Massively parallel measurements of molecular interaction kinetics on a microfluidic platform

Abstract

Quantitative biology requires quantitative data. No high-throughput technologies exist capable of obtaining several hundred independent kinetic binding measurements in a single experiment. We present an integrated microfluidic device (k-MITOMI) for the simultaneous kinetic characterization of 768 biomolecular interactions. We applied k-MITOMI to the kinetic analysis of transcription factor (TF)-DNA interactions, measuring the detailed kinetic landscapes of the mouse TF Zif268, and the yeast TFs Tye7p, Yox1p, and Tbf1p. We demonstrated the integrated nature of k-MITOMI by expressing, purifying, and characterizing 27 additional yeast transcription factors in parallel on a single device. Overall, we obtained 2,388 association and dissociation curves of 223 unique molecular interactions with equilibrium dissociation constants ranging from 2 × 10(-6) M to 2 × 10(-9) M, and dissociation rate constants of approximately 6 s(-1) to 8.5 × 10(-3) s(-1). Association rate constants were uniform across 3 TF families, ranging from 3.7 × 10(6) M(-1) s(-1) to 9.6 × 10(7) M(-1) s(-1), and are well below the diffusion limit. We expect that k-MITOMI will contribute to our quantitative understanding of biological systems and accelerate the development and characterization of engineered systems.

Fig. 1.

Schematic overview of the k-MITOMI platform. (A) Design drawing of the microfluidic device. Blue and grey lines represent flow and control channels, respectively. The four separately addressable “button” control channels (BF1 to BF4) are highlighted in red, cyan, green, and yellow. (B) Schematic of a unit cell. The capacitor is shown in grey. A “neck” valve, shown in red, separates the chamber from the detection area. Individual unit cells are separated from each other by a pair of “sandwich” valves (orange). “Button” membranes (green) are aligned to the center of the detection chamber. (C) K-MITOMI process. Controlled opening of the “button” allows for association or dissociation of fluorescently labeled DNA molecules to/from surface immobilized TF. (D) Actual association and dissociation traces of DNA molecules to/from the immobilized TF are shown with exponential fits.

Fig. 2.

Characterization of “button” actuation. (A) Response profiles of button actuation with a 60 ms pulse input. Movies of button actuation were recorded at 2,000 fps. (B) Pulse input signal versus measured pulse duration. A dashed blue line highlights the minimal pulse duration (see Inset). (C) Pulse durations measured across the entire device respond reliably and uniformly. Solid, horizontal lines indicate the expected pulse durations of 45, 95, and 195 ms.

Fig. 3.

Comprehensive kinetic measurements of transcription factor-DNA interactions. (A) Dissociation of Zif268-DNA complexes measured with a 750 ms pulse duration. Normalized, surface bound DNA traces are shown with exponential fits. Color code shows the separately measured K_d values (red to blue = high to low binding affinity). (B) Plot of K_d ranked Zif268 dissociation rate constants derived across different pulse durations, as well as extrapolated rate constants. (C) Association of two cognate DNA targets with Zif268 at two different pulse durations and comparable DNA concentration. (D) Plot of K_d ranked Zif268 association rate constants derived across different pulse durations. (E) Comparison of K_d values calculated from k-MITOMI association and dissociation rate constants to K_d values determined from saturation binding curves measured with MITOMI. (F) Zif268, Tye7p, Yox1p, and Tbf1p dissociation and association rate constants plotted against calculated K_d values.

Fig. 4.

Integrated kinetic measurements of a broad spectrum of transcription factors. (A) Surface bound cognate target DNA values are plotted against TF expression levels. (B) Dissociation rate constants of TF-DNA complexes for consensus and noncognate target DNAs. Color code highlights the different DBD families. (C) Dissociation rate constants of all TF-consensus DNA dyads are plotted against MITOMI derived relative affinity measurements.