Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Sci Rep. 2019 Apr 12;9(1):5976. doi: 10.1038/s41598-019-42509-y.
Synthetic biological circuits that can generate outputs with distinct expression dynamics are useful for a variety of biomedical and industrial applications. We present a method to control output dynamics by altering output mRNA decay rates. Using oscillatory expression of the transcription factor p53 as the circuit regulator, we use two approaches for controlling target gene transcript degradation rates based on the output gene's 3'-untranslated region (3'-UTR): introduction of copies of destabilizing AU-rich elements into the 3'-UTR or swapping in naturally occurring 3'-UTRs conferring different transcript stabilities. As a proof of principle, we apply both methods to control the expression dynamics of a fluorescent protein and visualize the circuit output dynamics in single living cells. We then use the naturally occurring 3'-UTR approach to restore apoptosis in a tunable manner in a cancer cell line deficient for caspase-3 expression. Our method can be readily adapted to regulate multiple outputs each with different expression dynamics under the control of a single naturally occurring or synthetically constructed biological oscillator.
能够产生具有不同表达动态的输出的合成生物电路在各种生物医学和工业应用中非常有用。我们提出了一种通过改变输出 mRNA 降解速率来控制输出动态的方法。我们使用转录因子 p53 的振荡表达作为电路调节剂，使用两种方法基于目标基因的 3'非翻译区（3'UTR）来控制靶基因转录本降解速率：在 3'UTR 中引入不稳定的 AU 丰富元件的拷贝，或交换赋予不同转录本稳定性的天然存在的 3'UTR。作为原理验证，我们将这两种方法都应用于控制荧光蛋白的表达动力学，并在单个活细胞中可视化电路输出动力学。然后，我们使用天然存在的 3'UTR 方法以可调节的方式恢复在 caspase-3 表达缺失的癌细胞系中的细胞凋亡。我们的方法可以很容易地适应在单个天然存在或合成构建的生物振荡器的控制下调节多个具有不同表达动态的输出。
