Strudies Of Fluorescent Biosensing And Living Cell Imaging Methods For Cancer-related MicroRNA

MicroRNAs(miRNAs)are a kind of noncoding endogenous RNA molecules found in eukaryotic cells and consist of 18-24 nucleotides.miRNAs regulate the gene expression by degrading target mRNA or repressing its post-transcriptional translation.miRNAs can regulate numerous biological processes,such as early development,immune response,hematopoiesis,cell differentiation,proliferation and apoptosis.Furthermore,increasing studies indicate that the abnormal expression of miRNAs is closely correlated with the initiation and progression of cancer.miRNAs have been used as important cancer diagnostic biomarkers and therapeutic targets.Therefore,specific and sensitive detection of miRNAs is crucial for diagnosis and therapy of cancer.miRNA has some intrinsic properties,such as short size,low-level expression,high-homology sequence among family members and vulnerable degradability.Based on this,some nucleic acid amplification strategies in vitro or in cells have been proposed.However,some limitations and challenges still remain in these strategies.1)Existing miRNA identification models can only play a role of identification in a limited functional region and can not discriminate the high-homology sequences,whose mismatched bases locate in varied positions;2)The enzyme-based signal amplification methods proposed for intracellular miRNA detection require the delivery of enzymes into cells.However,delivery of enzymes into cells requires cell fixation and dehydration,which might result in cell damage or death and could not satisfy the needs of miRNA detection in living cells;3)At present,the detection of miRNAs is mostly single detection.The detection of multiple miRNAs is vital for the accurate diagnosis and therapy of cancer;4)How to use miRNA as logical input to build logical platform to achieve cancer risk assessment and identification of different phenotypic cancer cells?In view of the above limitations and challenges,we have developed four strategies for biosensing detection and live cell imaging of miRNA as well as cancer risk assessment and cancer cell differentiation.The main contents are as follows:Chapter one is the introduction section,which mainly summarizes the characteristics,mechanism of action,biological significance and existing detection methods of miRNA.In addition,the limitations of existing detection methods are also summarized.In chapter two,a split recognition mode combined with cascade signal amplification strategy was developed for highly specific and sensitive detection of miRNA.Two recognition probes,hairpin probe(HP)with overhanging toehold domain and assistant probe(AP),were specially designed.Firstly,the toehold domain of HP and AP recognized part of miRNA simultaneously,accompanied with toehold-mediated strand displacement reaction to unfold the HP and form the stable DNA Y-shaped junction structure(YJS).Next,the AP in YJS could further act as primer to initiate the strand displacement amplification(SDA)reactions,which released a great number of trigger sequences.Finally,the trigger sequences hybridized with the padlock DNA which was specially designed with G-quadruplex complementary sequence and part of nicking site for Nt.BbvCI nicking endonuclease cleavage to initiate the circular rolling circle amplification(RCA)reaction and produced numerous G-quadruplex sequences.The G-quadruplex sequences could bind with N-methyl mesoporphyrin IX(NMM)to generate enhanced fluorescence responses for miRNA detection.In this strategy,the recognition sequence of miRNA was split on the two different function probes.No matter which position of the mismatched bases located in,the synergetic effect of rate changes of toehold-mediated strand displacement reaction and thermodynamic energies changes of direct hybridization made the YJS not be formed.The dual recognition effect of split recognition mode guaranteed the excellent selectivity to discriminate let-7b from high-homology sequences.Furthermore,owing to the high amplification efficiency of cascade signal amplification,this strategy could detect let-7b with a detection limit of 3.2 pM and the content of let-7b in total RNA sample extracted from HeLa cells was determined.These results indicated that this strategy would be a promising miRNA detection strategy in clinical diagnosis and disease treatment.In chapter three,an enzyme-free and label-free fluorescence biosensor based on cascade amplification of DNAzyme-powered 3-D DNA walker and hybridization chain reaction was developed for miRNA detection.Firstly,the walking probe hybridized with locking DNA to lock the DNAzyme designed in walking probe and form the blocked walking probes(BWPs).Then,the hairpin DNA substrates(HDSs)and the BWPs were modified on the magnetic nanobeads.Next,the miRNA hybridized with the locking DNA and initiated a strand displacement reaction to release the locking DNA from the BWPs and expose the DNAzyme.Then,the walking probes automatically moved along the 3-D tracks and catalyzed the cleavage of HDSs,releasing abundant triggers.Finally,the released triggers obtained through magnetic separation could initiate the hybridization chain reaction to form integrated G-quadruplex sequences,which could be inserted with NMM and yielded amplified fluorescence response signal for miRNA detection.Compared with the 3-D DNA walker powered by protein enzyme,this DNAzyme-powered 3-D DNA walker was simpler and more stable.All the DNA components were conjugated on the same magnetic nanobead,which greatly increased their local concentrations and reaction efficiency.Furthermore,the magnetic nanobeads could be separated to achieve a low background.The high amplification efficiency of 3-D DNA walker and hybridization chain reaction guaranteed a satisfactory sensitivity toward let-7a w ith a detection limit of 7.9 fM.Moreover,the content of let-7a in total RNA sample extracted from HeLa cells was determined.These results indicated that this biosensor offered an alternative strategy for the quantification of miRNAIn chapter four,a pH-responsive ZnO nanoprobe mediated DNAzyme signal amplification strategy was proposed for multiple miRNAs sensitive detection and imaging in living cells.The nanoprobe including ZnO nanoparticles(ZnO NPs)core,polydopamine(PDA)shell for adsorption the functional hairpin DNAs was designed PDA deposited on the surface of ZnO NPs by self-polymerization process under alkaline conditions.Hairpin DNAs were adsorbed on the PDA shell by ?-?interactions.When nanoprobe entered the cell through endocytosis,the acidic environment of the cell could decompose the ZnO NPs core to release the functional hairpin DNAs and Zn2+.The Zn2+ could act as cofactor for the DNAzyme cleavage amplification reaction,avoiding additional cell delivery processes.The recognition hairpin DNA contained the recognition sequence of the target miRNA and the DNAzyme,which was caged in the loop of recognition hairpin DNA.The reporter hairpin DNA contained the DNAzyme substrate and end-labeled fluorophore and quencher.The recognition hairpin DNA recognized the target miRNA and exposed the DNAzyme.Then,the exposed DNAzyme cleaved the substrate portion of the reporter hairpin DNA in the presence of Zn2+,causing the fluorophore and the quencher to move away from each other and the fluorescence recovery.The released hybrid double-strand of recognition hairpin DNA and miRNA continued to cleave the reporter hairpin DNA,producing enhanced fluorescent signal for sensitive detection of miR-21 and miR-373 with the detection limit of 54 pM and 38 pM,respectively.Different levels of miR-21 expression in three types of cells were distinguished.Furthermore,simultaneous imaging of miR-21 and miR-373 in same living cells was achieved.These results indicated that this strategy could have a potential application in miRNAs multiple assays for the accurate diagnosis and therapy of cancer.In chapter five,based on the MNAzyme cleavage amplification reaction,a DNA logic platform was developed for cancer risk assessment and cancer cells differentiation.The MNAzyme was formed through cleaving DNAzyme at their catalytic core and adding target sensor arms to create two part enzymes,Part A and Part B.In the absence of miRNA,Part A and Part B existed alone and could not form a complete catalytic center.The substrate could not be cleaved,ensuring a low background signal.When miRNA was present,Part A and Part B recognized part of miRNA simultaneously to form a MNAzyme complex.The MNAzyme complex could bind the substrate and cleave cyclically the substrate in the presence of Mn2+,producing enhanced fluorescent signal for cancer risk assessment and cancer cells differentiation.When this strategy was applied to the identification of cancer cells,MnO2 nanosheets were used as carriers for probe entry because the metal ions in the cells were insufficient to catalyze the MNAzyme cleavage amplification reaction.MnO2 nanosheets could be degraded under the intracellular glutathione(GSH)to produce Mn2+,which could be used as cofactor to catalyze the MNAzyme cleavage amplification reaction.In this strategy,the YES gates,AND gates,XOR gates and NOR gates were constructed for cancer risk assessment and cancer cells differentiation.These results indicated that this platform could provide an alternative logic gate construction method for cancer risk assessment and cancer cells differentiation.Chapter six is the conclusion section,which summarizes the innovation and prospect of this paper.