Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA.
Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA.
Nucleic Acids Res. 2020 Sep 18;48(16):e96. doi: 10.1093/nar/gkaa623.
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.
小 RNA 是一种非编码 RNA，在动植物的生命中发挥着重要作用。它们的长度为 21-24 个核苷酸，大小约为 10nm。由于其体积小、多样性高，因此开发具有足够分辨率和特异性的检测方法来进行多重和定量分析具有挑战性。我们创建了一种方法 sRNA-PAINT，通过将超分辨率方法 DNA 为基础的点在纳米级形貌中的积累（DNA-PAINT）与锁定核酸（LNA）探针的特异性相结合，来检测小 RNA，分辨率为 20nm。该方法依赖于设计针对小 RNA 的探针，这些探针将用于 PAINT 的 DNA 寡核苷酸（oligo）与用于杂交的含 LNA 的 oligo 结合；因此，我们开发了一个名为“用于原位杂交探针的 RNA 筛选和分析”（VARNISH）的在线工具用于探针设计。我们的方法利用了 DNA-PAINT 方法学的进展，包括用于定量的 qPAINT 和用于多重检测的 Exchange-PAINT。我们通过检测和定量早期发育阶段玉米花药中不同细胞层的小 RNA 来证明 sRNA-PAINT 的这些能力，这些小 RNA 对雄性生殖至关重要。
