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High-throughput Protein Expression Generator Using a Microfluidic Platform
Published on: August 23, 2012
RNA-protein binding kinetics in an automated microfluidic reactor
William K Ridgeway1, Effrosyni Seitaridou, Rob Phillips
1Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, MB33, La Jolla, CA 92037, USA.
Nucleic Acids Research
|September 18, 2009
Summary
A novel microfluidic Riboreactor automates RNA-protein binding assays, enabling long-time scale kinetic studies with single-molecule sensitivity. This technology advances the study of complex molecular assembly processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Bioengineering
Background:
- Microfluidic chips automate biochemical assays at the nanoliter scale, beneficial for RNA-protein binding reactions.
- Current microfluidic mixers struggle with complex reactions and long-time scale investigations.
Purpose of the Study:
- To design and construct a microfluidic 'Riboreactor' for studying RNA-protein complex formation kinetics over extended periods.
- To enable automated preparation and observation of binding reactions with high sensitivity.
Main Methods:
- Development of a computer-automated microfluidic reactor capable of preparing reactions from eight reagents.
- Integration of a two-photon microscope for observing 5-nl reactions over 1000 s with single-molecule sensitivity.
- Utilizing the Riboreactor for automated RNA-protein binding assays with a bacterial 30S ribosome fragment.
Main Results:
- The Riboreactor successfully prepared RNA-protein binding reactions in a fully automated manner.
- Observed binding rates were consistent with those from conventional assays.
- Demonstrated negligible photobleaching and high reproducibility in ultra-sensitive fluorescence detection.
Conclusions:
- The microfluidic Riboreactor combines automation, low sample consumption, and sensitive detection for reproducible biochemical assays.
- This platform is suitable for probing complex reaction networks in the assembly of large ribonucleoprotein complexes, such as ribosomes.
