Intellectual Merit: Different cell lines and tissue types are often characterized by the expression of distinct alternatively spliced transcripts, implicating epigenetic processes in establishing stable splicing patterns during mitotic cell divisions that accompany development and differentiation. This project will develop a powerful and exquisitely sensitive technology to detect and quantify low abundance mRNA transcripts in single cells invoking the "Plasmon nanoruler" concept using gold nanoparticle sensors. The method will be applied to test the hypothesis that pre-mRNA splicing patterns are mitotically inherited. Aims include: optimizing sensor design and delivery modules for the detection and quantification of multiple splice variants in single cells, and defining splice variant profiles and heritability of splicing patterns in single cells and their progeny. Expected deliverables are: development of a Plasmon nanoruler hyperspectral imaging platform to detect and quantify up to three splice variants in single cells without amplification steps; definition of experimental conditions for utilizing nanoprobes for intracellular mRNA quantification; and knowledge on the heritability of splicing. This high-risk approach will set the precedent for understanding and quantifying the heritability of splicing at single cell resolution. Moreover, once developed, the technology can be adapted by multiple investigators for future studies, including: detecting dynamic expression of transcripts from any desired gene in single cells across multiple genetic, developmental and environmental conditions; identifying rare phenotypes based on differential expression of splice variants; and visualization mRNA localization and trafficking.
Broader Impacts: Educational impact includes cross-disciplinary training of graduate and undergraduate students in the fields of biophysics, biochemistry, and biology. Graduate students will develop their professional and entrepeurship skills through one-on-one coaching from the PIs, as well as through seminars offered by Purdue's Instructional and Professional Development seminar series. Graduate students will hone their instructional and mentoring skills by supervising undergraduate research interns recruited through several ongoing programs at Purdue University.
