Nucleic Acids Research Advance Access published online on September 16, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp733
Methods Online |
RNA–protein binding kinetics in an automated microfluidic reactor
1Department of Molecular Biology, 2Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, MB33, La Jolla, CA 92037, 3Division of Engineering and Applied Science and Kavli Nanoscience Institute, California Institute of Technology,Pasadena, CA 91125 and 4Division of Natural Science and Mathematics, Oxford College of Emory University, Oxford, GA 30054, USA
*To whom correspondence should be addressed. Tel: +1 858 784 8740; Fax: +1 858 784 2199; Email: jrwill{at}scripps.edu
Received June 5, 2009. Revised August 1, 2009. Accepted August 19, 2009.
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.