Science of Synthesis

Prati, L.; Lucaroni, L.; Cazzamalli, S.; Neri, D.; Oehler, S., Science of Synthesis: DNA-Encoded Libraries, (2024) 1, 535.

In principle, most methodologies used to validate DEL-derived hits can be applied in the presence (on-DNA) or absence (off-DNA) of a DNA tag, as shown in Figure 10.[‌18‌,‌21‌] In this section, we will describe the experimental procedures for off-DNA hit validation which are frequently applied for the characterization of hit compounds derived from ESAC libraries. The use of a trifunctional linker to connect fragment pairs with a fluorescent moiety allows the performance of affinity measurements by fluorescence polarization (FP; Figure 10, part E; with fluorescein-bearing hits measured against target proteins in solution), enzyme-linked immunosorbent assay (small molecule ELISA; Figure 10, part F; with fluorescein-bearing hits measured against immobilized target proteins), and co-elution experiments.[‌10‌,‌20‌,‌22‌] Label-free affinity measurement techniques include surface plasmon resonance (SPR) measurements on a BIAcore instrument and isothermal titration calorimetry (ITC).[‌6‌,‌20‌] In on-DNA surface plasmon resonance measurements (Figure 10, part C), the DNA is used as analyte or immobilized on a streptavidin (SA) coated chip to inject the target protein as analyte. In off-DNA surface plasmon resonance (SPR) measurements (Figure 10, part D), the hit serves as analyte or is immobilized on the chip to be exposed to different protein concentrations.

Meeeeee eeeeeee eeeee eeee eee ee eeeeeeeee ee eeeeee eee eee ee eeeeeeeeee eee eeeeeeeeee eeeeeeeeee eeeeeee eeeeeeeeee eeeeeeeeeeeeee (MMM), eeeeeeeeeeee eeeeee ee eeee ee eeeeeeeee ee eeeeeeee eeeeee (eeeeee eeeeeeeee), eeeeeee eeeeeeee eeeeeeeee (MMM) eeeeeee, eee eeeeeeee eeeeeeeee eeee eeeeeeeeeeee (MMMM).[‌88‌–‌88‌]

‌Meeeee 88‌ Mee Meeeeeeeee Meeeeeeeeeeee Meeee Mee ee Meeeeeeee ee eee Meeeeeee (ee-MMM) ee Meeeeee (eee-MMM) ee e MMM Mee[‌88‌,‌88‌,‌88‌–‌88‌]

Meeeeeeeeeee Meeeeeeee

Meeeeeeeeeee Meeeeeeeeeee (MM):[‌88‌,‌88‌]

Mee eeeeeeeeeeeee eeeeeee eee eee eeeeeeeee ee eee eeeeeeeeee eeeeeee eeeeee ee eeeee e eeeeeeeeeeeee ee 8–888 eM. Meeeeeeee eeee eee eeeeeeeeee eeee eeeeeeeee ee MMMM eee eeeeeee ee eee eeeeeee eeeeee, eeeeee eee eeeee MMMM eeeeeeeeeeeeee eeeee 8% e/e. Meeeeeeee ee eee eeeeeeeee eeeeeeeeee eeeeee, eeeeeeeee eeeeeeeeeeee eee ee eeee, eeeeeeeee eeeeeeeeeee, MMMMMM-MMM-M, Me8M, eee Me8, eeeee eee eeee eeeeeeeee eee eeeeeeeeee. Mee eeeeeeee eeeeeeeeeeeee eeeeee ee eeeeee eeeee ee eeeeeeee eee eeeeeeee eeeeeeeeee. Meeeeeeee eeee eeee eeeeeeee ee eee eeeeee eeeeeee (MM ee eee eeeeeeeee eeeee) eee ee eeeeeeee ee eee eeeeeeee eeeeeeeeeeeeee (e.e., 8 eM eeeeeeee eeeee) eeeee eee eeeeeeee eeeeeeeee (MM ee eee eeeeeeeeee eeeee) eeeeee ee eeeeeeee ee eeee eeeeeeee eeeeeeeeeeeeee (e.e., 888 eM eeeeeeee eeeee). Meeeeeeeeeeeee ee eee eeeeeeee eeeee eee eeeeeeee eeee e Meeeeeee eeeeeeeeeeeeeeeee ee eee eeeeeeeeee eeeeee eee eeeeeeeeee eeeee ee eee eeeee eeeeeeeeee eeeeeeeeeee; eee eeeeeee: ε(MMMM) ee eeeeeeeee-eeeeeeee eeeeee (MMM) ee 888 ee: 88 888 ee−8 · M−8.

M eee-ee-eee eeeeee eeeeeeee ee eee eeeeeee eee eeeeeeee ee eeeee eeeeeeeeeee (e.e., 888-eeee eeeeeeeeee #888 888, Meeeeee Mee-Mee; eeeeeee eee eeee ee ee eeeeeeee eeee eeeeeee ee eee eeeeeeeeee) ee eeeeeeee ee eeeeeee eeeeee (8 µM) ee eee eeeee eeeeeee eee eeeee eeee, ee eeeee eeeeeee (88 µM) eee eeeee. 8 µM eeee eeeeeeeeeee eeee eee eeeee ee eee eeee eeee eeeeeeeeeeee, eeeeeeeee eeeeeeeee ee eee eeee eeeeee eeeeeeeeee ee eee eeee eeee. Meeeeeeeeeee, eee eeeeeeeeeeeee eeeeeee eee (8 µM) eee eeeee ee eeee eeee, eeeeeeee ee eeeeeeeeee eee 88 eee ee ee ee eee eeee. Me eeeeeee eeee eeeeeee, eee eeeee eee eeeeeee eeeeeeeeeee (8 eee ee 8888 eee) eeeeee eeeeeeeee eee eeeeeeeeee ee e eeeeeeeeee eeeeee ee eee eeeeeeeeee eeee (e.e., eeee MMMM: λMeeeeeeeee = 888 ± 88 ee, λMeeeeeee = 888 ± 88 ee).

Meeee-Meeeeeee Meeeee-Meeeee Meeeeeeeeeeee Meeee (MMMMM):[‌88‌]

Mee eeeeeee eee eeeee ee e eeeeeeeeeee eeee-eeeeeeee MeeeMeee (eeeeeeeeee eeeeeeeeeeeeee) ee MeeeeeeMeee (eeee eeeee eeeeeeeeeeee eeeeeeee) 88-eeee eeeee ee 888 µM (888 eM) eee eeee eee eeeeeeeeeeeeee eeeeeeeee ee 8 °M ee 8 e ee ee. Mee eeeeeeeeeee eee eeeeeeeee, eee eee eeeee eeee eeeeeee eeee 8% eeee eeeeee ee eeeeeeeee-eeeeeeee eeeeee (MMM; 888 µM) eee 88 eee ee ee. Mee eeeeeeeeeee eee eeeeeee, eee eee eeeee eeee eeeeee eeee MMM (8 × 888 µM). M eeeeee eeeeeeee ee eee eeeeeeeeeeeeeee eee ee MMM (e.e., 888 µM ee 8 eM, 8:8 eeeeeeee) eee eeeeeeee, eee 888 µM eee eeeee ee eeee eeee, eeeeeeee ee 88 eee eeeeeeeeee ee eee eeee ee ee. Mee eeeeeeeeeee eee eeeeeeeee, eee eee eeeee eeee eeeeee eeee MMM (8 × 888 µM). Meee, eeee-eeeeeeeeeee MeM (888 eM) ee eeMe (888 eM) ee MMM (888 µM) eeeeeeeeeeee eeee 8% eeee eeeeee eee eeeee eee eeeeeeeee eee 88 eee ee ee ee eee eeee. Mee eeeeeeeeeee eee eeeeeeeee, eee eee eeeee eeee eeeeee eeee MMM (8 × 888 µM). M 8:8888 eeeeeee eeeeeeeeeee eeeeeeeeee (MMM)-eeeeee eeeeeee (e.e., eeeeeee M-MMM ee eeee-Mee-MMM) ee MMM (888 µM) eeeeeeeeeeee eeee 8% eeee eeeeee eee eeeee ee eeee eeee eee eeeeeeeee eee 88 eee ee ee ee eee eeee. Mee eeeeeeeeeee eee eeeeeee, eee eee eeeee eeee eeeeee eeeee eeeee eeee MMM eeeeeeeeeeee eeee 8.8% Meeee-88 eee eeee eeeee eeeee eeee MMM. Meeeeee, 8,8′,8,8′-eeeeeeeeeeeeeeeeeeee (MMM; 88 µM) eee eeeee ee eeee eeee eee eeeeeeeee ee ee ee eee eeee eeeee eee eeeeeeeee ee e eeeeeee eeee eee eeeeeeee. Mee eeeeeeee eee eeeeeeee ee eeeeeeee ee 8 M ee M8MM8 (88 µM) eee eee eeeeeeeeee eee eeeeeeee ee 888 ee (ee 888 ee) eeee e eeeeeeeeee eeeeee.

Me-eeeeeee Meeeeeeeee eeee e Meeeee Meee Meeeeeeee Meeeee (e.e., MM-88 eeeeee):[‌88‌]

M 8 µM eeeee eeeeeeee ee eee eeeeeeeeeeeee eeeeeee eee (88 µM) eee e 88 µM eeeee eeeeeeee ee eee eeeeeee (88 µM), eeee ee eee eeeeeeeeee eeeeeee eeeeee, eeee eeeee eee eeeeeeeee eee 8 eee ee ee ee eee eeee. Mee eeeeeee eee eeeeee ee e eee-eeeeeeeeeeee MM-88 eeeeee. Meeeeee eeeeee eee eeeee ee eeeee e eeeeee eeee eee eeeeeeeee (888 µM, eeeeeeeeeeeee 8 eeeee) eeee eeeeeeeee. Mee eeeeeee eeeeeeeeeeeee ee eeee eeeeeeee eee eeeeeeee eeee e Meeeeeee eeeeeeeeeeeeeeeee eeeee eee eeeeeeeeeeee eee eeeeeeee ee e eeeeeeeeee eeeeee (e.e., eeee MMMM: λMeeeeeeeee = 888 ± 88 ee, λMeeeeeee = 888 ± 88 ee).

Meeeeee Meeeeee Meeeeeeee (MMM) Meeeeeeeeeee eeee MMMeeee Meeeeeeeeee:[‌88‌]

Mee eeeeee eeeeeee eee eeeeeeeeeee ee e eeeeee eeee MM8 (eeeeeeeeee eeeeeeeeeeeeee) eeeeeeeee eee eeeeeeeee eeeeeeeee eee “eeeee eeeeeeee” (MMM/MMM), eeeeee ee eeeee e eeeeee eeeee ee 8888–8888 MMe (MMMM: eee eeeeee eeeee eeee eeeeee ee eee eeeeee eeeee eeee eeeeee eee eeeeee ee eeeeeeeee eeeeeee). Me eee eeeeee eeeee eeeeee ee eeeeeee, eeeeee eeeeeeeeee eeeeeee eeeeeeeeeeeee eee eeeeee eee eeeeeee ee eeeeeee eeeeee eeee eeeee eM (eeeee eee eM ee eee eeeeeeeeee eeeeeee, ee eee eee Meee eeeeee). Meeeeeeee, MMM-MM eee eeee ee eeeeeee eeeeee eee eeeeeeeeeeeeee eeeeeeeeee ee MM8 eeeee. Mee eeeeeeeee eeee eee eeeee eeeeeeeeeee eeeeeeeee ee eee eeeeeee eeeeeeeee (eeeeeeeeee ee eeeeeeeee eeee eeee MMM/MMM, eeeeeeee ee eeeeeeeee ee eeeeeeeeeeee). Meeeeeeeeeeee, eeeeeeeeeeee eeeeeeee eeee eeeeeeeeeee ee e eeeeee eeee MM. Meeee, eee eeee eee eeeeeeeeeee ee eeeeeeeee 8 M MeMe ee 88 eM MeMM. Mee eeee eee eeeeeeeeeeee eeee eee eeeeeee eeeeee ee eeeeeee eeeeee eeeeeeeeee ee eee eeeeeeeeeeee eeeeeee ee eeeeeeeeee eeeeeeeeeeeee (e.e., 888 eM), eeeeeeee eeee eee eeeeeeeee eeeeeee ee eeeeeeeeeeee eeeeee eee eeeeeee eeeeeeeeee eeeee eeeeeeee eee eeeeeee eeeeee eeeee. Mee eeeeeeeee eeee eee eeeeeeee ee eeeeeeeee e 888 µM eeeeee eeeeeeee.

Meeee eeeeeeeeee eeeeeeeeeeeeee, eee eeee eee eeeeeeeeeeee eeee eee eeeeeeeeee eeeeeee eeeeee (8.88% Meeee-88 eee ee eeeee ee eee eeeeeee eeeeee ee eeeeeee eee-eeeeeeee eeeeeee). Mee eee eee eeeeeeeee ee eee eeeeeee eeeeee eee e eeeeee eeeeeeee eee eeeeeeee (eeeeeeeeee ee eeeeeeeee eeeee, eeeeeeeee ee eee eeeeeeee eee eeeeeeee eeeeeeee). Meee eeeee eee eee eeeeeee ee eee eeeeee eeee eeeeeeeee ee eeeeeeee eeeeeeee (e.e., MMMM) eee eeeeeeeeeeee eeeeeee ee eee eeeeeeeeee eeeeeee eeeeee. Meeeeee, eeeeeeee eeee e eeeeee eeeeee ee MMM eeeeeeeeeeee eee eeeeeee eeeeeeeeeee eeee eeee ee ee eeeeeeeee. Meeeeeeeee eeeeeeeeee ee eee eee ee eeeeeeeee eeeeeeeeeeeeee eeee eeeeeeeee eeeeeeeee ee eee eeeeeeeee eeeeeeee. Me eeeeeee, eeeeee eeee eee eeeeeeee eeeee, eeeeeeee ee eeeeeeeeee ee eee eee eeee eee ee eeee eeeeeeeeeeeeee. Meeeeee eeee, eeeeeeeeeeee eeee, eee eeee eeee eeeeee ee eeeeeeeee eeeee ee eeeeeeeeeeee eeeeeee. Meeeeeee eeeeeeeeeeee eeeeeeeeee eeeeee ee eeeeeeeeee ee ee eeee ee eee eeeeeeee eeeee eeee eeeeeeeee eeeeeee eeeeeeeeee eee eeeeeeeeeee eeeeeee (eeeeeee ee eeeeee eeeeeeee eeee eeeeeeeee eee eeee eeeeee eeeeeeeeeeeee).

Mee eeeeeee eeeeeeeee eee eeee ee eeeeeee, eeeeeeeeeeee eee eeeee eeeeeeee, eeeee ee eeeeeeeeeeeeee eeee e eeeeeeee eeeee (MM8 eeee) ee eeeeee (MM eeee), eee eeeeeeeee eeeeeeeee eeeeeeeeeeeeee ee eee eeeeeee ee eeeeeee.

Meeeeeeeee Meeeeeeee Meeeeeeeeee (MMM):[‌8‌]

M eeeee eeeeeeee ee eee eeeeee (e.e., 88 µM) eee e eeeee eeeeeeee ee eee eeeeeee (e.e., 888 µM) ee eee eeeeeeeeee eeeeeee eeeeee eeee eeeeeeee. Mee eeeeee eee eeeeeeee eeee eee eeeeeee eeeee eeeeeeeeee eeeeeeeeee eee eee eeee eeeeeeee eee eeeeeeee eeee eeee ee ee eeeeeeeeee eeeeeeeee eeeeeeeeeee.

References