Intelligent Fluorescent Encoded Probe For Multiplexed MicroRNAs Profiling In Living Cells

MicroRNAs(miRNAs)are endogenous,non-coding,single-stranded small RNAs of 18 to 23 nt.It is a key factor involved in the regulation of gene expression and plays an important role in various physiological processes,such as cell growth and development,endoplasmic reticulum stress,apoptosis and autophagy.miRNAs are abnormally expressed in the occurrence and progression of a variety of diseases(such as cancer,cardiovascular disease,degenerative diseases,diabetes,etc.),and is regarded by many researchers as a type of potential biomarker and new target for gene therapy.However,the detection of miRNAs is challenge owing to the unique characteristics such as short sequence,low abundance,high family homology,complex intracellular environment and cell expression heterogeneity of miRNAs.Especially,single miRNA detection is not reliable for diease diganosis and prognosis due the orthogonal regulation of the miRNA,and multiplexed miRNAs simultaneously detecion is imperatively needed.Thus,it is still key challenge for multiplexed miRNAs profiling in living cells:(1)At present,the most commonly used methods for multiplexed miRNAs detection are fluorescence quantitative PCR(qPCR)and gene chip technique,which require complex processes and professional operators especially it cannot reflect the in situ spatial information of miRNAs.(2)The multiple miRNA detection in living cells is limited by fluorescence spectrum overlap.and it is difficult to monitor more than 4 targets.(3)Although multiple in situ hybridization FISH(multi-FISH)method has been used by many researchers for multipleed RNAs in situ detection,this method cannot be extended to living cells because it involves highly cytotoxic permeability and elution process.(4)it is urgent to develop a high sensitivity,high selectivity and suitable multi-target in situ multiple detection system for miRNAs living cells.In view of the above considerations,our research work is as follows:1.Spatially resolved,error-robust multiplexed miRNAs profiling in single living cellsPrecise measuring of copy number and spatial profiling of multiple miRNAs in individual living cells is necessary to understand cellular behaviors and functions.However,simultaneous imaging of multiple miRNAs in individual living cells is challenging due to the lack of spectrally distinct encoded fluorophores and non-cytotoxic methods.We describe a multiplexed error-robust combinatorial fluorescent label encoding method,termed fluorophores encoded error-corrected label(FluoEL),enabling multiplexed miRNAs imaging in living cells with error-correcting capability.FluoELs comprise proportional dual fluorophores for encoding and a constant quantitative single fluorophore for errorcorrected quantification,simultaneously embedded in 260 nm core-shell silica nanoparticles modified with molecular beacon detection probes.FluoELs are low cytotoxicity and could accurately quantify and spatially resolve nine breast cancer-related miRNAs and evaluate their coordination.FluoELs provide a singlecell analysis platform to evaluate miRNA expression profiles and the molecular mechanisms underlying miRNA-associated diseases.2.Multiplexed organelles portrait barcodes for subcellular miRNAs array detection in living cellsDirect visualization and monitoring the change of multiple miRNAs at subcellular localization in the living cells would enhance the understanding of their spatio-temporal function information.However,it remains a key challenge owing to the fluorescent spectral overlap.Here,we introduce the Multiplexed Organelles Portrait Barcodes(MOPB)that consisted of heterochromatic fluorescent dye encoeded shell-core mesoporous silica nanoparticles modified with organelle targeting peptides and molecular beacons detection probe to achieve multiplexed miRNAs visualization in different organelles.Using this MOBP platform,we track as many as 8 miRNAs in mitochondria and endoplasmic reticulum,and their dynamic change are also monitored during the drug-induced Ca2+homeostasis broken down process.To the best of our knowledge,this is the first pioneering work for complex multipled miRNAs profiling in subcellular organelleof living cells.3.Orthometric multicolor encoded hybridization chain reaction amplifiers for multiplexed miRNAs profiling in living cellsMultiplexed fluorescent miRNA profiling for more than 4 types in living cells is challenging due to spectral overlap,representing a significant limitation to study the multiple regulatory elements interaction most related to occurrence and development of disease.We report a multiplexed fluorescent imaging strategy based on orthometric multicolor encoded hybridization chain reaction(HCR)amplifier named multi-HCR.This multi-HCR could self-assemble orthogonally due to the sequence specificity and programmability,allowing differentiation of 15 different combinations simultaneously by 4-colored chain amplifiers.It demonstrated excellent performance in monitoring 8 miRNAs’ changes relevant to the hypoxia-induced Mitochondria stress and endoplasmic reticulum stress process,leaving the apoptosis and autophagy fate in complicated cellular process.This multi-HCR provides a robust strategy for simultaneously profiling multiplexed biomarkers in living cells.