A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy

J Am Chem Soc. 2021 Sep 1;143(34):13782-13789. doi: 10.1021/jacs.1c05931. Epub 2021 Aug 23.

Abstract

Expansion microscopy (ExM) enables the nanoscale imaging of ribonucleic acids (RNAs) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents that allow the labeling and grafting of deoxyribonucleic acid (DNA) oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation in situ hybridization chain reaction RNA FISH (fluorescence in situ hybridization) techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multicolor staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ∼50 nm in ×10 ExM for both pre- and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable the rapid functionalization of DNA oligonucleotides for ExM.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry
  • Fluorescent Dyes / chemistry
  • Gene Expression
  • HeLa Cells
  • Humans
  • In Situ Hybridization, Fluorescence
  • Microscopy, Fluorescence
  • Nucleic Acids / chemistry*
  • Oligonucleotides / chemistry
  • Phosphoglycerate Kinase / genetics
  • Phosphoglycerate Kinase / metabolism
  • Staining and Labeling / methods*

Substances

  • Antibodies
  • Fluorescent Dyes
  • Nucleic Acids
  • Oligonucleotides
  • PGK1 protein, human
  • Phosphoglycerate Kinase