Critical Evaluation of Photo-cross-linking Parameters for the Implementation of Efficient DNA-Encoded Chemical Library Selections

Abstract

The growing importance of DNA-encoded chemical libraries (DECLs) as tools for the discovery of protein binders has sparked an interest for the development of efficient screening methodologies, capable of discriminating between high- and medium-affinity ligands. Here, we present a systematic investigation of selection methodologies, featuring a library displayed on single-stranded DNA, which could be hybridized to a complementary oligonucleotide carrying a diazirine photoreactive group. Model experiments, performed using ligands of different affinity to carbonic anhydrase IX, revealed a recovery of preferential binders up to 10%, which was mainly limited by the highly reactive nature of carbene intermediates generated during the photo-cross-linking process. Ligands featuring acetazolamide or p-phenylsulfonamide exhibited a higher recovery compared to their counterparts based on 3-sulfamoyl benzoic acid, which had a lower affinity toward the target. A systematic evaluation of experimental parameters revealed conditions that were ideally suited for library screening, which were used for the screening of a combinatorial DECL library, featuring 669 240 combinations of two sets of building blocks. Compared to conventional affinity capture procedures on protein immobilized on solid supports, photo-cross-linking provided a better discrimination of low-affinity CAIX ligands over the background signal and therefore can be used as a tandem methodology with the affinity capture procedures.

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

“…The library was used to isolate and characterize small molecule ligands against multiple target proteins. The strategic choice to encode the GB‐DEL with DNA in single‐stranded format opens the possibility to perform selection schemes based on photo‐crosslinking (e.g., by hybridization with oligonucleotide‐photosensitizer conjugates [ 9a,34 ] and to implement affinity‐maturation with oligonucleotide‐hit conjugates (Figure 4). Some of the hit compounds isolated from the GB‐DEL may be useful in practice and may deserve additional medicinal chemistry optimization.…”

Affinity Selections of DNA‐Encoded Chemical Libraries on Carbonic Anhydrase IX‐Expressing Tumor Cells Reveal a Dependence on Ligand Valence

“…Two distinct 48-mer oligonucleotides, differing in sequence only in the central portion and carrying a C6-amino modification at the 5' end, were individually conjugated to an acetazolamide (AAZ) derivative or to an acetyl group (Ac) [Figure S1]. AAZ is a high affinity CAIX binder (K D = 8.7 nM), [38] whose performance in selections could be compared to the one of Ac-DNA, serving as negative control of irrelevant specificity. An 18-mer oligonucleotide, carrying an AAZ moiety or an acetyl group at the 3' end, was used to assess the role of valence in selection experiments.…”

“…Selections with SKRC-52 additionally , a submicromolar CAIX binder (K D = 0.5 μM). [38] With SKRC-52, higher library input (5 × instead 1 × 10 6 copies per member per selection) and shorter library incubation time (1 min instead 1 h) yielded higher relative abundance of bivalent and monovalent CAIX binders as well as lower background noise [Figure 6G and H]. Selection results further improved when performing PCR amplification directly with cell suspension instead of using heat eluate [Figure 6I and Fig- ure S21].…”

“…[36,37] In a first step, we performed model cell selections using acetazolamide as known CAIX binder with a dissociation constant (K D ) to the target of 8.7 nM. [38] Quantitative PCR (qPCR) evaluation of selection results revealed that acetazolamide-linked DNA could be recovered with a~15-fold enrichment over negative controls in optimized experimental conditions (e. g. using 500 mM NaCl). Interestingly, the bivalent display on DNA of CAIX binders led to a substantial increase of ligand recovery and selectivity (~100-fold enrichment factor).…”

Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands

“…Synthesis and purification were performed following previously reported procedures [5] , yielding 23 mg, 28%. 1 78, 172.02, 171.00, 168.73, 167.61, 164.43, 161.32, 159.72, 152.11, 143.75, 141.55, 129.26, 126.74, 124.30, 112.81, 109.94, 102.45, 87.13, 80.14, 69.76, 69.31, 68.66, 45.98, 43.89, 30.48, 29.59 Synthesis and purification were performed following previously reported procedures [5] , yielding 30 mg, 41%. 1 56, 168.52, 165.15, 159.54, 151.90, 147.58, 147.02, 144.38, 141.33, 135.05, 132.04, 130.13, 129.13, 128.94, 128.11, 124.73, 116.38, 112.63, 109.74, 102.23, 82.26, 69.63, 68.80, 68.43, 43.66 12-mer LNA sequence:…”