Intracellular Biopsy Based MicroRNA Profiling for Dissecting the Temporal Dynamics of Cellular Heterogeneity
DescriptionHeterogeneity is an important and omnipresent feature of biological systems, and is reflected by the spatial and temporal variation of genetic, epigenetic, proteomic and functional phenotypes in a living organism. As one of the key factors, microRNAs (miRNA) are identified to be non-coding small RNAs that substantially contribute to cellular heterogeneity by binding to specific mRNAs to regulate their degradation or translation. Despite the increasingly recognized potential of using multidimensional miRNA profile for diagnostic and therapeutic applications, techniques for in-situ measurement of miRNAs in live cells are still limited.Towards this end, we aims to develop a novel and revolutionary miRNA profiling technique (inCell-biopsy) which is based on intracellular biopsy from live cells and is capable of dissecting the temporal dynamics of cellular heterogeneity. Technically, the platform will be grounded on the continuous development of a biosensing system using an array of diamond nanoneedles, which have previously been shown to facilitate intracellular delivery or isolation of various molecular targets via a reversible and minimum-invasive disruption of cellular membranes in living cells. Specifically for targeting intracellular miRNAs, the diamond nanoneedles will be functionalized with size-selective RNA-binding proteins to work as “fishing rods” to directly pull miRNAs out of cytoplasm while keeping the cells alive. Then, each nanoneedle will work as a separated reaction chamber for parallel, high-throughput, and highly sensitive analysis of the isolated miRNAs via in-situ amplification, visualization and quantification, which aims to detect miRNA of a single copy intracellular abundance.Following the technical development, we will further apply the inCell-Biopsy technique to study the cellular heterogeneity in normal and disease neural tissues by performing temperoal and spatial miRNA transcriptome anlaysis, which is not accessible by any existing RNA analytical methods. The large number of nanoneedles (around ten thousands per chip) provide quasi-single-cell miRNA profiling of a large population of cells. Such scattered miRNA expression data will be analyzed by a customized infomatic and machine learning approch to derive the intrinsic coherence of different cell subpopulations in a complex biosystem, each with a distinctive combinatorial miRNA expression. Temproal association of such profiles will be further used to reveal the transition of cellular status from normal to diseased condintions, and vice versa, in order to provide insight into mechanisms of action for poorly understood miRNA expression dynamics in cellular function. It is expected that success of this project will provide a revolutionary tool for miRNA profiling, and also has tremendous potential to assist the development and quality control of emerging therapeutic strategies involving cell components.
|Effective start/end date||1/01/21 → …|