Multitarget Affinity Specificity Screening in a High Throughput Format

Instrumentation for laboratory automation is increasingly becoming a ''plug and play'' operation. Many ancillary pieces of equipment, such as autosamplers, plate handlers, chromatographic instruments, and many classes of spectrometers, have built-in communication ports and/or the ability to trigger (or be triggered by) other pieces of equipment. Thus, it is now commonplace in most industrial analytical laboratories to utilize various user-customized combinations of these devices to increase sample throughput and facilitate unattended sample analysis. It is often the case that, once an analytical protocol is in place and can be manually performed by a technician, it can be automated with standard (or customized) robotics and control software and subsequently be performed more rapidly and more reliably via an automated protocol. Thus, it is not surprising that, following the initial success of the MASS assay to interrogate RNA-ligand binding in a multiply-parallel format, a key push in our laboratory has been to fully automate the approach and thereby, improve both the sample throughput and robustness of the assay. There are two aspects of any analytical scheme which one must consider when attempting to ''scale up'' a protocol from a research method to a high throughput assay: data acquisition and data analysis. Both are essential to operate in a true high throughput format [30].

Our general screening approach involves the simultaneous screening of three RNA targets against 11 compounds, or 33 analyses per well. As each well requires only 39 s per analysis and the assay can be run around the clock in an unattended manner, a single ESI-FTICR mass spectrometer can perform in excess of 67000 analyses per day. From the relative abundance of each noncovalent complex detected, a one-point Kd is calculated that gives a semi-quantitative indication of the binding for each ligand-substrate pair. For complexes that are not detected above a given threshold, a minimum value for the Kd is calculated based on the known concentration of the ligands and the targets - this is generally in the Kd b 2.5 mM regime. As described in detail elsewhere, we have automated the entire protocol from plate preparation to spectral acquisition to data interpretation. It has also been shown that these and related methods can be used to directly measure dissociation constants [14, 36-39].

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