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Critical Evaluation of Photo-cross-linking Parameters for the Implementation of Efficient DNA-Encoded Chemical Library Selections
journal contribution
posted on 2020-03-06, 13:45 authored by Alessandro Sannino, Adrián Gironda-Martínez, Émile M. D. Gorre, Luca Prati, Jacopo Piazzi, Jörg Scheuermann, Dario Neri, Etienne J. Donckele, Florent SamainThe
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.
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Keywords
tandem methodologylow-affinity CAIX ligandsbackground signalPhoto-cross-linking ParametersEfficient DNA-Encoded Chemical Library Selections669 240screening methodologieslibrary screeningcarbene intermediatescombinatorial DECL libraryDNA-encoded chemical libraries669 240 combinationsreactive naturediazirine photoreactive groupmedium-affinity ligandsbuilding blocksselection methodologiescarbonic anhydrasephoto-cross-linking processprocedureModel experiments3- sulfamoyl benzoic acidCritical Evaluationsingle-stranded DNArecoveryprotein binders
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