Enhanced nucleotide chemistry and toehold nanotechnology reveals lncRNA spreading on chromatin

Nat Struct Mol Biol. 2020 Mar;27(3):297-304. doi: 10.1038/s41594-020-0390-z. Epub 2020 Mar 10.

Abstract

Understanding the targeting and spreading patterns of long non-coding RNAs (lncRNAs) on chromatin requires a technique that can detect both high-intensity binding sites and reveal genome-wide changes in spreading patterns with high precision and confidence. Here we determine lncRNA localization using biotinylated locked nucleic acid (LNA)-containing oligonucleotides with toehold architecture capable of hybridizing to target RNA through strand-exchange reaction. During hybridization, a protecting strand competitively displaces contaminating species, leading to highly specific RNA capture of individual RNAs. Analysis of Drosophila roX2 lncRNA using this approach revealed that heat shock, unlike the unfolded protein response, leads to reduced spreading of roX2 on the X chromosome, but surprisingly also to relocalization to sites on autosomes. Our results demonstrate that this improved hybridization capture approach can reveal previously uncharacterized changes in the targeting and spreading of lncRNAs on chromatin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Pairing
  • Binding Sites
  • Biotinylation
  • Chromatin / chemistry*
  • Chromatin / metabolism
  • Chromosome Mapping
  • Chromosomes, Insect / chemistry
  • Chromosomes, Insect / metabolism
  • Drosophila Proteins / genetics*
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / metabolism
  • Heat-Shock Response
  • Nanotechnology / methods
  • Nucleic Acid Hybridization
  • Oligonucleotides / chemistry*
  • Oligonucleotides / metabolism
  • RNA, Long Noncoding / chemistry*
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • X Chromosome / chemistry*
  • X Chromosome / metabolism

Substances

  • Chromatin
  • Drosophila Proteins
  • Oligonucleotides
  • Pabp2 protein, Drosophila
  • RNA, Long Noncoding
  • RNA-Binding Proteins
  • locked nucleic acid