RESUMO
Despite tremendous success in understanding the chemical nature and the importance of cation-π interactions in a range of biological processes, particularly in epigenetic regulation, the design and synthesis of stronger cation-π interactions in living cells remain largely elusive. Here, we design several electron-rich Trp derivatives and incorporate them into histone methylation reader domains to enhance the affinity of the reader domains for histone methylation marks via cation-π interactions in living cells. We show that this site-specific Trp replacement strategy is generally applicable for the engineering of high-affinity reader domains for the major histone H3 trimethylation marks, such as H3K4me3, H3K9me3, H3K27me3, and H3K36me3, with high specificity. Furthermore, we demonstrate that engineered reader domains can serve as powerful tools for the enrichment and imaging of histone methylation, as well as for capturing the protein interactome at chromatin marks in living cells. Therefore, our study paves the way for the design of enhanced cation-π interactions in reader proteins in living cells for various biological applications.
Assuntos
Epigênese Genética , Histonas , Histonas/genética , Histonas/metabolismo , Cromatina , Metilação , Código GenéticoRESUMO
A positively charged aggregation-induced emission luminogen (AIEgen), TPE-P+, was constructed by linking a pyridyl cation to tetraphenylethylene (TPE) via a cyanoethylene bond. TPE-P+ can realize the identification of heparin (Hep) by aggregating with negatively charged Hep via electrostatic interactions. Upon addition of Hep, TPE-P+ exhibited 36-fold fluorescence enhancement in less than 5 s, exhibiting quick and sensitive response to Hep with a low detection limit down to 4 nM. Among various biological substances, even Hep analogs like chondroitin 4-sulfate and hyaluronic acid, TPE-P+ showed the most significant fluorescence enhancement to Hep only, demonstrating its excellent selectivity for Hep. In particular, with long-wavelength emission near 600 nm and large stocks shift (â¼160 nm), TPE-P+ enabled minimization of autofluorescence interference from a complex biological matrix and provided more accurate results. Finally, TPE-P+ was successfully applied for sensitive and selective detection of Hep in serum. Notably, there existed a good linear relationship in a serum assay when the Hep concentration ranging from 0 to 4 µM (R2 = 0.9934) covered the clinical dosage level during both cardiovascular surgery and long-term care, suggesting the potential clinical practice for quantifying Hep in serum. Moreover, TPE-P+-Hep complex can be further disaggregated by protamine (PRTM) due to the stronger affinity between Hep and PRTM, thereby leading to further detection of PRTM effectively. Last, but not least, the "off-on-off" system designed for both Hep and PRTM detection proved to be reversible.
Assuntos
Corantes Fluorescentes/química , Heparina/sangue , Piridinas/química , Estilbenos/química , Animais , Bovinos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Espectrometria de FluorescênciaRESUMO
Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule in many physiological processes. Relevant investigations indicated that H2S plays a cytoprotective effect under endoplasmic reticulum stress. Currently, it is still a challenge to design effective methods for ratio detection of endoplasmic reticulum H2S. Herein we are the first to construct a ratiometric near-infrared fluorescent probe (M-H2S) for sensing H2S in the endoplasmic reticulum. M-H2S has high selectivity and sensitivity toward H2S (LOD = 39.1 nM). Additionally, M-H2S possessed an excellent endoplasmic reticulum targeting ability. Moreover, M-H2S was successfully utilized to visualize exogenous/endogenous H2S in HeLa cells and zebrafish. Particularly important, endogenously produced H2S was observed under endoplasmic reticulum stress induced by tunicamycin.
Assuntos
Retículo Endoplasmático/química , Corantes Fluorescentes/química , Sulfeto de Hidrogênio/análise , Imagem Óptica , Animais , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura Molecular , Peixe-ZebraRESUMO
Cysteine oxidation by H2O2, generating either cysteine sulfenic acid (CSOH) or disulfide (CSSC), is involved in redox homeostasis and signaling. Compared with quantification of the cysteine content, monitoring the cysteine dynamics in real-time, in particular, takes on even greater importance. However, existing fluorescent probes suffer from low specificity or irreversible recognition mechanisms. In the present work, we have successfully developed a reversible fluorescent probe for the cycle between cysteine and H2O2 based on the Michael addition-elimination reaction. This probe features a specific and quantitative response to cysteine. The reversible detection was realized repeatedly with the addition of cysteine and H2O2 in order. We also demonstrated its usage for monitoring exogenous and endogenous cysteine in living cells. Eventually, this probe was capable of imaging cysteine dynamically in real-time.
Assuntos
Cisteína , Corantes Fluorescentes , Cisteína/metabolismo , Peróxido de Hidrogênio , Oxirredução , Transdução de SinaisRESUMO
As an important organelle, the endoplasmic reticulum (ER) participates in the synthesis and secretion of various proteins, glycogen, lipids and cholesterol in eukaryotic cells. In this work, an endoplasmic reticulum-targeted reversible fluorescent probe (ER-Se) was designed and synthesized. The probe, based on a selenide group, shows high sensitivity and good selectivity toward HClO (LOD = 0.85 µM). In addition, the probe has reversible capability towards HClO/GSH. Most importantly, co-location experiment results indicated that the probe exhibited a great ability to target the endoplasmic reticulum. Furthermore, the probe was successfully applied to detect exogenous and endogenous HClO in ER and monitored the redox status changes during ER stress.
Assuntos
Retículo Endoplasmático/química , Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Compostos Organosselênicos/química , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura Molecular , Imagem Óptica , Compostos Organosselênicos/síntese química , Oxirredução , Tunicamicina/farmacologiaRESUMO
We report a sensitive fluorescence probe Ratio-ALP for in vivo ratiometric imaging of alkaline phosphatase (ALP). The probe was designed by conjugating a naphthalimide with a phosphate group as the responsive moiety for enzymatic reactions. The fluorescence intensity ratio displayed a linear relationship against the concentration of ALP in the concentration range from 0 to 200 U/L, its LOD is as low as 0.25 U/L. Ratio-ALP has good selectivity and sensitivity, high cell permeability, and low cytotoxicity. Furthermore, probe Ratio-ALP was also successfully applied to image endogenous ALP activity in living cells.