James Eberwine, PhD, the Elmer Holmes Bobst Professor of Systems Pharmacology and Experimental Therapeutics, at the Perelman School of Medicine at the University of Pennsylvania, was named one of 16 finalists in the first phase of the Follow that Cell Challenge funded by the National Institutes of Health (NIH). The competition was run by crowdsourcing company Innocentive and 687 designated "solvers" entered initially.
Eberwine's group was one of five top-prize winners selected to receive monetary awards totaling $88,000. The prize winners will now advance, along with the other 11 finalists from Phase 1, to the challenge's second phase, which requires proof-of-concept data over the next two years, to compete for a single, final prize of $400,000.
This competition is the first time NIH has commissioned a challenge such as this. The goal is to stimulate the development of new tools and methods that will enable researchers to predict the behavior and function of a single cell in complex tissue over time. This ability could help reveal valuable information such as how cells transition from a healthy to diseased state or identify changes that influence a cell's responsiveness to treatment. In addition, non-destructive methods for monitoring single cells could assist with early disease detection and allow doctors to better tailor therapies to cells as they evolve throughout the course of a disease.
"The hope of NIH and all entrants is that this new type of competition will eventually attract funding from commercial or foundation sources," explains Eberwine. "The idea of being able to do live cell analysis is of course the goal for many of us and if our technology works the way we envision then it will be of significant benefit to fundamental science research and also in the clinical arena."
The Eberwine team's proposal aims to develop live-cell, real-time RNA expression profiling, called BLINKER, to assess the transcription of genes in live mouse and human neurons as it happens. The in vivo assay (within a living cell) is designed to assess synthesis over the course of tens of millisecond intervals as RNA synthesis occurs at about 80 bases (DNA building blocks) per second.
"We will assess neurons in cell culture as well as in live preparations to identify RNAs being transcribed from the cell's DNA," Eberwine explains. "We have generated preliminary data showing the viability of this technology in vitro [in test tubes], and it will be transitioned into live cells."
The technique relies on a multi-purpose molecule built with several working parts - a sequence that will bind to the capture sequence engineered into the RNA polymerase enzyme; a cell-penetrating addition to transport it into the cell; a part that moves the peptide into the cell's nucleus; and a peptide nucleic acid (PNA) cassette that will permit interaction with RNA as it is being synthesized. Upon binding of this sequence to RNA, a change in fluorescing signal can be detected as a blink in live cells using fluorescent light sheet microscopy.
"The physical distances between blinks on the cell's strand of DNA are unique for any particular RNA, and using a set of algorithms that we are developing, we can identify the gene that is being transcribed," explains Eberwine.
Preliminary in vitro data suggests that this technology will have the ability to profile 100,000s of sequences from single cells in minutes, decreasing the time and cost of RNA sequencing significantly. "We want to develop BLINKER as a means to assess changes in gene expression that correlate with long-term potentiation, which is a physiological correlate of learning and memory, as well pharmacological responsiveness of mouse and human neurons.
Other members of the BLINKER Team include:
Jai-Yoon Sul, an assistant professor of Systems Pharmacology and Translational Therapeutics, Penn;
Ulo Langel, a professor of Neurochemistry, Stockholm University and Tartu University, Estonia;
David Capelleri, an assistant professor of Mechanical Engineering, Purdue University; and
Junhyong Kim, the Patricia M. Williams Professor of Biology, Penn.
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