Symmetry-breaking malachite green as a near-infrared light-activated fluorogenic photosensitizer for RNA proximity labeling.

Cellular RNA is asymmetrically distributed in cells and the regulation of RNA localization is crucial for proper cellular functions. However, limited chemical tools are available to capture dynamic RNA localization in complex biological systems with high spatiotemporal resolution. Here, we developed a new method for RNA proximity labeling activated by near-infrared (NIR) light, which holds the potential for deep penetration. Our method, termed FAP-seq, utilizes a genetically encoded fluorogen activating protein (FAP) that selectively binds to a set of substrates known as malachite green (MG). FAP binding restricts the rotation of MG and rapidly activates its fluorescence in a wash-free manner. By introducing a monoiodo modification to MG, we created a photosensitizer (MG-HI) with the highest singlet oxygen generation ability among various MG derivatives, enabling both protein and RNA proximity labeling in live cells. New insights are provided in the transcriptome analysis with FAP-seq, while a deeper understanding of the symmetry-breaking structural arrangement of FAP-MG-HI was obtained through molecular dynamics simulations. Overall, our wash-free and NIR light-inducible RNA proximity labeling method (FAP-seq) offers a powerful and versatile approach for investigating complex mechanisms underlying RNA-related biological processes.

Mussel-inspired monomer - A new selective protease inhibitor against dentine collagen degradation.

OBJECTIVES: To evaluate the inhibitory effect of a novel mussel-inspired monomer (N-(3,4-dihydroxyphenethyl)methacrylamide (DMA) on the soluble and matrix-bound proteases. METHODS: The inhibitory effect of DMA (0, 1, 5, and 10 mM) and 1 mM chlorhexidine (CHX) dissolved in 50% ethanol/water on soluble recombinant human matrix metalloproteinases (rhMMP-2, -8, and -9), as well as cysteine cathepsins (B and K) were evaluated using both fluorometric assay kits and molecular docking. The effect of CHX and DMA on matrix-bound proteases was examined by in situ zymography, and the fluorescence intensity and relative area were calculated by Image J software. All data obtained were analyzed by one-way ANOVA followed by Tukey test (α = 0.05). RESULTS: The anti-proteolytic ability of DMA increased in a dose-dependent manner except that of rhMMP-9. Inhibitory effect of 1 mM DMA against rhMMP-2, - 8, - 9, as well as cathepsin B and K was all significantly lower than 1 mM CHX (p < 0.05). The molecular docking analysis was in good agreement with the experimental results, that the binding energy of DMA was lower than CHX for all proteases. In situ zymography revealed that all DMA- and CHX-treated groups significantly inactivated the matrix-bound proteases, with a dramatic reduction of the fluorescence intensity and relative area compared with the control group (p < 0.05). SIGNIFICANCE: Under the prerequisite condition that the overall inhibitory performance on matrix-bound proteases was comparable by DMA and CHX, the more selective property of DMA could avoid inducing potential negative effects by suppressing MMP-9 when applied in dental treatment compared with CHX.