Nanoscale Imaging of RNA with Expansion Microscopy

The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

[1]  Bin Wu,et al.  Single β-Actin mRNA Detection in Neurons Reveals a Mechanism for Regulating Its Translatability , 2014, Science.

[2]  J. Chang,et al.  Expansion microscopy , 2018, Journal of microscopy.

[3]  Jean-Marie Rouillard,et al.  Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes , 2012, Proceedings of the National Academy of Sciences.

[4]  E. Lander,et al.  The Xist lncRNA Exploits Three-Dimensional Genome Architecture to Spread Across the X Chromosome , 2013, Science.

[5]  Le A. Trinh,et al.  Programmable in situ amplification for multiplexed imaging of mRNA expression , 2010, Nature Biotechnology.

[6]  O. Steward,et al.  Compartmentalized Synthesis and Degradation of Proteins in Neurons , 2003, Neuron.

[7]  Long Cai,et al.  Single cell systems biology by super-resolution imaging and combinatorial labeling , 2012, Nature Methods.

[8]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[9]  R. Jaenisch,et al.  X Chromosome Inactivation Is Mediated by Xist RNA Stabilization , 1997, Cell.

[10]  Edward S. Boyden,et al.  Expansion microscopy , 2015, Science.

[11]  S. Nakagawa,et al.  Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy. , 2016, Methods.

[12]  Viviana Gradinaru,et al.  Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing , 2016, Development.

[13]  Sanjay Tyagi,et al.  Neuronal mRNAs travel singly into dendrites , 2012, Proceedings of the National Academy of Sciences.

[14]  Junhyong Kim,et al.  Cytoplasmic Intron Sequence-Retaining Transcripts Can Be Dendritically Targeted via ID Element Retrotransposons , 2011, Neuron.

[15]  George M. Church,et al.  Highly Multiplexed Subcellular RNA Sequencing in Situ , 2014, Science.

[16]  G. Seelig,et al.  Dynamic DNA nanotechnology using strand-displacement reactions. , 2011, Nature chemistry.

[17]  F. Del Bene,et al.  Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy , 2004, Science.

[18]  Arjun Raj,et al.  Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. , 2010, Methods in enzymology.

[19]  Timur Zhiyentayev,et al.  Single-cell in situ RNA profiling by sequential hybridization , 2014, Nature Methods.

[20]  D. Holdstock.  Past, present--and future? , 2005, Medicine, conflict, and survival.

[21]  John N. Hutchinson,et al.  An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. , 2009, Molecular cell.

[22]  F S Fay,et al.  Visualization of single RNA transcripts in situ. , 1998, Science.

[23]  Oswald Steward,et al.  Synaptic Activation Causes the mRNA for the IEG Arc to Localize Selectively near Activated Postsynaptic Sites on Dendrites , 1998, Neuron.

[24]  J. Rinn,et al.  Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution , 2015, Genome Biology.

[25]  K. Matyjaszewski,et al.  Bright Fluorescent Nanotags from Bottlebrush Polymers with DNA-Tipped Bristles , 2015, ACS central science.

[26]  Allan R. Jones,et al.  Genome-wide atlas of gene expression in the adult mouse brain , 2007, Nature.

[27]  J. Flanagan,et al.  RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. , 2012, The Journal of molecular diagnostics : JMD.

[28]  Erin M. Schuman,et al.  The Local Transcriptome in the Synaptic Neuropil Revealed by Deep Sequencing and High-Resolution Imaging , 2012, Neuron.

[29]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[30]  Carolina Wählby,et al.  In situ sequencing for RNA analysis in preserved tissue and cells , 2013, Nature Methods.

[31]  Joshua C Vaughan,et al.  Expansion microscopy with conventional antibodies and fluorescent proteins , 2016, Nature Methods.

[32]  X. Zhuang,et al.  Spatially resolved, highly multiplexed RNA profiling in single cells , 2015, Science.

[33]  T. Creighton.  Methods in Enzymology , 1968, The Yale Journal of Biology and Medicine.

[34]  Dmitri A. Nusinow,et al.  Xist RNA and the mechanism of X chromosome inactivation. , 2002, Annual review of genetics.

[35]  Robert H. Singer,et al.  Fluorescence in situ hybridization: past, present and future , 2003, Journal of Cell Science.

[36]  Byung C. Yoon,et al.  Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair , 2012, Nature Reviews Neuroscience.

[37]  D. Balding,et al.  HLA Sequence Polymorphism and the Origin of Humans , 2006 .

[38]  Niles A. Pierce,et al.  Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability , 2014, ACS nano.

[39]  Scott A. Rifkin,et al.  Imaging individual mRNA molecules using multiple singly labeled probes , 2008, Nature Methods.

[40]  Edward S Boyden,et al.  Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies , 2016, Nature Biotechnology.

[41]  Michael Unser,et al.  A pyramid approach to subpixel registration based on intensity , 1998, IEEE Trans. Image Process..

[42]  I. Amit,et al.  Comprehensive mapping of long range interactions reveals folding principles of the human genome , 2011 .