Novel Nanoflares For MiRNA Analysis In Tumor Cells

The ability to visualize and detect biomolecules in tumor cells is of great significance for advancement in molecular biology,early disease diagnosis and furt her understanding of cancer related signaling pathways.It not only requires the probe to pass through the cell membrane barrier,but also has a certain stability and good biocompatibility in the complex cell environment.Nanoflare s have been widely used in biomedical detection,disease diagnosis and treatment.However,due to complex intracellular environment and low abundance mi RNA in cytoplasm,it still faces some challenges in the detection of intracellular mi RNA with high accuracy and sensitivity.On the one hand,probe may be early-activated by non-targets,thus leading to non-specific signals and insufficient accuracy due to complex intracellular environment.On the other hand,when the abundance of intracellular mi RNA is very low,probes will face challenge owing to ?one-to-one?signal-triggered model(one target triggers one signal),which could lead to inadequate sensitivity.Therefore,in order to improve the accuracy and sensitivity of intracellular mi RNA detection based on nanoflares,a variety of novel nanoflares have been constructed by combination with multi-color imaging,ratiometric signal output,spatiotemporal control and m RNA-powered amplification technology for the analysis of intracellular mi RNA.The specific research contents are as follows:1.Two-color-based nanoflares for two mi RNA simultaneous imaging in colon cancer cellsDue to the insufficient accuracy of single target detection,two-color-based nanoflares have been developed for simultaneous detection of two mi RNA in live cells.Flare strands labeled with fluorophore FAM and Cy5 are hybridized with the corresponding recognition strands,respectively and functionalized on Au NP by Au-S bond.In this conformation,the close proximity of the fluorophore to the Au NP surface leads to quenching of the fluorescence.However,when target mi RNA bind to the corresponding recognition sequence,respectively and form long and stable duplex,flare strands will be released from Au NPs and significant fluorescence enhancement can be detected.Compared to the traditional single-color-based nanoflares,the two-color-based nanoflares could offer more reliable and practical information for cancer detection,improving the accuracy of early disease diagnosis.The confocal fluorescence imaging demonstrated that two-color-based nanoflares could simultaneously detect two mi RNA in colon cancer cells.2.2'-O-methyl modified nanoflares for specific mi RNA imaging and inhibition in breast cancer cellsInhibiting the function of mi RNA by anti-sense oligonucleotides(ASOs)can induce cells growth inhibition and apoptotic cells death.Here,a 2'-O-methyl modified nanoflare is created for specific mi RNA imaging and inhibition in breast cancer cells.ASOs are delivered into cells by Au NP and modified by 2'-O-methyl for improving stability and binding affinity.Au NPs are functionalized with anti-mi RNA modified by2'-O-methyl that are hybridized with FAM-labeled flare strand.Due to the close proximity of the FAM to the Au NP surface,its fluorescence is quenched.However,when a specific mi RNA binds to anti-mi RNA,on the one hand,the concomitant displacement of the flare can be detected as an increase in fluorescence for specific mi RNA imaging.On the other hand,the function of mi RNA can be supressed owing to the binding with anti-sense strand,achieving mi RNA imaging and inhibition.QRT PCR and MTT data showed that 2'-methyl modified ASOs could effectively enter cells and inhibit mi RNA expression,leading to cell death.3.Photocaged FRET nanoflares for intracellular mi RNA precise imagingThe initial activity of nanoflare above-mentioned is uncontrollable,so it will directly undergo reaction with target once they meet in transit during delivery or endocytosis,leading to unwanted early activation of the probe and generating nonspecific signals and low accuracy.Thus,a photocaged FRET nanoflare is constructed for mi RNA imaging in live cells.A short inhibitor strand containing a group of photocleavable linkers(PC-linkers)and a hairpin flare strand labelled with donors(Cy3)and acceptors(Cy5)are hyb ridized with recognition strand.Afterwards,they are modified on the surface of the Au NP.Flares are captured by binding with recognition,separating Cy3 and Cy5,resulting in a low FRET efficiency.Here,probes remain in an inert stage.However,upon UV irradiation at 365 nm,the PC-linkers are quickly cleaved and released two DNA fragments,thereby exposing a toehold domain to target mi RNA and flares are gradually displaced from recog nition strands to form stable hairpin structures that bright Cy3 and Cy5 into close proximity,resulting in a high FRET efficiency.The results of electrophoresis,fluorescence and confocal imaging experiments showed that only in the presence of light and mi RNA could generate significant FRET signal,which improves the detection accuracy.4.Endogenous m RNA-powered FRET nanoflares for intracellular mi RNA amplification imagingWhen the abundance of intracellular targets is very low,above-mentioned FRET nanoflare will face challenge owing to the ?one-to-one? signal-triggered model,which could lead to inadequate sensitivity.An endogenous m RNA-powered FRET nanoflare has been established for intracellular mi RNA imaging.After being triggered by mi RNA,it can autonomously work with the fueling of endogenous m RNA inside living cells without any auxiliary additives.In the presence of a small amount of mi RNA and a large number of m RNA,mi RNA is used to trigger the nanoflares to run then recycle as a catalyst and lots of m RNAs as fuels can release numerous intermediate s,which continuously recycle the mi RNA and generate multiple hairpins labeled with Cy3 and Cy5 from the Au NPs,resulting in significantly amplified FRET signals.In such a way,the ?one-to-more? signal amplification model is finally realized.It employs endogenous m RNA molecules as driving force to amplify the detection of low-abundance molecules,avoiding distributing system of exogenously addition and simplifying the operation procedures.In additio n,due to the participation of m RNA,the sensitivity is about 3 orders of magnitude higher than that of FRET nanoflares without amplification.5.Spatiotemporal control of m RNA-powered nanoflares for mi RNA imaging in live cellThe initial activity of amplified FRET nanoflares is not controllable at desirable time and localition,these probes may directly interact with targets once they meet in transit,leading to early activation and nonspecific signals and poor detection accuracy.A spatiotemporal control of m RNA-powered nanoflares is proposed for mi RNA imaging in live cells.The nanoflare is silent in the process of cell internalization.However,after entering the cell,it can be selectively activated by light irradiation at defined time point.In the presence of a small amount of mi RNA and a large number of m RNA,mi RNA is used to trigger the nanoflares to run and lots of m RNAs as fuels can continuously generate numerous intermediates,which are able to release and recycle mi RNA and further liberate multiple hairpins labeled with Cy3 and Cy5 from the Au NPs,resulting in significantly amplified FRET signals and achieving?one-to-more?signal amplification model.It employs illumination as external stimulus to spatiotemporally control over initial ac tivity of nanoflares,avoiding undesirable early-activation and reducing nonspecific signals,improving detection accuracy and spatiotemporal resolution.In addition,endogenous m RNA is employed as fuel strand to amplify signal,which detection limit could be as low as 3.5 pM.