Abstract
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.
Highlights
In plants, 21- to 24-nucleotide, non-coding small RNAs regulate many important biological processes [1]
Plant and animal genomes encode various small RNAs (sRNAs) that can be divided into two major categories: microRNAs and small interfering RNAs [2]. miRNAs are derived from long, hairpin precursor RNAs processed by precise cleavage, yielding mature miRNAs which influence gene transcript levels and translation [3]
To perform sRNA-Points Accumulation for Imaging in Nanoscale Topography (PAINT), we designed a probe that is composed of three sequences: the probe backbone sequence with locked nucleic acid (LNA) bases, the DNA-PAINT docking strand sequence, and a linker sequence that connects those two (Figure 1)
Summary
21- to 24-nucleotide (nt), non-coding small RNAs (sRNAs) regulate many important biological processes [1]. Plant EVs are populated with miRNAs as well as with fragments as small as 10 to 17 nt, so-called tiny RNAs [2] All of these sRNAs vary in abundance from low to extremely high, as measured by sequencing, which typically utilizes gram quantities of plant tissue, a process that substantially limits spatial analyses. Nanometer resolution imaging for the localization and analysis of sRNAs, we created a detection method called sRNA-PAINT. This method combines the high resolution and precise quantification of DNA-PAINT with the efficiency and specificity of LNA-based in situ hybridization. We have demonstrated that it can be combined with qPAINT to localize and quantify distinct sRNAs in fixed biological sample, and with Exchange-PAINT for multiplexed target detection
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