Quantitative, super-resolution localization of small RNAs with sRNA-PAINT
Quantitative, super-resolution localization of small RNAs with sRNA-PAINT
复制标题
用sRNA-PAINT对小RNA进行定量、超分辨率定位
DOI：
10.1093/nar/gkaa623
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发表时间：
2020-09-18
影响因子：
14.9
通讯作者：
Caplan, Jeffrey L.
中科院分区：
文献类型：
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作者：
Huang, Kun;Demirci, Feray;Caplan, Jeffrey L.
小分子RNA是一类非编码RNA，在动物和植物的生命中发挥着重要作用。它们的长度为21- 24-nt，大小约为10 nm。它们的小尺寸和高多样性使得开发具有足够分辨率和特异性的检测方法以进行多重和定量具有挑战性。我们创建了一种方法，sRNA-PAINT，通过结合超分辨率方法，基于DNA的纳米级拓扑学点积累（DNA-PAINT），以及用于原位检测多个小RNA的锁核酸（LNA）探针的特异性，用于检测20 nm分辨率的小RNA。该方法依赖于设计探针以靶向小RNA，所述小RNA将用于PAINT的联合收割机DNA寡核苷酸（oligos）与用于杂交的含LNA的寡核苷酸组合;因此，我们开发了一种用于探针设计的在线工具，称为“用于原位杂交探针的RNA的审查和分析”（VARNISH）。我们的方法利用了DNA-PAINT方法的进步，包括用于定量的qPAINT和用于多路复用的Exchange-PAINT。我们通过检测和定量早期发育阶段玉米花药不同细胞层中对雄性有性生殖重要的小RNA来证明sRNA-PAINT的这些能力。
Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and similar to 10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called `Vetting & Analysis of RNA for in situ Hybridization probes' (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.