Quantitative Assessment of Affinity Selection Performance by Using DNA‐Encoded Chemical Libraries

Abstract

DNA‐encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA‐barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid‐phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub‐micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 104 copies per library member are used as the input.

Ultra-large chemical libraries for the discovery of high-affinity peptide binders

“…8 and 9). This result is consistent with the work of Sannino and coworkers, who observed low recoveries for micromolar affinity ligands in the context of DELs 35 . We tested the use of increased magnetic bead concentration to recover lower affinity ligands; however, this parameter had little effect on recovery, perhaps suggesting that these ligands are lost during the washing step ( Supplementary Fig.…”

The Impact of Variable Selection Coverage on Detection of Ligands from a DNA-Encoded Library Screen

“…It has been previously reported that high-affinity ligands for a model protein can be detected via sequencing within the context of a small library (~3E+05 compounds) when sufficiently represented (>10 4 copies/library member) in the input. 16 Our sequencing data reported here confirm these findings within the context of a larger library (>1E+08 compounds) and additionally demonstrate how the amount of selection coverage for a given sample can determine whether or not lower-affinity ligands, such as those with micromolar affinity, can be detected. These lower-affinity ligands could in fact be an excellent medicinal chemistry starting point, particularly when probing a difficult target.…”

A Single‐Stranded DNA‐Encoded Chemical Library Based on a Stereoisomeric Scaffold Enables Ligand Discovery by Modular Assembly of Building Blocks

“…The display of a photocrosslinker on the complementary strand may help stabilize complex formation and facilitate the recovery of preferential binders. [ 9c,41 ]…”