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A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity
Joe C Liang1, Andrew L Chang, Andrew B Kennedy
1Division of Chemistry and Chemical Engineering, 1200 E. California Blvd., MC 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Nucleic Acids Research
|July 20, 2012
Summary
Researchers developed a rapid screening method for RNA control devices. This high-throughput system enables precise engineering of ribozyme devices for complex cellular networks.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Biochemistry
Background:
- RNA-based control devices offer potential for programming cellular functions.
- Quantitative tailoring of these RNA devices has been a significant challenge, limiting their application in cellular networks.
Purpose of the Study:
- To develop a high-efficiency, high-throughput screening strategy for rapidly generating ribozyme-based control devices.
- To enable quantitative tailoring of RNA devices with user-specified regulatory activities for cellular networks.
Main Methods:
- A two-color fluorescence-activated cell sorting (FACS) based screening strategy was developed.
- Large libraries (over 10^6 variants) of ribozyme devices were screened.
- Individual components within the ribozyme device platform were randomized to generate diverse libraries.
Main Results:
- The screening strategy efficiently isolated functional RNA sequences within two sorting cycles.
- New ribozyme device sequences exhibited up to 10.5-fold increased in vitro cleavage rates.
- Device sequences showed up to 2-fold increased in vivo activation ratios, with a titratable window correlating in vitro and in vivo activities.
Conclusions:
- The developed FACS-based screen provides a generalizable strategy for quantitative tailoring of RNA genetic control elements.
- This approach facilitates broader integration of engineered RNA devices within biological networks.
- Optimizing RNA device activity directly within the cellular environment is crucial for effective gene regulation.
