Development Of Fluorescence Methods For CircRNAs Assay

Circular ribonucleic acids(circRNAs)are a novel class of endogenous noncoding RNAs biomolecules in eukaryotes that form covalently closed loop structures,without a 5’cap or a3’Poly A tail,are formed via backsplicing,and are predominantly localized in the cytoplasm or stored in exosomes.CircRNAs have the characteristics of high abundance,specificity,highly conserved and stability in cells,making them a hot topic in the medical field for developing disease diagnostic biomarkers and researching drug treatment targets.Recent studies have shown that circRNAs function as competing endogenous RNAs or micro RNA sponges that regulate transcription and splicing,binding to proteins,and translation.Moreover,circRNAs have become a valuable biomarker in biomedical research and clinical diagnosis,which is conducive to the development of new circRNA therapeutic targets and potential circRNA vaccines.It is critical to develop highly sensitive,more specific,label-free circRNA detection methods.Traditional methods for detecting circRNA mainly include circRNA sequencing,microarray analysis,Northern blotting,and RT-q PCR.However,they have drawback of sophisticated probe design,low sensitivity,lengthy operation time,poor specificity limit their wide applications.To address these issues,a number of novel circRNA assays have recently emerged,such as fluorescence analysis,electrochemical analysis,single molecule detection,fluorescence in situ hybridization.These methods increase the sensitivity and specificity of circRNA detection.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.Such as rolling circle amplification,hybridization chain reaction,loop-mediated isothermal amplification,exonucleases-assisted cycling enzymatic amplification,catalytic hairpin assembly.In this theresis,we develop a variety of fluorescence biosensors based on fluorescence detection technology of nucleic acid amplification,combined with advanced biochemical analysis methods for the quantiffcation of circRNA with high sensitivity and selectivity.Specific research contents include:1.We construct a label-free fluorescent biosensor for ultrasensitive analysis of circRNAs based on the integration of target-initiated cascade signal amplification strategy with a light-up G-quadruplex.This assay involves only one assistant probe that targets the circRNA-specific back-splice junction.When circRNA is present,it hybridizes with the assistant probe to initiate the duplex-specific nuclease(DSN)-catalyzed cyclic cleavage reaction,producing abundant triggers with 3’-OH termini.Then,terminal deoxynucleotidyl transferase(Td T)catalyzes the addition of d GTP and d ATP at the 3’-OH termini of the resultant triggers to obtain abundant long G-rich DNA sequences that can form efficient G-quadruplex products.The addition of Thioflavin T(Th T)can light up G-quadruplex,generating an enhanced fluorescence.This assay may be performed isothermally without the involvement of any nucleic acid templates,exogenous primers,and specific labeled probes.Importantly,this biosensor can discriminate target circRNA from one-base mismatched circRNA and exhibits good performance in human serum.Moreover,it can accurately detect circRNA in cancer cells at a single-cell level and even differentiate the circRNA levels in the tissues of healthy persons and nonsmall cell lung cancer(NSCLC)patients,with promising applications in circRNA-related cancer diagnosis and therapeutics.2.We develop a new fluorescent biosensor with zero background for label-free and sensitive detection of circRNAs activity based on target-triggered helicase-dependent amplification(HDA).When circRNA is present,it hybridizes with the probe-1 and probe-2 to initiate the Splint R ligase-catalyzed proximity ligation reaction,producing abundant template for HDA amplification.Notably,the high amplification efficiency of HDA generates numerous double-stranded DNA products,which can be easily detected by SYBR Gold in a label-free manner.This assay can be carried out in a zero-background manner within 40 min under an isothermal condition(65 °C),which is the rapider and simpler method reported so far for the circMTO1 assay.Notably,this biosensor exhibits has the advantages of high sensitivity,high specificity,and high efficiency,and it can be further used to discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues.Moreover,it can determine circMTO1 at single-cell level and distinguish the expressions of circMTO1 in cell of normal persons and cancer cell,holding great promise in circRNA-related biological research and clinical diagnosis.3.We demonstrate the construction of label-free fluorescent G-quadruplex nanowires for label-free and accurate monitoring of circular RNAs in breast cancer cells and tissues by integrating proximity ligation-rolling circle amplification cascade with lighting up G-quadruplex.The presence of target circRNA facilitates the Splint R ligase-mediated ligation of padlock probe.Upon the addition of primers,the ligated padlock probe can serve as a template for the initiation of subsequent RCA,generating numerous long G-quadruplex nanowires that can incorporate with Thioflavin T(Th T)to generate a remarkably improved fluorescence signal.Benefiting from good specificity of Splint R ligase-mediated proximity ligation reaction and exponential amplification efficiency of RCA,this strategy can sensitively detect target circRNA with a limit of detection of as low as 4.65 a M.Furthermore,this method can accurately measure cellular circRNA expression with single-cell sensitivity,and discriminate the circRNA expression between healthy para-carcinoma tissues and breast cancer tissues,holding great potential in studying the pathological roles of circRNA and clinic diagnostics.4.We developed a labeling free dual-color light up RNA aptamers nanosensor,the integration of target initiates three-way junction(3WJ)structure-driven T7 transcription cascade amplification machinery with fluorescent light-up aptamers(FLAPs)biosensor for simultaneous measurement of different circRNAs in breast cancer(BC)tissues.The binding of target circRNAs to their corresponding 3WJ-template and 3WJ-primer initiates the polymerization amplification reaction,generating many double strands which can initiate the T7 transcription amplification for the production of more fluorescent light-up aptamers.Malachite green aptamer(MGA)and baby spinach aptamer can bind with fluorophores malachite green(MG)and((Z)-4-(3,5-difluoro 4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one)(DFHBI),respectively,to generate significantly enhanced fluorescence signals.The fluorescence signals can be simply counted by FLS1000,with MG fluorescence intensity indicating circMTO1 and DFHBI fluorescence intensity indicating circCDYL.This method can simultaneously quantify circMTO1 and circCDYL in living cells and BC cancer tissues,and it can distinguish the expression of circRNAs between BC patients and healthy persons.The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction.