Tracking hundreds of molecular interactions at once
September 24, 2012 § Leave a comment
Scientists have been delving into systems biology now for more than a decade, but a major drawback in the field has been a “dearth of quantitative data,” says Sebastian Maerkl at the Ecole Polytechnique Federale de Lausanne in Switzerland. So in this week’s Proceeding of the National Academy of Science, Maerkl and his colleagues describe a tool that gets quantitative measurements of hundreds of molecular interactions at once.
Systems biology takes a more holistic and integrated view of biological systems, trying to understand how different molecular and cellular pathways are connected to one another. However to date, Maerkl says most of our understanding of molecular interactions on a systems scale, such as protein-protein or protein-nucleic acid ones, is qualitative and binary. The information is usually the yes/no kind, without a glimpse of the details of the binding events.
So to be able to get to these details, Maerkl and colleagues used microfluidics to design a special assay platform. Called k-MITOMI, the device is an extension of an instrument Maerkl developed with Stephen Quake at Stanford University in 2007. It is based on microfluidics, which allows nanoliter volumes of samples and reagents to be manipulated in various ways inside channels that are micrometers in diameter.
k-MITOMI can “freeze” interactions, which decouples the number of reactions being followed from the sampling frequency of the readout method used. This makes the device capable of monitoring hundreds of interactions. Other methods of measuring rate constants, such as surface plasmon resonance and electrophoretic mobility shift assays, can’t simultaneously track these many reactions.
The investigators tackled the simultaneous measurement of rate constants for 768 interactions between transcription factors and DNA. These interactions are the basis of gene regulation and expression. The interactions between transcription factors and DNA are particularly difficult ones to measure by conventional methods, says Maerkl, because they have fast on-and-off-rates.
But with k-MITOMI, the investigators were able to establish that the affinities of several mouse and yeast transcription factors for DNA were largely governed by their offstates.
Because the device can measure several orders of magnitude in binding affinity, from nanomolar to micromolar, Maerkl says, “We think the platform will be applicable to a large number of physiologically relevant interactions.”