Title: Metabolic RNA Labeling-Enabled Time-Resolved Single-Cell RNA Sequencing

Authors: Kun Yin, Shichao Lin*, Chaoyong Yang*

Abstract: Gene expression of cells is a highly heterogeneous and dynamic program that changes over time in various biological processes such as embryogenesis, disease progression, and response to stimuli. Understanding the molecular mechanisms of heterogeneous and dynamic gene expression is crucial for advancing our knowledge of health and disease. The recent development of single-cell RNA sequencing (scRNA-seq) technologies has offered a great opportunity to dissect cellular heterogeneity by profiling the transcriptomes of individual cells. However, scRNA-seq captures only static snapshots of gene expression and fails to temporally resolve the RNA dynamics. Therefore, the rapid changes in transcription, the coordinated regulation of RNA synthesis and degradation rates, and the cellular interactions driving cell fate decisions remain poorly understood. In the past few years, metabolic RNA labeling-based scRNA-seq has emerged as a cutting-edge chemical tool to tackle these challenges. Nucleoside analogs are applied to label newly transcribed RNAs and distinguish them from pre-existing RNAs. This time-resolved technology unbiasedly captures the true RNA dynamics for thousands of genes in each of the individual cells, providing unprecedented insight into the regulation of heterogeneous and dynamic gene expression in diverse biological processes.In this Account, we highlight the recent advances achieved by our group and other laboratories in metabolic RNA labeling-enabled time-resolved scRNA-seq. First, we summarize the recent development of time-resolved scRNA-seq by integrating metabolic RNA labeling (e.g., 4-thioridine labeling) with various scRNA-seq platforms. We highlight our size-exclusion and locally quasi-static hydrodynamics-based Well-TEMP-seq method, which greatly improves the performance of time-resolved scRNA-seq (higher throughput, higher cell barcoding efficiency, and RNA recovery rate) and lowers the cost. Next, we extend the labeling strategy from single nucleoside labeling to double nucleoside labeling and develop scDUAL-seq The sequential (pulse-pulse) labeling by two different nucleosides in scDUAL-seq addresses the limitation of single nucleoside labeling in the simultaneous monitoring of RNA synthesis and degradation processes and accurate measurement of RNA kinetics. The ability of scDUAL-seq to discriminate between different cell states also allows the unveiling of the interplay between RNA synthesis and degradation that controls distinct RNA regulatory strategy transitions during dynamic processes. Then, we discuss the further development of in vivo metabolic RNA labeling-based scRNA-seq by our laboratory (Dyna-vivo-seq) and others, which advances the time-resolved scRNA-seq studies from cultured cells to animal models. This innovation opens new avenues to reveal single-cell RNA dynamics in living organisms. Finally, we introduce our attempts to integrate time-resolved scRNA-seq with spatial transcriptomics, adding a spatial dimension to temporal RNA dynamics. This new paradigm allows the dissection of the spatiotemporal regulation of gene expression and cell fate decisions through cell-cell interactions in the tissue microenvironment, which holds great promise for biomedical applications.Our perspectives on the current limitations of the chemical tools for single-cell RNA dynamics profiling and the future directions for improvement are also provided. We anticipate that this Account will inspire chemists to develop advanced chemical tools to profile the heterogeneous and dynamic gene expression programs and offer transformative insights into the molecular landscape of RNA dynamics in health and disease.

Full-Link: https://pubs.acs.org/doi/10.1021/acs.accounts.6c00010