Confocal Nanoscanning, Bead Picking (CONA): PickoScreen Microscopes for Automated and Quantitative Screening of One-Bead One-Compound Libraries

Abstract

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

Towards mimicking short linear peptide motifs: identification of new mixed α,β-peptidomimetic ligands for SLAM-Associated Protein (SAP) by confocal on-bead screening

“…The on-bead screens of the mixed αβ phosphopeptide libraries were carried out following previously described procedures [8]. Briefly, confocal nanoscanning, CONA, is a fully automated and quantitative on-bead screening process.…”

“…Based on the distribution of hit beads and the analysis of the fluorescence intensity of bead-bound Cy5-SAP within the sublibraries, a total of 48 beads from 13 wells were isolated using the bead-picking device on the PS04 instrument [8]. All retrieved hit beads were placed into separate glass vials for subsequent fluorescence labelling.…”

High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections

“…The small (10 μm) TentaGel beads employed for library construction both facilitate large library synthesis (each gram of resin contains 1000-fold more 10-μm beads than conventional 90-μm beads) and the use of FACS-based screening, which quantitatively analyzes and collects several thousand compound beads per second. This represents a vast improvement over manual bead picking, which is slow, manual, and subjective in the absence of custom screening technology 29 . The greatly enhanced throughput of NGS-based structure elucidation uniquely provided rapid and deep analysis of hit structures, critical for matching the throughput of FACS 30 .…”

Combinatorial/Library Peptide Synthesis

“…After completion of the scan, the hits are ranked by their intensities, and the best are picked by an actuated capillary and placed into individual containers for structural evaluation. The bead picking process is slower and can retrieve about 1 bead/min [280][281][282]. Resynthesis and retesting of the active compounds has shown that surface inhomogeneity prevents precise ranking of the ligand quality based on the solid support fluorescence reading (Figure 12.10).…”

“…Observation of increased ring intensity represents the binding to macromolecular fluorescently labeled target. Fluorescence in the middle of the bead represents autofluorescence of the bead material or synthesized compound and is irrelevant to the biological interaction with the target[280].…”