1. o Goal: Engineer synthetic RNA devices for the reversible arrest of mammalian cell populations in G 0/1. o Previous methods: Currently available small molecule inhibitors of the cell cycle tend to broadly disrupt cell function o Our method: o A switchable RNA platform allows inducible arrest through specific endogenous gene targets with potentially any small molecule effector by modular replacement of the target or aptamer-based sensing region. o This platform represents a general class of synthetic biology tools for modular, dynamic, and multi-input control over endogenous protein levels in mammalian cells. [1] Morgan, David O. The Cell Cycle: Principles of Control. 1999-2007 New Science Press. [2] http://en.wikipedia.org/wiki/Hoechst_stain [3] Pozarowski, P (2004) Analysis of cell cycle by flow cytometry. Methods Mol Biol. [4] http://www.cellsignal.com/common/content/content.jsp?id=pathways-cc-g1s [5] Pfeiffer, B.D., Truman, J.W. et al. (2012) Using translational enhancers to increase transgene expression in Drosophila. PNAS. [6] Wei, K.Y., Chen Y.Y. et al. (2013). A yeast‐based rapid prototype platform for gene control elements in mammalian cells. Biotech & Bioeng. This research was made with Government support under and awarded by DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a, Siebel Foundation. System Design Engineering synthetic ligand-responsive RNA devices for controlling the cell cycle Kathy Y. Wei, Christina D. Smolke Dept. of Bioengineering, Stanford University Contact: kywei@stanford.edu, christina.smolke@stanford.edu Methods o Motivation: Tools for the control of mammalian programming is a relatively underdeveloped area. We propose to use simple, general-purpose components (an RNA ribozyme or hairpin coupled to an RNA aptamer) to control a complex phenomenon (such as regulation of the cell cycle). o Applications: Increase heterologous protein production. Better control of signal processing in the self-renewal versus differentiation decision. Increase the reliability of mammalian genetic integration techniques. Cell cycle signaling network is controlled by a few key regulatory nodes Small molecule control of G 0/1 arrest through RNA switches The signaling network controlling cell progression from G 1 to S phase is large and interwoven with other processes in the cell. While our understanding of the network is incomplete, we do know that a very small set of key protein regulators act as nodes. The regulatory nodes act such that changes in the level of that node results in measurable population level changes in cell cycle progression. Some key nodes are boxed. Combining proteins does not appear to have an additive effect Ribozymes are RNA sequence capable of self-cleavage. These can be made ligand-responsive by incorporating an aptamer such that ligand binding to the aptamer effects ribozyme cleavage. Specifically, this results in an inducible switch where absence of ligand results in an active ribozyme and low gene expression and presence of ligand results in inactive ribozyme and high gene expression. For more information, see [6]. RNA switches that control G 0/1 arrest Cells were transfected with plasmids overexpressing potential regulatory protein nodes to identify which are capable of inhibiting progression of G 0/1 to S. mCherry served as a non-disruptive control and cyclin D1 served as a negative control. The most promising key regulators are p16, p21, and p27. ueGFP CMVHSVTKpA protein promoterpA Tuning the expression of key regulator p27 using syn21 and IRES improved the degree of control over cell cycle arrest. The syn21 sequence is 21 nt that is inserted prior to the start codon and was identified to increase expression in Drosophila [5]. Introducing a second copy of p27 should further increase the amount of expression. While linking the two proteins with a cleavable peptide (P2A) did not improve the amount G 0/1 arrest (possibly due to lack of cleaving and thus mis-folding), linking with an IRES was effective for increasing the amount of control over the cell cycle. protein promoterpA protein P2A/IRESsyn21 Mechanism of small molecule inducible RNA switches h AAA n h S G2 M G1 Ribozyme active No inducer Gene expression off Basal %G 0/1 High %G 0/1 Inducer Ribozyme inactive S G2 M G1 RNA (rz) switches were inserted behind the optimized expression construct for the key cell cycle regulatory node p27 and integrated into cell lines. “OFF only” is a cleaving, non-switching control and “ON only” is a non-cleaving, non-switching control. Both Switch A and Switch B induce arrest of cells in G 0/1 with the addition of a small molecule inducer. Both switches show small molecule depend control of cell cycle and function best at 0.75 mM induction (p-value < 0.05). protein promoter pA protein P2A/IRES syn21 rz switch Optimize expression of cell cycle regulatory nodesKey regulators control cell cycle signaling network p16, p21, and p27 identified as key regulators in G 0/1 arrest [1] [4] Many of the regulatory proteins in the cell cycle signaling pathways naturally work together or in parallel branches to achieve their effect. Therefore, combining the expression of many regulatory nodes has the potential to increase the amount of arrest in G 0/1 compared to expression of a single node. In these particular combinations however, the effect of using multiple nodes did not exceed that of overexpressing a single key regulator. ueGFP CMVHSVTKpA protein promoterpA protein promoterpA protein promoterpA Gene expression on OR Future Work Future work includes identifying regulatory nodes that are key to arresting cells in other phases such as G 2 /M (shown here) and S. Putting such nodes under RNA switches that are sensitive to different inducers and combining with RNA switches that alter G 0/1 arrest will create a sophisticated controller that can provide multiple different phenotypic outputs given different combinations of small molecule inputs. A) DNA stain to determine phase A)Cell cycle phase is determined by the amount of DNA staining using an intercalating dye such as propidium iodide (PI). B)Flow cytometry is used to measure the proportion of cells in G 0/1 by examining a histogram of cell frequency vs. DNA staining. Measurements are normalized to a control (either empty or fluorescent) to allow easy comparison across experiments and reported as:  %G 0/1 = (%G 0/1 sample) – (%G 0/1 control) [2] [3] Cell cycle arrest measured by DNA staining B) Measure by flow cytometry Multi-input/multi-output RNA switch control Tuning regulatory node expression via synthetic sequences, cleavable peptides, and IRES