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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.

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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.

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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.