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WIREs Nanomed Nanobiotechnol
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Beyond quantification: in situ analysis of transcriptome and pre‐mRNA alternative splicing at the nanoscale

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In situ analysis offers a venue for dissecting the complex transcriptome in its natural context to tap into cellular processes that could explain the phenotypic physiology and pathology yet to be understood. Over the past decades, enormous progress has been made to improve the resolution, sensitivity, and specificity of single‐cell technologies. The continued efforts in RNA research not only facilitates mechanistic studies of molecular biology but also provides state‐of‐the‐art strategies for diagnostic purposes. The implementation of novel bio‐imaging platforms has yielded valuable information for inspecting gene expression, mapping regulatory networks, and classifying cell types. In this article, we discuss the merits and technical challenges in single‐molecule in situ RNA profiling. Advanced in situ hybridization methodologies developed for a variety of detection modalities are reviewed. Considering the fact that in mammalian cells the number of protein products immensely exceeds that of the actual coding genes due to pre‐mRNA alternative splicing, tools capable of elucidating this process in intact cells are highlighted. To conclude, we point out future directions for in situ transcriptome analysis and expect a plethora of opportunities and discoveries in this field. WIREs Nanomed Nanobiotechnol 2017, 9:e1443. doi: 10.1002/wnan.1443 This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Scope of this review article. (a) The lifecycle of mRNA. (b) A brief summary of the mainstream methods for RNA detection and the ISH techniques that are covered in this review.
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Schematic of the fluorescence in situ RNA‐Seq (FISSEQ). Cellular mRNAs are in situ reverse‐transcribed to cDNA in the presence of aminoallyl‐dUTP followed by cross‐linking to the cellular protein matrix. The cDNA fragments are then circulated and PCR amplified (rolling‐circle amplification). Thereafter, a ‘nano‐ball’ containing thousands of copies of the original mRNA template is formed and subjected to SOLiD sequencing. The imaging readouts are postprocessed for library construction and bioinformatics analysis. (Reprinted with permission from Ref . Copyright 2015 Nature Publishing Group)
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Proposed barcoding fluorescence in situ hybridization for detection of mRNA variants generated from alternative splicing. A simplified four‐exon gene is used for illustration. For a single‐color imaging strategy, individual exons can be separately imaged in a predetermined round of sequential hybridization. In comparison, the multiple‐color strategy enables identification of each exon in one‐time hybridization. Signal missing in a specific round or color channel would indicate the splicing out of the alternative exon.
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Simultaneous detection of numerous RNA species in single cells by multiplexed error‐robust fluorescence in situ hybridization (MERFISH). In the barcoding FISH technique, a target RNA sequence is assigned a unique signal pattern, which is a serial binary code of ‘1’ and ‘0’ in MERFISH achieved with sequential hybridization and imaging. Here, ‘1’ and ‘0’ represent whether or not a fluorescence signal is detected from the target RNA in a specific round of hybridization. In theory, with N rounds of hybridization, 2N‐1 RNA species can be artificially barcoded. Data obtained from barcoding FISH not only enable the inspection of the multigene expression networks but also provide the spatial information of each transcript. (Reprinted with permission from Ref . Copyright 2015 The American Association for the Advancement of Science)
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Raman in situ hybridization for multiplex detection of BRCA1 variants. The capturing strands (CS) are immobilized to gold‐coated glass slide, and the probing strands (PS) are conjugated to 40 nm gold nanoparticles (AuNPs) that have Raman tags. A sandwich complex is formed in the presence of a target variant (TS) and the Raman tag gives out strong surface enhanced spectroscopy signals due to the local field enhancement between AuNPs and the gold substrate. Using different Raman tags, up to four BRCA1 variants could be simultaneously quantified at the fM level. (Reprinted with permission from Ref . Copyright 2008 American Chemical Society)
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Plasmonic in situ hybridization for real‐time monitoring of spliced BRCA1 variants. (a) Gold nanoparticle (AuNP)‐based probes flanking alternative splicing sites of BRCA1 are microinjected into living cells. Upon hybridization, the probe pair would form dimer structures with distinct interparticle distances depending on the inclusion/removal of the alternative exons. (b) The plasmon peak of AuNPs is sensitive to the interparticle distance and would shift to longer wavelengths when the probe pair is close enough. As demonstrated, a pair of probes respectively targeting exons 8 and 12 can be used to monitor the BRCA1 variants of ∆(11q) and ∆(9, 10, 11q). Deletion of exon 11q places the probe pair adjacent and the plasmonic coupling moves the plasmon peak from 538 to 584 nm, while an additional deletion of exons 8 and 9 further moves the plasmon peak to 612 nm. All these characteristic changes can be imaged with hyperspectral dark‐field microscopy. (c) With this method, three BRCA1 variants were quantified in MCF‐7 and MDA‐MB‐231 breast cancer cells that were either asynchronous or synchronized to the G1/S boundary in cell cycle. (Reprinted with permission from Ref . Copyright 2014 Nature Publishing Group)
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Gold nanoparticle (AuNP)‐based molecular beacons. (a) Hairpin DNA is attached to AuNP surface through a hexane thiol linker. In the absence of target mRNA, the probe sequence forms a self‐complementary hairpin structure and the fluorescent dye is quenched in the proximity of AuNP. (Reprinted with permission from Ref . Copyright 2015 Springer) (b) In a similar design, 13‐nm‐sized AuNPs are conjugated with fluorescent ‘flare’ DNA, dubbed nanoflares. Before hybridization to its target, the ‘flare’ DNA is stuck on the AuNP due to a weak base‐paring with the surface DNA layer. In the presence of target mRNA, the ‘flare’ DNA is released to bind with the target where more bases are matched. As a proof‐of‐concept, the β‐actin mRNA was imaged. (Reprinted with permission from Ref . Copyright 2015 National Academy of Sciences)
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Concept of transient absorption microscopy in situ hybridization (TAM‐ISH) for background‐free RNA imaging. (a) The scheme of improving signal‐to‐background ratio in single cells. (b) Energy diagram of the transient absorption (left) and stimulated Raman scattering (right) processes. DOS, density of states. (c) Background‐free TAM imaging of gold nanoparticles (AuNPs). Without a temporal delay between the pump and probe pulses, cells can be imaged at 2950 cm−1 by SRS while AuNPs can be imaged by TAM. With a temporal delay of 2 ps, only the TAM signal from AuNPs is retained. (Reprinted with permission from Ref . Copyright 2016 Royal Society of Chemistry)
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Detection of human epidermal growth factor receptor 2 (HER2) mRNA at the single‐copy resolution by second‐harmonic generation in situ hybridization (SHG‐ISH). (a) Formation of a dimer structure is required to correctly identify a target mRNA. (b) Implementation of SHG‐ISH in three cell lines with different HER2 expression. Compared to regular second‐harmonic imaging microscopy (SHIM), the developed SHaSM could better resolve the clustered HER2 mRNAs within a diffraction‐limited spot. (Reprinted with permission from Ref . Copyright 2014 American Chemical Society)
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Concept and working mechanism of the second‐harmonic super‐resolution microscopy (SHaSM). (a) Barium titanium oxide (BTO) nanocrystals show heterogeneous second‐harmonic generation (SHG) signals under polarized illumination. (b) The simulated and measured SHG emission from a single BTO nanocrystal confirmed its polarization‐dependent property. (c) In SHaSM, by varying the polarization of excitation the centroid of each BTO can be precisely located. (Reprinted with permission from Ref . Copyright 2014 American Chemical Society)
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