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4.3.1 Experimental Procedures

DOI: 10.1055/sos-SD-241-00249

Cai, B.; Krusemark, C. J.Science of Synthesis: DNA-Encoded Libraries, (20241600.

In the crosslinking-based approach (Scheme 1, part A and Scheme 2),[‌8‌] a protein target with a SnapTag purification tag is expressed on HEK293 suspension cells. Upon binding, an electrophilic crosslinker (RG-ssDNA) is added to covalently trap protein–ligand interactions by affinity labeling. Benzylguanine-biotin (BG-biotin) is co-incubated with cells to covalently label SnapTag. After lysis of the cells, biotinylated proteins are captured on streptavidin beads. Stringent washes are applied to remove non-binders. Enriched ligands are identified by qPCR or DNA sequencing.

Me eee eeeeee-eeeeeeee eeeeeeeee eeeeeeee eeeeeeee (Meeeee 8, eeee M eee Meeeee 8),[‌8‌] e eeeeeee eeeeee eeeee eeee MeeeeMM ee eeeeeeeee ee MMM888 eeeeeeeeee eeeee. MMM-eeeeee eeeeeee eeee e eeee eeeee eeeeee ee eee eeeeeeee MMM eeeeee (M8M-eeMMM) eee eeeeeeeee eeee eeeee. Meeeeeeee-eeeeeee eeeeeeeeeeeee ee eee eeeee-eeeeeeee MMM eeeeee ee e eeeeee ee eee eeeeeee–eeeeee eeeeeee, eeeeeeee eee eeeeeeeeee ee eee eeeeee eeee eee-eeeeeee eeeee eeeeeeeeeeee eeeee.

Meeeee 8 Meeeeeeeee eee MMM Meeeeeeeee ee Meee-Meee Meeeeeee[‌8‌,‌8‌]

Meeeeee eeee MeeMeeeee.eee.

Meeeee 8 MMM Meeeeeeee Meeeee[‌88‌]

Meeeeee Meeeeee: Meeeee 8, eeeee M, eeeee eee eeeeeeee eee eeeeeeeeee eeeeee (888-eeee) eeee eee eeeeeeeee eeeeeee MeeeMee-δ eeeeee eeeeeeee (MMM), eeeee e MMM-eeeeee eeee-eeeeeeee eeeeee (Mee-Mee-Mee, MMM)[‌88‌] ee eeeeeeee eeeeeee eee MMM-eeeeee eeeeeee eeee ee eeeeeeee eeeeeee (eee-eeeeee). Meeeeeee ee eeeee ee eee eeeeee ee MMM eeeeeeeee eeee eee eeeeeee MMM eeeeeee; eeeeeeeeee ee eeeee ee eee eeee eeeeee ee eeeeeeee ee eeeeee–MMM eeeeeeeee eeeeeeee ee eee eee-eeeeee–MMM eeeeeeeee. Meeeeeeeee eeeeeee eee eeee eeeeee eeeee eee eeeeee-eeeeeeee eeeeeeeee eeeeeeee eeee eee MeeeeMM eee eeee e eeeeeeee eeeeeeee eeeeeeee ee MMM-eeeeee eeeeeee eeeeeeee ee eeeeeeeeeeee eeeeeeeeee (Meeeee 8, eeeee M).[‌8‌] Mee eeeeeeee ee MMM eee eeeeeeee ee eeee eeee 88-eeee eeeee eee MeeeeMM-eeeee eeeeeeeee eeeeee, eeeee eee eeeeeee eeeeeeeeee eee eeeeeeeeee. M eeeeee eeee eeeeee eeeeeeee, Mee-Mee-MM, eee eeeeeeeeeee eeeeeeeee ee eeee eee eeeeee 88-eeee eeeeeeeeee eeee eee eee-eeeeee eeeeeee.

Meeeee 8, eeee M, eeeee eee MMM eeeeeeeeee eeeeeee eeee e eeeeeeeee eeeeeee MeeeMee-MMM eeeee e 88-eeeeeeee MMM eeeee ee e eee-eeeeeeee eeeeeeee eeeeeee eee e eeee-eeeeeeee eeeeeeee eeeeeee. Meeee eeeeeee eeee eeeeeeee (Me) eeeeeee eeee 8.8 eM ee 888 eM eeee eeeeeeeeee.[‌8‌] Mee eeeee eeeee eeeeeeeeee eee eee88 eeeeeeeeee eeeeeeee ee e eee-eeeeee eeeeeee. Meeeeee, eee eeeeeeee ee eee eeeeeeee eeee eeeeeee ee eeee 88 eeeee eeeeee eeee eee eeeeeeeee eeeeeeee-eeeeeee eeeeeeeeeeeee,[‌8‌] eeeeeeeeeeeee eee eeeeeeeeeee ee eeeee eeeeeeee eeeeeeeeeeee ee e eeeeee ee eeeeeeeeeeeeeee eeee eeeeeee eeee eee-eeeeeee eeeeeee eeeeeee eeeeeee ee eeee eeeee.

Meeeee 8 Meeeeeeee ee MMM-Meeeeee Meeeeeeee eeeeeee MMM ee Meee-Meee Meeeeeee[‌8‌,‌8‌]

Meeeeeeeee eeee eeeeeeeeeeeee eeee Mee[‌8‌] eeee eeeeeeeeee eeee eee Meeeeeee Meeeeeee Meeeeee, eee eeee Mee[‌8‌] eeee eeeeeeeeee eeee eee Meeee Meeeeee ee Meeeeeeee.

Meeeeeeeeeee Meeeeeeee

Meeeeeeee Meeeeeeeeeee ee eee Meeeeee ee Meeeeeee (MMM) ee Meee Meeee:[‌8‌,‌8‌]

Me eeeeeee MMM eeeeeeeeee eeeeeee eeeeeee eeeeeee ee eeeee, eee eeeeeeeeeeee-eeeee eee eeeeeeeee eeeeee eeeeeee eeeee eeeeeeeeee eeeeeee eee eeeeeeeeee ee eee MMM eeee e MeeeMee ee MeeeeMM eeeeee eee, eeeeeeeeeeee, ee Meee888M eeeee (Meeeee Meeeee Meeeeeeeee, M88 888). Meeeeeeee eeeeeeeeeeee eee eeeeeeeee ee eeeeeeeee Meee888M eeeee ee e 88-eM Meeeeeeeee eeeee eeee Meee888 Meeeeeeeee Meeeee (8 eM). Meeeee eeee eeeeee ee 888 eee eeeee ee eeeeeee eeeeee (MM-888 MM) eeeeee ee e 88 °M eeeeeeeee eeee 8% MM8 eee >88% eeeeeeee eeeeeeee. Me eee eee ee eeeeeeeeeeee, eeeee eeee e eeeeeee ee 8–8 eeeeeee eeeeee eeeee/eM eeee eeeeeee ee e eeeee eeeeeee ee 8.8 eeeeeee eeeeee eeeee/eM eee eeeeeeeeeee eeee e eeeeeeee eeeee-eeeee eeeeeeeeeeee eeeeeee, Meeeeeeeeeeee 888 (Meeeee Meeeee Meeeeeeeee, M88 888), eeeeeeeee eee eeeeeeeeeeeeʼe eeeeeeee. Mee eeeeeeeee eeeeeeeeeee, eeeee eeee eeeeeeeee eeeeeeeee 8–8 eeee eeee-eeeeeeeeeeee ee eeeeeeee eeeeee-eeeeeee eeeeeeeeee.

Meeeeeeeeee ee MMM Meeeeeeeee:[‌8‌]

MMM eeeeeeeeee eeee eeeeeeeeeee ee e 8′-eeeee-eeeeeeee eeeeee eeeeeee eeeeee. Meee 88-eee eeeee eeeeeeee e eeeeeee eeeee, e 88-eee eeeeee eee eeeeeeeeeeeee eeeee MMM eeeeeeeee, e MMM eeeeee, eee e 88-eee eeeeee eee eee ee e MMM eeeeee [8′ eeeee eeeeeeee M88, eeeeeeeeeee eeeeee eeeeee (Mee Meeeee 8, eMM8, Meeeeeeeee MMM Meeeeeeeeeee (MMM)] (eee Meeeee 8). Meee eeeeeeeee eee eeeeeeee ee e eeeeee eeMMM eeeeeeee eeeeeeee ee MMM eeeeeeeeeeee (8e MeeeeMee Meeeee, 8.8 ee/μM eeeeeeee, 8.8 µM eeeeeee eeeeee–88 eee–eMM8 eeeeee eeeeeee eeeeee, 8.8 µM eeeeee eeeeeee eeeeee, 8.8 eM eMMMe, eee 8.888 M/μM MeeeeMee MMM eeeeeeeeee). Mee eeeee eeeeeeeeee eeeeeee ee e 88-eee eeMMM eeee e 88-eee eeMMM eeeeeeeee e eeeeeee eeeeee, eeeee eee ee eeeeeeeeee ee e eeeeeeee eeeeeeee (eee eeeeeeeeeeee-eeeee eeeeeeee) ee e eeee eeeee eeeee (eee eeeeee-eeeee eeeeeeee) eeeeee ee eee 8′ eee ee eee eeeeeeee 88-eee eeMMM eeeeee. Mee 88-eee eeeeeee eeeeeee (Meeeee 8) eee eeeeeee eeeeee eee eeeeee eee eeeeeee.

Meeeeeeee eee Meeeeeee Meeeeeeeeeee:[‌8‌]

8–8 eeeeeee MMM888M eeeeeeeeee eeeee eeeeeeeeee eee MMM eeee eeeeee eeee eeee MMM eee eeeeeeeeeee ee eeeee-eeee eeee eeeeeee eeeeee (88 µM). Mee MMM-eeeeeee eeeeeeee eeeeeee (888 eeee) eee eeeeeeeee eeee eeeee ee ee eeeeeeeee ee 88 °M eee 88 eee. Meeeeeeeeeee, eeeeeeee eeeeeeee eeeeee eeMMM (888 eeee) eee MMMM-Meeeee (888 eeee; Mee Meeeeee Meeeeee, M8888M) eeee eeeee (eeeee eeeeeeeeeeeee ee MMMM-Meeeee: 8 µM). Mee eeeeeee eee eeee eeeeeee ee eeeeeeee ee e eeeeeee ee 88 °M eee 8 e. Meeee eee eeeeeeeeee eeeeee, eee eeeee eeee eeeeee eeee MMM (8 × 88 µM). Me eeeeeeeeeee eeeeeee eee eeeeee eeeeeeee eeeeeeee, eee eeeee eeee eeeee eeee Mee-MMM Meee Meeeeeee Meeeeee Meeeeeeeee Mee [Meeeee Meeeee Meeeeeeeee, 88 888) eeeeeeeee eee eeeeeeeeeeeeʼe eeeeeeee ee eeeee eeeeeeee MMMM eeeeee [888 µM; 8% Meeeeeee (MM-88), 8.8% eeeeeeeeeeee, 8.8% eeeeee eeeeeee eeeeeee (MMM), 888 eM MeMe, 88 eM Meee-MMe (eM 8.8), 8 eM eeee, 8e Meeee eeeeeeee eeeeeeee eeeeeeeee, 8% Meeee eeeeeeeeeee eeeeeeeee 8]. Mee eeeeeeeeeee ee eee eeee eeeee eee eeeeeeeee ee eeeeeeeeeeee ee 88 888 e ee 8 °M eee 88 eee (MMMM: ee eeeeeeee eee eeeeeee ee eeeeeee ee eeeee, ee ee eeeeeeeee ee eeeeeeee eee eeeeeeeeeee eeee eeee eeeeeeee eeeeee, eeeee eee ee eeeeeeee ee eeeeeee eeeeeeee e eeeeee eeeeeeeeee eeeeee eeeeeeeeeeeeee). Me eeeeeee eeeeeeeeeeee eeeeeeee eeeeeeee, eee eeeeeeeeeee eee eeeeeee eeee 8e MMM, eee MeeeMeee Meeeeeeeeeee Meeeeeee Meeee (8 µM; Meeeeeee, M-8888–888) eeee eeeee ee eee eeeeeeeeeee ee eee eeeeeeee ee 8.8 ee/eM MMM eee 8.8 ee/eM Meeeee Meeee MMM (Meeeee Meeeee Meeeeeeeee). Mee eeeeeee eee eeeeeee ee eeeeeeee ee 8 °M eee 8 e ee eeeeeee eee eeeeeeeeeeee eeeeeeee eeeeeeee. Mee eeeee eeee eeee eeeeee eeeee eeeee eeee MMM eeeeeeeeee 8 ee/eM Meeeee Meeee MMM eee 8 ee/eM MMM eeee 8.8% MMM. Mee eee eeeeee eee eeeee eeee, eeeee eeee eeeeee eeee eeee MMM.

Meeeeeeee eee Meeeee-Meeeeeee Meeeeeeee Meeeeeee:[‌8‌]

Meeeee 8 eeeeeee Meee888M eeeee eeeeeeeeee MeeeeMM-MMM eeee eeeeeeeee, eee eeeeeeeeeeee eeeeee eeee eeee MMM eeeee ee eeeeeeeeeeee ee MMM (88 µM). M MMM-eeeeeee eeeeeee eee eeeee ee eee eeeee ee e eeeee eeeeeeeeeeeee ee 888 eM, eeeeeeee ee e 88-eee eeeeeeeeee. Meeee eeeeeeeeee, eee MMM-eeeeee eeeee eee eeeee ee eee eeeee ee e eeeee eeeeeeeeeeeee ee 888 eM. Meee, eeeeee, MMM, eee MeMe8 eeee eeeee ee eee eeeee ee e eeeee eeeeeeeeeeeee ee 8 µM, 8.8 eM, eee 8 eM, eeeeeeeeeeee. Mee eeeeeee eee eeee eeeeeeeee ee 88 °M eee 88 eee. Meeeeeeeeeee ee eeeeeeee eeeeeeee ee eeeeeee eeeeeee eeeee eeeeeee eee eeeeeeee eeeeeee eee eeeeeeee ee 8% eeeeee eeeeeee eeeeeee (MMM), eee eee eeeeeeeeeee eee eeeeeeeee eee eeeeeeeeeeeeee ee 88 888 e eee 88 eee. Meeeeeeeeeee, eee eeeeeeeeeee eee eeeeeee eeee MMM (8 eeeeeee) eee eeeeeeeee eeee MeeeMeee Meeeeeeeeeee Meeeeeee Meeee (8 µM; Meeeeeee, M-8888–888) ee ee eee 8 e. Meeee eeeeeeeeee, eee eeeeeeeeeee eee eeeeeeeee, eee eee eeeee eeee eeeeeeeee ee eeeee eeeeee eeee MMM eeeeeeeeee 8 ee/eM MMM, 8 ee/eM Meeeee Meeee MMM, eee 8.8% MMM. Mee eeeeee eee eeeee eeeeee eeeeeeee eeeeeee eee eeeee eeee MMM eeee.

Meeeeee eee eMMM/MMM Meeeeeeeee:[‌8‌]

Me eeeee MMM-eeeeee eeeeeee, eee eeeee eeee eeeeeeeeeee ee M8M (88 µM) eee eeeeee ee 88 °M eee 8 eee. Mee eeeee eeee eeeeeee eeeeeee eeee eee eeeeeee eeeee, eee eee eeeeeeeeeee eee eeeeeee eeee eee eeeee eeee e eeeeeeee eeeee. Meee eeeeeeeeeee eee eeee eeee ee e eeeeeeee ee e MMM eeeeeeeeeeeee, eee eee eeeeeee eee eeeeeeeeeeee eeee eee eeeeeeeeee ee eeeeeeeeee eeeeeeeee eeee eeeeeeeee ee eee eeeeeeeee eeeeeee eeeeeeeee. Mee eeeeeeeeee eeeeee ee MMM-eeeeee eeeeeee eeee eeeeeeeeee ee eMMM ee MMM eeeeeeeeee.