Automated screening for small organic ligands using DNA-encoded chemical libraries

Abstract

DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d.

Figure 1

Comparison of different DECL types. (a) Single-pharmacophore two building block “DNA-recorded” library. (b) Single-pharmacophore three building block “DNA-templated” library. (c) Dual-pharmacophore two building block ESAC library. (d) Dual-pharmacophore two building block PNA/DNA hybrid library.

Figure 2

Overview of the screening process. In Part I, binding library members are identified from the multiplicity of library members. Part II describes the amplification of the eluted library members` DNA tags, followed by Illumina HTDS. The sequencing results are analyzed in Part III using a C++ program and displayed in MATLAB.

Figure 3

Plate loading scheme. The upper panel shows two KingFisher 200 µl plates from above. As the magnetic particle processor transfers all magnetic beads contained in a row (e.g., A1 to A12) during each step, the wells are loaded in a row-wise fashion (e.g., target proteins in row B of plate 1). The respective solutions are filled into the wells as depicted in the lower panel: 200 µl per well for all washing steps and 100 µl of washed beads, target protein, library and Tris buffer. The numbers above the arrows indicate the incubation time of the beads at each step. In this set-up, each column allows the performance of an independent selection. While the handling by the magnetic particle processor is identical for all plate columns, individual selection parameters may be varied in terms of target protein, DECL type and general buffer composition.

Figure 4

Layout of the two-step PCR. In the first PCR reaction, selection specific codes (code 1, code 2) are added. These reactions are purified, pooled, and used as template for the second PCR reaction, which introduces DNA sequences required for Illumina sequencing.

Figure 5

Data processing workflow. Illumina HTDS raw data is converted from *.fastq to *.fasta. HTDS data, the structure file and the code lists are provided as input for the C++ program, which generates three output files. The normalized output file is imported into MATLAB. The selection fingerprint is obtained using a MATLAB script.

Figure 6

Selection fingerprints. (a) Fingerprint of a 2-building-block DECL selection against horseradish peroxidase. NSC: normalized sequence count. Sequence counts normalized to 100; cutoff level = 1000. Binding building blocks have an elevated NSC value and are visible as lines. The crosspoints of binding building blocks feature the highest enrichment, indicating that both building blocks contribute to the binding (chelate effect). (b) Unselected, naïve 2-building block DECL library. (normalized to 100, cutoff level = 200). Before the affinity selection, all library members are present in comparable amounts. (c) Graphical representation of a 3-building block DECL selection. The axes represent the three sets of building blocks, while dot colour and size represent the sequence counts of a compound according to the heat scale given at the right.