RNA-protein binding kinetics in an automated microfluidic reactor.
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.
Ridgeway
William K
WK
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, MB33, La Jolla, CA 92037, USA.
Seitaridou
Effrosyni
E
Phillips
Rob
R
Williamson
James R
JR
D002138:Calibration; D004867:Equipment Design; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D017785:Photons; D012313:RNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000032:analysis / Q000378:metabolism
D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D012313:RNA; D008274:Magnesium