EAGER: qRNA-PAINT as a method for high-throughput, quantitative, single molecule analysis of cellular RNAs and their networks

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RNA molecules are used by cells to transfer and decode information stored in DNA. This includes messenger RNAs that encode data for making proteins and small RNAs that play important regulatory roles. Plant biologists continue to discover exciting, novel roles for small RNAs during plant growth, development, and responses to the environment. Like nearly all molecules in cells, the ability of small RNAs to carry out their functions depends on their location and quantity. However, due to their small size, it has been particularly challenging to detect and locate small RNAs. In plants, current methods can only detect one, or at most, two small RNAs at a time. This project develops a new technique called qRNA-PAINT to detect, quantify, and localize tens to hundreds of small RNAs in a single sample, simultaneously. qRNA-PAINT makes it possible to examine the complex relationships of small RNAs inside of a plant cell with super-resolution precision. Due to the wide-ranging role of small RNAs, this technique will have broad impacts on scientists seeking to answer fundamental biological questions in both plants and animals. Insights into the roles of small RNAs in plant signalling pathways may be translatable to increasing crop plant productivity and resistance to environmental stress and pathogens.In the current age of genomics, there is a wealth of expression data for small RNAs. However, methods to examine the subcellular, cellular, and even tissue level localization of small RNAs are limited. This has made it challenging to examine the relationship of different RNAs, such as miRNAs, siRNAs, mRNAs, and miRNA targets. This proposal will fully develop the qRNA-PAINT method to examine the relationship of tens to hundreds of different small RNAs. qRNA-PAINT is a modification of the exchange points accumulation for imaging in nanoscale topography (exchange-PAINT) method, which has been used to detect up to ten different protein targets. It accomplishes this by using the stochastic binding of dye labeled imager oligonucleotides to docking strands attached to antibodies. The qRNA-PAINT method does not use antibodies, but rather just locked nucleic acid (LNA) probes connected to the docking strands for exchange-PAINT. Consequently, it potentially can be massively multiplexed for numerous small RNAs, to examine signaling networks in single cells and tissue types. The maize anther will be used as the model system and qRNA-PAINT data will complement the already available extensive set of sequencing data. Furthermore, qRNA-PAINT will be combined with exchange-PAINT (for proteins) to examine the relative localization of protein machinery to study the biogenesis of different types of small RNAs. Ultimately, it will be used to answer outstanding questions on the spatio-temporal regulation of small RNA biogenesis and signaling networks.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.