Abstract CRISPR‐LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg 2+ to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans‐cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn 2+ and Ca 2+ could replace Mg 2+ as cofactors of LbuCas13a. In contrast, Ni 2+ , Zn 2+ , Cu 2+ , or Fe 2+ inhibits the cis‐ and trans‐ cleavage activity, while Pb 2+ does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans‐cleavage activity. Finally, we showed that combination of Mg 2+ and Mn 2+ can further enhance the trans‐cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.
A 3,4-dihydroisocoumarin derivative fused with dihydrothiophene, talarolactone A (1), and two known compounds, terreusinone (2) and 4,6-dihydroxy-5-methylphthalide (3), were isolated from Talaromyces sp. associated with Xanthoparmelia angustiphylla. The structure of 1 was deduced from extensive spectroscopic data, electronic circular dichroism calculations, and X-ray diffraction analyses. A plausible biosynthetic pathway of 1 was further proposed. Compound 1 showed selective antimigratory activity in a wound-healing assay without appreciable cytotoxic activity.
Targeted degradation of membrane proteins represents an attractive strategy for eliminating pathogenesis‐related proteins. Aptamer‐based chimeras hold great promise as membrane protein degraders, however, their degradation efficacy is often hindered by the limited structural stability and the risk of off‐target effects due to the non‐covalent interaction with target proteins. We here report the first design of a covalent aptamer‐based autophagosome‐tethering chimera (CApTEC) for the enhanced autophagic degradation of cell‐surface proteins, including transferrin receptor 1 (TfR1) and nucleolin (NCL). This strategy relies on the site‐specific incorporation of sulfonyl fluoride groups onto aptamers to enable the cross‐linking with target proteins, coupled with the conjugation of an LC3 ligand to hijack the autophagy‐lysosomal pathway for targeted protein degradation. The chemically engineered CApTECs exhibit enhanced on‐target retention and improved structural stability. Our results also demonstrate that CApTECs achieve remarkably enhanced and prolonged degradation of membrane proteins compared to the non‐covalent designs. Furthermore, the CApTEC targeting TfR1 is combined with 5‐fluorouracil (5‐FU) for synergistic tumor therapy in a mouse model, leading to substantial suppression of tumor growth. Our strategy may provide deep insights into the LC3‐mdiated autophagic degradation, affording a modular and effective strategy for membrane protein degradation and precise therapeutic applications.
Targeted degradation of membrane proteins represents an attractive strategy for eliminating pathogenesis‐related proteins. Aptamer‐based chimeras hold great promise as membrane protein degraders, however, their degradation efficacy is often hindered by the limited structural stability and the risk of off‐target effects due to the non‐covalent interaction with target proteins. We here report the first design of a covalent aptamer‐based autophagosome‐tethering chimera (CApTEC) for the enhanced autophagic degradation of cell‐surface proteins, including transferrin receptor 1 (TfR1) and nucleolin (NCL). This strategy relies on the site‐specific incorporation of sulfonyl fluoride groups onto aptamers to enable the cross‐linking with target proteins, coupled with the conjugation of an LC3 ligand to hijack the autophagy‐lysosomal pathway for targeted protein degradation. The chemically engineered CApTECs exhibit enhanced on‐target retention and improved structural stability. Our results also demonstrate that CApTECs achieve remarkably enhanced and prolonged degradation of membrane proteins compared to the non‐covalent designs. Furthermore, the CApTEC targeting TfR1 is combined with 5‐fluorouracil (5‐FU) for synergistic tumor therapy in a mouse model, leading to substantial suppression of tumor growth. Our strategy may provide deep insights into the LC3‐mdiated autophagic degradation, affording a modular and effective strategy for membrane protein degradation and precise therapeutic applications.
Abstract DNAzyme‐based fluorescent probes for imaging metal ions in living cells have received much attention recently. However, employing in situ metal ions imaging within subcellular organelles, such as nucleus, remains a significant challenge. We developed a three‐stranded DNAzyme probe (TSDP) that contained a 20‐base‐pair (20‐bp) recognition site of a CRISPR/Cas9, which blocks the DNAzyme activity. When Cas9, with its specialized nuclear localization function, forms an active complex with sgRNA within the cell nucleus, it cleaves the TSDP at the recognition site, resulting in the in situ formation of catalytic DNAzyme structure. With this design, the CRISPR/Cas9‐inducible imaging of nuclear Zn 2+ is demonstrated in living cells. Moreover, the superiority of CRISPR‐DNAzyme for spatiotemporal control imaging was demonstrated by integrating it with photoactivation strategy and Boolean logic gate for dynamic monitoring nuclear Zn 2+ in both HeLa cells and mice. Collectively, this conceptual design expands the DNAzyme toolbox for visualizing nuclear metal ions and thus provides new analytical methods for nuclear metal‐associated biology.
DNAzyme-based fluorescent probes for imaging metal ions in living cells have received much attention recently. However, employing in situ metal ions imaging within subcellular organelles, such as nucleus, remains a significant challenge. We developed a three-stranded DNAzyme probe (TSDP) that contained a 20-base-pair (20-bp) recognition site of a CRISPR/Cas9, which blocks the DNAzyme activity. When Cas9, with its specialized nuclear localization function, forms an active complex with sgRNA within the cell nucleus, it cleaves the TSDP at the recognition site, resulting in the in situ formation of catalytic DNAzyme structure. With this design, the CRISPR/Cas9-inducible imaging of nuclear Zn
Coronavirus disease 2019 (COVID-19) poses an extremely serious global impact on public healthcare for individuals of all ages, including children. Increasing evidence has shown that liver abnormalities are commonly found in children with COVID-19, and age-related features in innate and adaptive response have been demonstrated. However, there are few reports and studies on COVID-19 related liver injury in children, and the data are scattered. So that many contradictions have arose. This situation is not only due to the serious ethical issues in studying pediatric patients with COVID-19, but also because of the short duration and wide coverage of the COVID-19 epidemic, the severity and complexity of clinical cases varied, as did the inclusion criteria for case reporting and patient outcomes. Therefore, we totaled the incidences, characteristics and pathomechanism of liver injury in children since the COVID-19 outbreak. The etiology of COVID-19-related liver injury is divided into three categories: (1) The direct mechanism involves severe acute respiratory syndrome coronavirus 2 binding to angiotensin-converting enzyme 2 in the liver or bile duct to exert direct toxicity; (2) the indirect mechanisms include an inflammatory immune response and hypoxia; and (3) COVID-19-related treatments, such as mechanical ventilation and antiviral drugs, may cause liver injury. In summary, this minireview provides fundamental insights into COVID-19 and liver dysfunction in children.
The level of 25-hydroxyvitamin D3 [25(OH)VD3] in human blood is considered as the best indicator of vitamin D status, and its deficiency or excess can lead to various health problems. Current methods for monitoring 25(OH)VD3 metabolism in living cells have limitations in terms of sensitivity and specificity and are often expensive and time-consuming. To address these issues, an innovative trident scaffold-assisted aptasensor (TSA) system has been developed for the online quantitative monitoring of 25(OH)VD3 in complex biological environments. Through the computer-aided design, the TSA system includes an aptamer molecule recognition layer that is uniformly oriented, maximizing binding site availability, and enhancing sensitivity. The TSA system achieved the direct, highly sensitive, and selective detection of 25(OH)VD3 over a wide concentration range (17.4–12,800 nM), with a limit of detection of 17.4 nM. Moreover, we evaluated the efficacy of the system in monitoring the biotransformation of 25(OH)VD3 in human liver cancer cells (HepG2) and normal liver cells (L-02), demonstrating its potential as a platform for drug–drug interaction studies and candidate drug screening.
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