High-throughput single-molecule optofluidic analysis

Abstract

We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

Figure 1

A microfluidic formulator for high-throughput single-molecule FRET measurements. (a) Device image (left) with the mixing ring highlighted (arrow). Scale bar, 5 mm. The schematic (right) depicts critical features of the control and flow layer (control and flow channels). (b) A schematic plot representing smFRET measurements as a two-dimensional histogram of FRET versus stoichiometry. The subpopulations of interest are hybridized poly(dT) (low FRET population; dsDNA) and unhybridized poly(dT) (high FRET population; ssDNA). (c) Heat map of hybridization efficiency (ratio of dsDNA to total DNA) for various concentrations of NaCl and complementary strand. (d) Matrix of FRET-stoichiometry scatter plots with contour overlay of fits used to generate heat map shown in c.

Figure 2

RNAP activity measured with smFRET. (a) A schematic of the assay depicts RNAP transcribing the template, thus producing complementary transcript that hybridizes to the poly(dT) probe. (b) A matrix of FRET-stoichiometry scatter plots (as in fig. 1) depicting hybridization of the poly(dT) probe to newly produced transcript upon titrating RNAP and glutamate. (c) Heat map showing quantification of the data in b. Amount of transcript is plotted for various concentrations of RNAP and glutamate.