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.

Implementation of cell-free biological networks at steady state

“…Our microfluidic device contains eight independent 33-nL reactors (SI Appendix) and functions similarly to previous devices (19)(20)(21). Dilutions occurred in discrete steps, where each dilution step added fresh ITT mix and template DNA, displacing part of the old reaction volume (Fig.…”

Microfluidic sensing: state of the art fabrication and detection techniques

“…3, typical optical detection methods comprise the direct detection by monitoring the light properties including fluorescence, 74,[81][82][83] absorbance, [84][85][86] and luminescence-based [87][88][89] methods, and the light property modulation detections such as surface plasmon 78 and optical waveguides. 79,132 Photonic crystals, optical cavity structures, and several optical techniques have also been reported to be integrated with the microfluidic system for sensing purposes, using one-and two-dimensional surface PCs (guided mode resonance filters), [133][134][135][136] , or three-dimensional photonic crystals, 137,138 optical cavities, 139,140 whispering gallery mode resonators, [141][142][143][144][145][146][147] and optical tweezers for cell related monitoring or fluidic rheological measurements [148][149][150] (Fig.…”

Next generation microfluidic platforms for high-throughput protein biochemistry

“…FRET can thus be used as a molecular ruler, as the transfer between donor and acceptor is strongly dependent on the distance between the two. Ridgeway et al have used a microfluidic device and FRET to measure the kinetics of binding of a rRNA and ribosomal protein [21 ] (the authors also used FCS, which is described in the next section). Because FRET is very sensitive to distance it generally requires precise knowledge of the molecule and is much more readily applied to DNA or RNA containing complexes than pure protein-protein interactions.…”

“…Chou et al quantitated epidermal growth factor receptor (EGFR) and its interaction with Src and STAT3 [22]. Ridgeway et al implemented a two-photon detection mechanism to reduce the amount of photobleaching of the sample and with single molecule sensitivity [21 ].…”