Affinity labeling of DNA-linked ligands for high throughput ligand binding assays and the uses thereof

An assay method for small molecule screening to determine apparent affinity constant of a small molecule ligand through ligand displacement or direct binding comprising

• a. preparing a DNA-linked probe, a complementary strand DNA-linked cross-linker, a free ligand, and a target protein of interest, wherein said DNA-linked probe comprises a known protein ligand appended at the 5′ end of an encoding DNA along with an electrophilic or photoreactive crosslinker attached opposite to the ligand on the 3′ end of the encoding DNA;
• b. applying said free ligand to said target protein, the DNA-linked probe, and the complementary DNA-linked crosslinker in a compatible medium;
• and
• c. running the assay, collecting and analyzing the data to afford said assay result information;
• d. wherein in a ligand displacement assay, variation of the concentration of the target protein of interest or the free ligand results in a dose dependent variation in crosslinking yield, which enables the determination of the apparent affinity constant of said ligand by curve fitting of the data because the crosslinking yield is proportional to the affinity constant of the ligands;
• e. wherein in a direct binding assay, direct determination of a compound's IC50 is determined with reference to the bound ligand;
• and
• f. wherein said assay method is capable of being configured for high throughput screening, generation of structure-activity relationships, and development of selective chemical probes with a high sensitivity and a low cost.

A DNA based protein ligand binding assay for characterization of protein-ligand interactions of multiple ligands comprising;

• a. incubating a protein target with a DNA-linked probe, wherein the DNA-linked probe comprises a known protein ligand appended at the 5′ end of an encoding DNA along with an electrophilic crosslinker or photo reactive group appended to the same end or opposite end from the ligand, such that the DNA-linked probe can be crosslinked to the target protein;
• b. where the electrophilic crosslinker is selected from the group of sulfonyl fluoride or N-hydroxysuccinimide ester, and the photoreactive group is selected from diazirine or aryl azide benzopenome;
• c. where multiple assay samples are screened as a pool, by using multiple DNA-linked probes, each of which has a distinct DNA sequence which becomes a barcode to encode the identity of that specific probe ligand;
• d. following incubation to allow crosslinking, labeling by the probe is quenched by addition of excess free ligand,
• e. wherein co-incubation with a competing free ligand will displace the DNA-linked probe, resulting in decreased affinity labeling of the protein and lower DNA recovery in subsequent purification and isolation of the protein;
• f. collected components from all samples are pooled;
• g. target protein is then purified;
• h. the relative recovery of each DNA probe is determined by DNA sequencing and indicates the crosslinking yield in each sample, where a dose dependent reduction in crosslinking is observed as the concentration of free ligand increases, enabling the calculation of IC50 values.