Traditional methods to study MTase activity require a laborious and costly DNA labeling process. In this article, we report a simple, colorimetric, and label-free methylation-responsive DNAzyme (MR-DNAzyme) strategy for MTase activity analysis. This new strategy relies on horseradish peroxidase (HRP) mimicking DNAzyme and the methylation-responsive sequence (MRS) of DNA which can be methylated and cleaved by the MTase/endonuclease coupling reaction. Methylation-induced scission of MRS would activate the DNAzyme that can catalyze the generation of a color signal for the amplified detection of methylation events. Taking Dam MTase and DpnI endonuclease as examples, we have developed two colorimetric methods based on the MR-DNAzyme strategy. The first method is to utilize an engineered hairpin-DNAzyme hybrid probe for facile turn-on detection of Dam MTase activity, with a wide linear range (6-100 U/mL) and a low detection limit (6 U/mL). Furthermore, this method could be easily expanded to profile the activity and inhibition of restriction endonuclease. The second method involves a methylation-triggered DNAzyme-based DNA machine, which achieves the ultrahigh sensitive detection of Dam MTase activity (detection limit = 0.25 U/mL) by a two-step signal amplification cascade.

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

A generic sensing strategy that integrates CRISPR-Cas12a with a DNA circuit is proposed for amplified detection of microRNA.

Highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) were prepared by a simple and green route employing sodium alginate as a carbon source and tryptophan as both a nitrogen source and a functional monomer. The as-synthesized N-CNPs exhibited excellent water solubility and biocompatibility with a fluorescence quantum yield of 47.9%. The fluorescence of the N-CNPs was intensively suppressed by the addition of ascorbic acid (AA). The mechanism of the fluorescence suppression of the N-CNPs was investigated, and the synergistic action of the inner filter effect (IFE) and the static quenching effect (SQE) contributed to the intensive fluorescence suppression, which was different from those reported for the traditional redox-based fluorescent probes. Owing to the spatial effect and hydrogen bond between the AA and the groups on the N-CNP surface, excellent sensitivity and selectivity for AA detecting was obtained in a wide linear relationship from 0.2 μM to 150 μM. The detection limit was as low as 50 nM (signal-to-noise ratio of 3). The proposed sensing systems also represented excellent sensitivity and selectivity for AA analysis in human biological fluids, providing a valuable platform for AA sensing in clinic diagnostic and drug screening.

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A Ti3C2QD-based fluorescent probe for an ALP activity assay and embryonic stem cell identification was developed by taking advantage of the inner filter effect.

Small-molecule regulation is a powerful switching tool to manipulate cell signal transduction for a desired function; however, most available methods usually require genetic engineering to endow cells with responsiveness to user-defined small molecules. Herein, we demonstrate a nongenetic approach for small-molecule-controlled receptor activation and consequent cell behavior manipulation that is based on DNA-mediated chemically induced dimerization (D-CID). D-CID uses a programmable chemical-responsive DNA nanodevice to trigger DNA strand displacement and induce the activation of c-Met, a tyrosine kinase receptor cognate for hepatocyte growth factor, through dimerization. Through the use of various functional nucleic acids, including aptamers and DNAzymes, as recognition modules, the versatility of D-CID in inducing c-Met signaling upon addition of various small-molecular or ionic cues, including ATP, histidine, and Zn , is demonstrated. Moreover, owing its multi-input properties, D-CID can be used to manipulate the behaviors of multiple cell populations simultaneously in a selective and programmable fashion.