Single-cell RNA Profiling Based On Nucleic Acid Isothermal Amplification

Cell is the basic structural and functional unit of organism.As every cell is unique,the development of analysis tools for studying single-cell transcriptome is vital for investigating the gene regulatory network,the heterogeneity of cell behavior and the complexity of disease development.In the present dissertation,we focused on developing single-cell RNA imaging methods by using isothermal nucleic acid amplification techniques,enabling highly multiplexed in situ detecting RNAs with single-molecule and single-base resolution.These methods will be single-cell analysis for studying the mechanism of gene transcription and transcription regulation process.The main sections of this dissertation are as follows:1.MiRNAs are non-coding RNAs that play crucial roles in regulating gene expression.The abnormal expression of miRNAs may associate with serious diseases,such as cancers,cardiovascular disease,and neurological disorders.Visualizing intracellular miRNAs is challenging because of their small size,sequence homology and sometimes low-expression.By designing a structure-switchable dumbbell probe,a toehold-initiated strand-displacement process was used to initiate rolling circle amplification of specific miRNAs,an approach that achieves both stringent recognition and in situ amplification of the target miRNA.Compared to the padlock probe based method,this assay,termed toehold-initiated rolling circle amplification(TIRCA)possesses higher specificity,which can be utilized to identify miRNAs let-7a,let-7f and let-7c with single-base difference in the same family.The single-cell miRNA profiling data clearly shows that the genetically identical cells exhibit significant cell-to-cell variation in miRNA expression,indicating that single-cell analysis is important to study the cell behaviors and functions.2.We developed a robust method for the direct detection of mRNA termed target RNA-initiated rolling circle amplification,enabling imaging of mRNA with single-molecule and single-nucleotide resolution in single cells.By utilizing a Splint R ligase capable of efficiently catalyzing the ligation of padlock probe by the target RNA,the method can enable efficiently detecting mRNA without reverse transcription(detection efficiency over 20%).Meanwhile,attributed to the ligation-based recognition process,the method confers specificity sufficient to genotype mRNAs with one-nucleotide variations,thus could be applied for analyzing SNPs.Single-cell mRNA imaging data show that mRNAs with different functions may represent with distinct spatial patterns,and genes with relevant functions may express coordinately.The method has enabled spatial mapping and correlation analysis of gene expression in single cells which could help us to elucidate the gene functions and regulatory mechanisms.This method offers mRNA profiling ability with high spatial resolution and sequence specificity,thus is expected to be a single-cell analysis platform for not only investigating gene expression,but also potential for analyzing single-nucleotide variants or mRNA alternative splicing at single-cell level.3.The multiplexing ability for RNA profiling by fluorescence imaging is limited by the number of spectrally distinct fluorophores.Considering the sequence programmability and well-understood hybridization thermodynamics,we developed a DNA sequence-encoded fluorescence barcoding method,termed sequence-encoded amplicon(SeqEA),enabling highly multiplexed imaging of RNAs in single cells with single-molecule resolution.SeqEA confers mutiplexed RNA profiling ability with single-molecule and single-nucleotide resolution.Thus it is expected to be a single-cell analysis technique both for investigating the gene expression regulatory network,and parallelly analyzing single nucleotide sequence variants or alternative splicing at single-cell level.