| Autor: |
Machyna M; Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.; Chemical Biology Institute, Yale University, West Haven, CT, USA., Kiefer L; Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.; Chemical Biology Institute, Yale University, West Haven, CT, USA., Simon MD; Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA. matthew.simon@yale.edu.; Chemical Biology Institute, Yale University, West Haven, CT, USA. matthew.simon@yale.edu. |
| Abstrakt: |
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. |
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