title: 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.
authors.affiliation: Department of Molecular Biology and NCCR program Frontiers in Genetics, University of Geneva, Geneva 4, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Geertz
authors.forename: David
authors.initials: D
authors.lastname: Shore
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
mesh_terms: D000465:Algorithms; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C079376:TBF1 protein, S cerevisiae; D014157:Transcription Factors; C080459:Yox1 protein, S cerevisiae; D004247:DNA