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 ∼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.

中文翻译：

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使用sRNA-PAINT对小RNA进行定量，超分辨率的定位。

小RNA是非编码RNA，在动植物的生活中都起着重要作用。它们的长度为21至24 nt，尺寸约为10 nm。它们的小尺寸和高多样性使开发具有足够分辨率和特异性以进行多路复用和量化的检测方法面临挑战。通过结合超分辨率方法，纳米级地形中基于DNA的点积累（DNA-PAINT）和锁定核酸的特异性（LNA），我们创建了一种用于检测20 nm分辨率小RNA的方法sRNA-PAINT。 ）原位探针检测多个小RNA。该方法依赖于设计探针以靶向小RNA，这些小RNA将用于PAINT的DNA寡核苷酸（寡核苷酸）与包含LNA的寡核苷酸结合进行杂交；因此，我们开发了一种在线工具，称为“用于原位杂交探针的RNA检验和分析”（VARNISH），用于探针设计。我们的方法利用了DNA-PAINT方法的先进性，包括用于定量的qPAINT和用于复用的Exchange-PAINT。我们通过检测和定量玉米花药早期不同细胞层中的小RNA展示了sRNA-PAINT的这些功能，这对于雄性有性繁殖很重要。