Many high-throughput methods allow the function of proteins to be probed, but very rarely can more than the presence of a specific interaction be observed. The new microlaboratory allows quantitative measurement of biomolecular interactions allowing the functional differentiation of proteins by virtue of how strongly they bind to different biomolecules giving new insights into which genes proteins regulate.
The device may eventually find application in drug target discovery and studying the interactions between drug and target, especially in looking for off-target drug interactions.
There are two major problems associated with measuring biological interactions; firstly that any interaction is governed by a logistically challenging number of variables; the other is that these interactions are often transient or weak. These factors are particularly problematic for high throughput methods such as yeast two-hybrid and tandem affinity mass spectrometry as well as protein-protein and protein-DNA binding microarrays where weak interactions are often missed.
The new technique, described by researchers at the Howard Hughes Medical Institute in the January 12 issue of Science, involves trapping a type of protein known as a transcription factor to measure the binding energy between the protein and specific DNA sequences.
According to the lead author of the paper, Dr Stephen Quake, measuring the binding energy of a transcription factor and a single DNA sequence is not enough. It is far more meaningful to know the energy involved in a transcription factor binding to many different DNA sequences, giving researchers a more complete picture of the binding energy landscape.
To this end, the authors created an apparatus consisting of 2,400 individual reaction chambers less than a nanolitre in volume, each controlled by three valves and including a button membrane. The mechanically induced trapping molecular interactions (MITOMI) device fits over a 2,400-unit DNA microarray.
The apparatus is constructed by producing a silicon mold using the same photolithography process used to make the microarrays before casting the elements of the MITOMI device in rubber and bonding them to the array.
Transcription factors are then pumping into the chambers that each contain a slightly varied DNA sequence anchored to the microarray chip before the button membranes expel any unbound molecules from the chambers and ensure that no bound material is washed away from the array.
A DNA array scanner then allows quantification of the binding energy of the trapped transcription function.
Quake emphasized that this technique is can be used to measure the interactions between any two proteins as well as between a protein and DNA. The measurements allowed the researchers to predict the biological function of two different yeast transcription proteins Pho4p and Cbf1p that regulate a range of cellular processes including cell proliferation, development and metabolism; all of which play a role in cancer growth.
According to Quake: "We discovered a wealth of interesting things - to me the most important being that by using the binding data for different sequences we could predict which genes the yeast transcription factors would regulate."