A small molecule inducible microRNA-based cell cycle controller

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Kathy Wei, Christina D. Smolke

Bioengineering Department, United States

In order to enable the engineering of cellular function in living organisms, it is necessary to develop tools that are both genetically encoded and capable of regulating endogenous protein levels in response to user-specified molecular signals. The goal of this project is to engineer a set of microRNA regulatory networks with integrated modular ligand sensors for the reversible arrest of living mammalian cell populations in G0/1, S, G2, and M. Unlike currently available small molecule inhibitors of the cell cycle that broadly disrupt cell function, a switchable microRNA platform allows inducible cell cycle arrest through regulation of specific endogenous gene targets with potentially any small molecule effector by modular replacement of the microRNA targeting sequence or aptamer-based sensing region. We have identified promising RNAi targets for G0/1 arrest in a human cell line, measuring over 76% knockdown of mRNA levels by qRT-PCR and accumulation of over 92% of cells in G0/1 by flow cytometry. By creating cell lines with stably integrated microRNA regulatory networks, we have quantitatively measured mature microRNA and target mRNA levels by qRT-PCR, as well as target protein levels and cell cycle distribution by flow cytometry, providing a detailed picture of the changes in gene expression that lead to a measurable phenotypic change. The creation of a model cell line that can be easily and reversibly paused at specific cell cycle phases has potential applications for increasing heterologous protein production, better control of signal processing in the self-renewal versus differentiation decision, and increasing the reliability of mammalian genetic integration techniques. More broadly, these ligand-responsive microRNAs represent a general class of synthetic biology tools that can be adapted for sophisticated control of complex cellular processes in higher organisms.