A Ratio-based Fluorophore-RNA Complex Based On Click Chemistry And Tris(2-carboxyethyl)phosphine Near-infrared Fluorescent Probe

Organic small molecule fluorescent probes have become an indispensable tool in modem biology because they can provide dynamic information concerning the localization and concentration of target molecules.Fluorescence has great potential in the detection of biological molecules.At present,the developed fluorescent molecules can be used not only as fluorescence labeling of biological molecules,but also as stimulus-responsive fluorescent probes for the analysis of characteristic analytes.In this paper,ratiometric fluorescent probe and near-infrared fluorescent probe were synthesized by modifying coumarin and hemicyanine fluorophore,and the sensitive detection of RNA in vitro transcription and tri(2-carboxyethyl)phosphine(TCEP)were achieved respectively.This paper is carried out in the following two aspects:1.Ratiometric RNA aptamer/fluorophore complex for RNA synthesis detectionRNA synthesis play a critical role in most cellular and developmental processes.Establishing a quantification method for RNA in vitro synthesis with low background without requiring translation has important application prospects for the study of RNA synthesis process.The approach has been reported for RNA in vitro transcription quantification based on the principle that fluorescence intensity is enhanced by binding of DFHBI fluorescent dyes with RNA aptamers.Since intensity-based fluorescence detection can be influenced by variations in an excitation intensity,emission collection efficiency,local probe microenvironment changes and other factors in complex biological samples.Therefore,ratiometric fluorescence measurement-based RNA aptamer/fluorophore complex would benefit the RNA in vitro transcription quantification.In this study,we have designed and synthesized a new fluorophore containing coumarin and DFHBI for quantifying the in vitro synthesis of RNA using ratiometric RNA aptamer-fluorophore complex.We added triazolyl-coumarin(CM)module into the 3,5-difluoro-4-hydroxybenzylidene imidazolmone(DFHBI)molecule via Cu(?)-catalyzed azide-alkyne cycloaddition.DFHBI exhibits low background fluorescence itself under the excitation at 447 nm,and when DFHBI was modified with coumarin,DFHBI-CM exhibits bright fluorescence upon excitation at 337 nm with or without RNA,and displays the characteristic emission band of quantitative triazolyl-coumarin centered at 420 nm.When DFHBI-CM is combined with RNA aptamer Spinach2 to generate Spinach2/DFHBI-CM fluorescence complexes,under the excitation wavelength of 447 nm,the fluorescence intensity at 502 nm is significantly enhanced.By comparing the emission intensity at 420 nm and 502 nm,ratiometric fluorescence measurement is achieved.Based on this principle,DFHBI-CM was applied as a fluorophore in RNA aptamer-fluorophore complex for ratiometrically detecting RNA in vitro synthesis.Our design has the following advantages:(1)ratiometric fluorescence can minimize the environmental inferring factors and thus,more accurate and effective detection can be achieved;(2)compared with run-off transcription detection of RNA,this ratiometric fluorescent probe detection speed is faster.2.A selective and sensitive azido near-infrared fluoresent probe for tris(2-carboxyethyl)phosphine quantitative detection and its application for E.coli determinationTris(2-carboxyethyl)phosphine(TCEP)is a hydrophilic trialkylphosphine reducing agent that are widely used in biochemistry.TCEP has been found to play an important role important in anti-cancer and retinal therapy.TCEP can significantly promote the reaction of cisplatin toward zinc finger proteins as a novel strategy for anti-cancer proposes.In retinal therapy,TCEP protects retinal ganglion cells and reduces oxidative damage to photoreceptors,which is related to photic injury and diseases.Therefore,it is particularly important to establish a specific and sensitive quantitative detection method for TCEP.We designed and synthesized a sensitive near-infrared fluorescent probe,azido hemicyanine(HC-N3),for the detection of tris(2-carboxyethyl)phosphine(TCEP).TCEP can react with azido fluorophore and induce fluorescence change,achieving the lowest LOD(92 nM).HC-N3 has good selectivity for TCEP among thiols and 20 kinds of natural amino acid.The quantification of E.coli(101-103 cfu mL-1)demonstrated its practicability in complicated biosystems.