Repair-driven DNA tetrahedral nanomachine combined with DNAzyme for 8-oxo guanine DNA glycosylase activity assay, drug screening and intracellular imaging.

8-oxo guanine DNA glycosylase (8-oxoG DNA glycosylase), a crucial DNA repair enzyme, is essential for maintaining genome integrity and preventing diseases caused by DNA oxidative damage. Imaging 8-oxoG DNA glycosylase in living cells requires a dependable technique. In this study, we designed a DNAzyme-modified DNA tetrahedral nanomachine (DTDN) powered by 8-oxoG restoration. Incorporating a molecular beacon probe (MB), the constructed platform was used for amplified in situ monitoring of 8-oxoG DNA glycosylase. Under normal conditions, duplexing with a complementary strand modified with two 8-oxoG sites inhibited the activity of DNAzyme. The restoration of DNAzyme activity by the repair of intracellular 8-oxoG DNA glycosylase on 8-oxoG bases can initiate a signal amplification reaction. This detection system can detect 8-oxoG DNA glycosylase activity linearly between 0 and 20 U mL-1, with a detection limit as low as 0.52 U mL-1. Using this method, we were able to screen 14 natural compounds and identify 6 of them as 8-oxoG DNA glycosylase inhibitors. In addition, a novel approach was utilized to assess the activity of 8-oxoG DNA glycosylase in living cells. In conclusion, this method provides a universal tool for monitoring the activity of 8-oxoG DNA glycosylase in vitro and in living cells, which holds great promise for elucidating the enzyme's functionality and facilitating drug screening endeavors.

Reduced graphene oxide quenched peptide probe for caspase-8 activity detection and cellular imaging.

Cysteinyl aspartate-specific protease 8 (caspase-8) plays a key role in various biological processes by regulating apoptosis. Therefore, this makes accurate detection and intracellular imaging of caspase-8 of great importance for drug screening, disease diagnosis, and prognostication. Here, by designing a reduced graphene oxide (rGO) quenched peptide probe, we constructed a new biosensing system for monitoring in vitro and intracellular caspase-8 activity. In this system, a fluorophore-labeled peptide and rGO were used as the substrate of caspase-8 and the fluorophore quencher, respectively. The hydrolysis of caspase-8 on the polypeptide probe substrate can generate two fragments with different lengths. The release of the short fragment labeled with the fluorophore causes recovery of the fluorescence signal (Ex/Em = 520/576 nm). Under the optimized conditions, the proposed fluorescence method exhibited a linear response range of 0.2 to 5 U·mL-1 for caspase-8 with a limit of detection (LOD) of 0.2 U·mL-1 in vitro. Furthermore, this method has been successfully applied to monitoring the upregulation of intracellular caspase-8 activity caused by tert-butyl hydroperoxide (TBHP) and fluorouracil. Flow cytometry assay indicated the positive relation between the upregulation of intracellular caspase-8 activity and cell apoptosis rate. In summary, the above results demonstrated the practical application of this method for apoptosis-related cell imaging.

The Effect of Nucleating Agents on Enzyme-Induced Carbonate Precipitation and Corresponding Microscopic Mechanisms.

Plant urease has the advantages of high activity and small size in enzyme-induced calcium carbonate precipitation (EICP). However, there area lack of nucleation sites for calcium carbonate in EICP. Sucrose and sorbitol, which are readily available and inexpensive, have the potential to provide nucleation sites for EICP as nucleating agents. To explore the effects of the two nucleating agents on EICP, the productivity of calcium carbonate, unconfined compressive strength (UCS) and microscopic mechanisms were tested. It is found that the productivity of EICP can be increased as much as 5.1% by the addition of sorbitol with an optimal content of 5%, and the productivity of EICP can be increased as much as 12.3% by the addition of sucrose with an optimal of 4%. The UCS of EICP-treated sand increases by 2.2 times after being improved by sorbitol with a content of 5.2%, the CaCO3 content of EICP-treated sand with sorbitol added increased by 1.5% compared to conventional EICP-treated sand. These results show that the two nucleating agents are effective for improving EICP. The SEM images verify that sorbitol/sucrose can compensate for the lack of nucleating sites in EICP and explicate the effect of nucleating agents on EICP.

A radar-like DNA monitor for RNase H-targeted natural compounds screening and RNase H activity in situ detection.

Ribonuclease H is essential for the research and development of complex pathema. The high rigidity and versatility of DNA tetrahedrons means they are often used in biosensing systems. Inspired by "radar" technology, we proposed a radar-like monitor to detect RNase H activity in vitro and in situ by integrating DNA tetrahedral elements. The structure of a radar-like monitor was self-assembled from five customized single nucleic acid strands. Four DNA strands were assembled as DNA tetrahedrons with a long strand labeled by Dabcyl (quencher) at one of the apexes, while the fifth strand (DNA-RNA heterozygous strand) was labeled with a FAM (Fluorophore) hybrid with a long strand. The fluorescence was quenched because the fluorophore and the quencher were very close. In the presence of RNase H, the RNA chain was hydrolyzed and the fluorophore released, resulting in fluorescence recovery. The radar-like monitor was used to detect the RNase H activity in vitro with a detection limit of 0.01 U mL-1. Based on the RNase H activity detection and the inhibitory effect of natural-compounds-targeting RNase H, three inhibitors were obtained among 35 compounds extracted from Panax japonicus. Therefore, the radar-like monitor was successfully used to detect RNase H activity in situ due to the long-term anti-DNase I effect of the RNA/DNA hybrid structure and DNA tetrahedrons structure. Overall, this radar-like monitor can effectively avoid false-positive signals and significantly improve the accuracy, precision, and reliability of detection. It is expected that the development of such an intelligent nano-platform will open the door to cancer diagnosis and treatment in clinical systems.