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DNA Encoded Library Screening and hit validation HitGen
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DNA-encoded libraries can be screenedagainst target proteins and preferential binders can be identified according toa general procedure. In a typicalscreening experiment, a protein of interest is immobilized on a solid supportand subsequently incu-bated with the DNA-encoded library. After several washingsteps, binding molecules attached to DNA will remain bound to the targetprotein, whereas non-binders should be washed away. In a conceptually similarapproach, DNA-encoded libraries can be pre-incubated with affinity-taggedproteins in solution before the capture step on the solid phase. Preferential binders can now be eluted fromthe solid phase by heat-induced protein denaturation and identified by PCRamplifi-cation of the DNA-barcode, followed by high-throughput DNA sequencing.Owing to the PCR amplification process, even very low copy numbers of the boundDNA–small-molecule conjugates can be efficiently detected. As a consequence,DNA encoding can be used for the synthesis and screening of combinatoriallibraries, containing very large numbers of compounds, individually present atvery low concentrations. Furthermore, libraries can be stored as frozenaliquots and can be interrogated against numerous protein targets using roboticprocedures. After decoding, hit compounds are validated by re-synthesisprocedures and affinity measurements. It is particularly convenient tore-synthesize binders as fluorophore conjugates, thus enabling affinitymeasurements in solution using fluorescence polarization (FP) as the readout.Conveniently, the fluorophore can be replaced by short fluorescently labeledDNA-tags, which can facilitate the re-synthesis procedure.  筛选流程DNA Encoded Library.png |
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