Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.
Chemical Biology Institute, Yale University, West Haven, CT, USA.
Nat Struct Mol Biol. 2020 Mar;27(3):297-304. doi: 10.1038/s41594-020-0390-z. Epub 2020 Mar 10.
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.
理解长非编码 RNA（lncRNA）在染色质上的靶向和扩散模式需要一种能够同时检测高强度结合位点并以高精度和高可信度揭示全基因组扩散模式变化的技术。在这里，我们使用带有 toehold 结构的生物素化锁核酸（LNA）寡核苷酸来确定 lncRNA 的定位，该寡核苷酸能够通过链交换反应与靶 RNA 杂交。在杂交过程中，保护链竞争性地置换污染物，从而实现对单个 RNA 的高度特异性 RNA 捕获。使用这种方法分析果蝇 roX2 lncRNA 表明，与未折叠蛋白反应不同，热休克导致 roX2 在 X 染色体上的扩散减少，但令人惊讶的是，它也导致重新定位到常染色体上的位置。我们的结果表明，这种改进的杂交捕获方法可以揭示染色质上 lncRNA 靶向和扩散的以前未表征的变化。
