Project Summary/Abstract Each step-change in increasing resolution on RNA identification and quantification (i.e., transcriptome profiling) has been transformative to our understanding of cell biology. As the most recent technological revolution, the ability to map the spatial distribution of RNA molecules has revealed the importance of subcellular RNA localization, advanced our understanding of cell and developmental biology, and transformed the single cell transcriptomics field. However, despite the recent development, current RNA-mapping tools are limited to molecules with long specific sequence (mostly > 500 nt). Many other important RNA species (such as splicing isoforms, miRNA, and RNA editing), with much shorter specific sequence motif, remain inaccessible in situ. Our goal is to make this next resolution jump in profiling transcriptomics, i.e., RNA-mapping with high transcript- selectivity and single base-sensitivity. We plan to achieve the goal by developing a new tool, named SMOOTHY- FISH (Single-nucleotide specificity, Minimum Of Off-Target effects, and High Yield), which enables spatially mapping all types of RNA molecules in individual cells. In this proposal, we will demonstrate SMOOTHY-FISH from two aspects: technique development and one example application test. More specifically, for the technical development, we anticipate achieving a platform that can suggest robust and reliable SMOOTHY-FISH probe- sets to any assigned RNA target sequence, a user-friendly system that will be widely accessible. To achieve the goal, we will incorporate the unique power of magnetic tweezers (a single molecule tool with nanometer and milliseconds resolution) to systemically quantify the dynamics and kinetics of nucleic acid hybridization under various conditions, to reveal how fast and how stable the designed SMOOTHY-FISH probe-sets can form the needed secondary structure, and to build a mechanistic modeling framework to describe and predict the hybridization dynamic process. As for the example application test, we will use SMOOTHY-FISH to study the mystery biogenesis process of circular RNA with isoform-specificity (i.e., co-existence with its linear isoforms) for the first time. We anticipate revealing the potential to modulate circular RNA expression quantitatively and efficiently, i.e., lay a foundation for therapeutic strategies in the future. Together, this work will provide a next- generation tool for in situ RNA identification and quantification, allow us to localize and quantify many important, but currently inaccessible RNA species at the single molecule level in individual cells, thus help open a new era of RNA biology, from basic science research to clinical applications.