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Published September 16, 2009 | Published
Journal Article Open

RNA–protein binding kinetics in an automated microfluidic reactor

Abstract

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

Additional Information

© The Author(s) 2009. Received June 5, 2009. Nucleic Acids Research Advance Access originally published online on September 16, 2009. Revised August 1, 2009. Accepted August 19, 2009. The authors would like to thank E. Kompfner for expression of the His-tagged S15 construct, Y. Gambin, R. Gόmez-Sjöberg, C. Hansen, A. Karnaukhov, H.-J. Lee and S. Quake for helpful discussions, and D. Millar and J. Tainer for access to laboratory equipment. National Institutes of Health (R37-GM-53757 to J.R.W., DP-OD000217 to R.P.); National Science Foundation Graduate Research Fellowship (to W.K.R.). Funding for open access charge: National Institutes of Health (R37- GM-53757 to J.R.W.).

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