Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery.

The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.

Nanotheranostic Application of Fluorescent Protein-Gold Nanocluster Hybrid Materials: A Mini-review.

The gold nanoclusters (Au NCs) are a special kind of gold nanomaterial containing several gold atoms. Because of their small size and large surface area, Au NCs possess macroscopic quantum tunneling and dielectric domain effects. Furthermore, Au NCs fluorescent materials have longer luminous time and better photobleaching resistance compared with other fluorescent materials. The synthetic process of traditional Au NCs is complicated. Traditional Au NCs are prepared mainly by using polyamide amine type dendrites, and sixteen alkyl trimethylamine bromide or sulfhydryl small molecule as stabilizers. They are consequently synthesized by the reduction of strong reducing agents such as sodium borohydride. Notably, these materials are toxic and environmental-unfriendly. Therefore, there is an urgent need to develop more effective methods for synthesizing Au NCs via a green approach. On the other hand, the self-assembly of protein gold cluster-based materials, and their biomedical applications have become research hotspots in this field. We have been working on the synthesis, assembly and application of protein conjugated gold clusters for a long time. In this review, the synthesis and assembly of protein-gold nanoclusters and their usage in cell imaging and other medical research are discussed.

Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro).

The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.