Printing chemical libraries on microarrays for fluid phase nanoliter reactions

Abstract

Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots͞cm 2 . Using aerosol deposition, subsequent reagents and water were metered into each reaction center to rapidly assemble diverse multicomponent reactions without crosscontamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease-substrate interactions, and highthroughput screening reactions. An inhibitor of caspases 2, 4, and 6 was identified by using a 352-compound combinatorial library microarrayed in quadruplicates on 100 slides and screened against caspases 2, 4, and 6, as well as thrombin and chymotrypsin. From one printing run that consumes <1 nanomole of each compound, large combinatorial libraries can be subjected to numerous separation-free homogeneous assays at volumes 10 3 -10 4 smaller than current high-throughput methods. DNA chips and microarrays for genotyping and expression profiling give no information about the activities of enzymes that can be regulated by posttranslational modifications or cleavage state. Protein microarrays have used the capture of proteins to libraries of immobilized DNA sequences, peptides, antibodies, chemical motifs, or other proteins(1-6). The three major formats for protein arrays currently use plain glass slides (1), 3D gel pad chips (7) (''matrix'' chips), or nanowell chips (2, 3). All of the formats require pretreatment for immobilization, which is a laborious and potentially protein-altering process. Also, none of these three formats have used soluble fluorogenic substrates to quantify multicomponent reactions common to high-throughput screening or to quantify numerous enzymes in a sample like plasma. Microarray-based activity assays of kinases require the use of immobilized substrates (2, 6) and cannot be easily implemented for the direct screening of soluble compounds from a combinatorial library. Assays performed in well plates before arraying onto chips for detection do not exploit the scale down or liquid handling power of microarray printing (6). Thus, a need exists to create localized reaction volumes in an array-based format as well as to create a method of rapidly delivering small volumes of fluid to each reaction. Additionally, evaporation effects typically prevent the extreme scale down of well-plate reactions to nanoliter volumes (8). We have developed a technique that performs enzymatic assays at nanoliter volumes in the liquid phase with minimal evaporation, no crosscontamination, and high reproducibility, and that has the capability to rapidly assemble multicomponent reactions with minimal sample usage in a microarray format. Methods. An OmniGrid Accent and OmniGrid contact microarrayers (Gene Machines, San Carlos, CA) were used for arraying. Arrays were printed on the OmniGrid Accent by using a 1 ϫ 1 pin protocol or on the OmniGrid with a 4 ϫ 8 pin protocol. All glass slides (Erie Scientific, Portsmouth, NH) were washed in dry ethanol and vacuum dried before arra...

Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry

“…9. The library was screened for inhibitors of human CTSL at 1 M in 400 mM NaCl, 20 mM malonate buffer, and 1 mM EDTA, pH 5.5, with fluorogenic substrate Z-Phe-Arg-AMC (Bachem) at 1 mM for detection.…”

“…After the addition of enzyme and substrate, the reactions were incubated for 4 h before imaging the slide, as described in ref. 9.…”

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

“…Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highly parallel and miniaturized format.…”

“…Methods-The fluorogenic substrate library was synthesized and printed according to protocols previously described (5,6,9). The P 1 ϭ Arg and P 1 ϭ Lys sublibraries, along with calibration standards (unacylated 7-amino-4-carbamoylmethylcoumarin (ACC), acetylcapped ACC, and blanks), were printed at either 50 or 100 M in a 16 ϫ 24 format equivalent to a 384-well plate on polylysine-coated glass slides (Erie Scientific, Portsmouth, NH) using a 1 ϫ 1 pin (Telechem, Sunnyvale, CA) protocol on an OmniGrid Accent (Gene Machines, San Carlos, CA) microarrayer.…”

“…The proteases were delivered to the substrate arrays as described previously and underwent ϳ30-fold dilution from the initial delivery concentration after mixing in the microspot (5,6). The slides (n ϭ 6 per enzyme) were incubated at 37°C for 30 min to 6 h. This incubation time resulted in ϳ5-25% cleavage of the best substrate on each array, assuring that the assays were run within the linear range.…”