Establishment Of π-FISH Fluorescence In Situ Hybridization Method And Its Potential Application

Pathogenic microorganisms such as bacteria,viruses,and parasites are the leading causes of animal diseases and deaths.The precise detection and molecular mechanism research of pathogenic microorganisms in animals will provide an essential basis for disease prevention and control.Although current biotechnology has a good detection effect on some pathogenic molecules,most microRNA,lncRNA,and splicing variants with important functions have poor detection effects or cannot be detected due to the lack of effective antibodies.In addition,single-cellRNA sequencing(scRNA-seq)technology has greatly promoted the analysis of cell functions in the interaction between pathogenic microorganisms and hosts.However,it lacks tissue microenvironment and spatial information,which hinders the interpretation of cell-cell and tissue structure-function information in pathogen-host interactions.Fluorescence in situ hybridization(FISH)can overcome these deficiencies and provide an effective means for pathogenic microorganisms’detection and molecular research.FISH can accurately quantify the nucleic acid copy number of pathogenic microorganisms and accurately detect the nucleic acid sequence lacking effective antibodies at the single-cell and single-molecule levels.It can also reveal the spatial information of the interaction between pathogenic microorganisms and host cells in situ.Although the development ofFISH technology has made great progress in recent years,there are still some limitations.There was no excellent detection effect for short nucleic acid sequences less than 1 kb.Biological molecules such asRNA,DNA,and proteins cannot be detected simultaneously.It still needs to be improved to achieve high signal intensity while maintaining low background noise.Therefore,multiplexπ-FISH andπ-FISH+methods were developed to overcome the above shortcomings.This method has the advantages of high hybridization efficiency,strong signal amplification ability,low background noise,and wide application range.It can detect different biomolecules(DNA,RNA,protein,and small molecule)individually or simultaneously and perform qualitative,quantitative,and localization analyses of long and short nucleic acid sequences.The main findings are as follows:1.Establishment of a novel fluorescence in situ hybridization method--π-FISHIn situ detection of nucleic acid sequences can be achieved by hybridization ofπtarget probe,secondary amplification probe,tertiary amplification probe,and fluorescence signal probe.In this study,the design of theπtarget probe makes its hybridization efficiency higher than that of the traditional split probe.The secondary and tertiary amplification probes of U-shaped have stronger signal amplification levels than the traditional L-shaped probes.The detection of high,medium,and low expression genes(ACTB,PPIA,B2M,and MTOR)was compared with other fluorescence in situ hybridization methods.The results showed thatπ-FISH is better than the current mainstream methods smFISH(single-molecular fluorescence in situ hybridization)and HCR(hybridization chain reaction).Hybridization efficiency,fluorescence intensity,and specificity are higher,and the background noise is lower.Furthermore,the background level ofπ-FISH in cells and tissues was verified with singleπtarget probes,noπtarget probe,RNase treatment,and nonspecificπtarget probes(bacterial dap B gene).By detecting lncRNA MALAT1 in He La cells,the two reported subcellular localization patterns were verified.The third localization pattern of MALAT1was detected for the first time in this study,indicating the efficiency and subcellular localization ability ofπ-FISH.The accuracy ofπ-FISH was verified by comparing the expression patterns of Cux2 and Pcp4 genes in mouse brain tissues as described by the Allen Institute for Brain Science(AIBS).The specificity of theπ-FISH signal was verified by detecting mutually exclusive neuronal marker genes.Finally,the reliability ofπ-FISH detection results was demonstrated by detecting single molecule colocalization betweenπ-FISH and HCR.2.Application ofπ-FISH in situ annotation of cell types and in situ verification after single-cell sequencingIn this study,we detected 21 neuronal marker genes in the same mouse brain tissue slice after two rounds of hybridization byπ-FISH.The spatial distribution of each sublayer of the S1 cortex was mapped according to the expression peaks of nine excitatory neuron marker genes,and the spatial patterns of L1,L2-3,L4,L5,and L6layers were reproduced,and L5a and L6b sublayers were accurately distinguished.Based on single-cell sequencing data,π-FISH in situ verified the spatial distribution of 13 Gad~+neuron subclasses in the S1 cortex.Among them,Int 9 and Int 10 subclasses were mainly distributed in the superficial layer(L2/L3),while Int 6 subclass was mainly distributed in the deep layer(L5-L6).3.Application ofπ-FISH in the detection of genomic loci and chromosomal variationIn this study,π-FISH was used to detect the repeats of telomeres and centromeres,and the proportion of polyploidy in He La cells was calculated.Single-channel and dual-channel detection of ACTB genomic loci,where the signals of both channels overlap,indicates the feasibility ofπ-FISH for multiplex DNA detection.In this study,the inversion translocation sites in DLO Hi-C sequencing data of THP-1 cells were verified in situ.The results showed thatπ-FISH could provide an effective method for detecting animal chromosomal diseases.4.Application ofπ-FISH in the detection of multiple proteins and small moleculesIn this study,various pathogenic proteins and corresponding regulatory mechanism proteins can be detected simultaneously using antibody-conjugated nucleic acid andπ-FISH detection of corresponding nucleic acid sequences,which is not limited by secondary antibodies.Compared with conventional immunofluorescence,π-FISH has a stronger signal,higher specificity,and lower background noise.Theπ-FISH assay also detected IFNG(interferon-gamma)-flag,CSFV(Classical swine fever virus)E2,PCV2(porcine circovirus 2)Cap and pol II(RNA)polymerase II,which delineated the expression pattern of these four proteins in a single cell.The distribution of inhibitory neurotransmittersγ-aminobutyric acid(GABA)and Neu N protein in mouse cerebral cortex was also detected byπ-FISH,indicating thatπ-FISH is effective in detecting proteins and small molecules in tissues.5.Application ofπ-FISH in the co-detection of DNA,RNA,and proteinsIn this study,co-π-FISH was a strategy for the co-detection of multiple biomolecules byπ-FISH.co-π-FISH demonstrated the co-localization of lncRNA NEAT1 and Pol II protein at MAPK15 genomic loci,indicating that it can co-detect DNA,RNA,and proteins,further demonstrating the potential ofπ-FISH technology in multi-omics studies.6.Application ofπ-FISH in the detection of short nucleic acid sequences--π-FISH+π-FISH+was developed by combiningπ-FISH with HCR technology in this study.Only oneπtarget probe could achieve ultra-high signal amplification.The principle ofπ-FISH+is as follows:Based onπ-FISH,the fluorescence signal probe is replaced with the HCR split-initiator probe and self-folding hairpin probe,and the other steps and sequences are unchanged.In this study,π-FISH+was used to verify the signal colocalization of mi R-145 and sponge lncRNA MALAT1 in situ,identify the ARV7splicing variant highly similar to other variants,and detect the deletion of short DNA sequence.7.Application ofπ-FISH in the detection of various pathogenic microorganismsIn animal husbandry,microorganisms such as bacteria,viruses,and parasites are harmful to animal health.Accurate detection and identification can avoid economic losses in the livestock industry as soon as possible.In this study,pathogens such as M.tuberculosis,African swine fever virus(ASFV),and T.gondii were detected.π-FISH identified M.tuberculosis 16S rRNA from cultured alone and infected THP1 cells,detected ASFV B646L gene expression in porcine alveolar macrophages,and mapped the distribution of T.gondii SAG1 and ROP2 genes.The results indicated thatπ-FISH technology could be widely used to detect pathogenic microorganisms such as bacteria,viruses,and parasites.