In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT

Abstract

OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila).

Keywords: Chromatin; Chromosomes; DNA-PAINT; FISH; Genome; OligoDNA-PAINT; OligoSTORM; Oligopaint; STORM; Single-molecule; Super-resolution.

Fig. 1

Implementation of OligoSTORM and OligoDNA-PAINT using Oligopaints. A) The Mainstreets and Backstreets of Oligopaint oligos consist of nongenomic sequences that can be used for a variety of purposes. For example, inclusion of primer sequences enables oligo amplification, including selective amplification of subsets of the oligo library. B) Oligopaint oligos can be directly labeled with a fluorophore (red circle) via amplification with primers conjugated with a fluorophore. C) Oligopaint oligos can be indirectly labeled via hybridization of labeled secondary oligos to Mainstreet and/or Backstreet. D-F) OligoSTORM can be achieved via a variety of strategies, including labeling Oligopaint oligos (D) or secondary oligos (E) with a photoswitchable reporter (red) or pairing an activator (yellow) conjugated to Mainstreet or Backstreet with a reporter (red) that is carried by a secondary oligo hybridized to Mainstreet or Backstreet (F). G) OligoDNA-PAINT can be achieved via the repeated, transient binding to Mainstreet or Backstreet of short imager strands conjugated with a fluorophore.