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      Nanoscale Imaging of RNA with Expansion Microscopy

      Nature Methods

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          Abstract

          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 (ExM) 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, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) 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.

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          Most cited references21

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          Semiconductor nanocrystals as fluorescent biological labels.

          Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. Compared with conventional fluorophores, the nanocrystals have a narrow, tunable, symmetric emission spectrum and are photochemically stable. The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing fluorophores.
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            RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.

            Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~10(4) to 10(6) pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.
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              Dynamic DNA nanotechnology using strand-displacement reactions.

              The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. This has enabled the construction of a diverse and rapidly growing set of DNA nanostructures and nanodevices through the programmed hybridization of complementary strands. Although it had initially focused on the self-assembly of static structures, DNA nanotechnology is now also becoming increasingly attractive for engineering systems with interesting dynamic properties. Various devices, including circuits, catalytic amplifiers, autonomous molecular motors and reconfigurable nanostructures, have recently been rationally designed to use DNA strand-displacement reactions, in which two strands with partial or full complementarity hybridize, displacing in the process one or more pre-hybridized strands. This mechanism allows for the kinetic control of reaction pathways. Here, we review DNA strand-displacement-based devices, and look at how this relatively simple mechanism can lead to a surprising diversity of dynamic behaviour.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                27 May 2016
                04 July 2016
                August 2016
                04 January 2017
                : 13
                : 8
                : 679-684
                Affiliations
                [1 ]Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [2 ]Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [3 ]McGovern Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [4 ]Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA
                [5 ]Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [6 ]Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA
                [7 ]Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA
                [8 ]Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
                [9 ]Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                Author notes
                Correspondence should be addressed to E.S.B ( esb@ 123456media.mit.edu )
                [10]

                These authors contributed equally to this work.

                Article
                NIHMS790112
                10.1038/nmeth.3899
                4965288
                27376770
                72a8a4b2-6ef5-45af-afd4-49f6fbbf7812

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