Science of Synthesis

Huang, Y.; Li, X., Science of Synthesis: DNA-Encoded Libraries, (2024) 1, 594.

Using DPAL, our laboratory has developed several photo-crosslinker-based DEL selection methods. A “labeling-then-digestion” strategy has been employed for selection against unmodified and in-solution proteins.[‌8‌] As shown in ‌Scheme 2‌, part (a), a short DNA bearing a photo-crosslinker (PC-DNA) is hybridized at the common primer binding site of the DEL to establish a covalent linkage between the potential binders and the target protein. The DNA tag of the binder is stabilized in the form of a pseudo-hairpin protein–DNA complex, so that it can still survive the following nuclease digestion, while the DNA tags of the non-binders are digested. As a result, only the DNA barcodes linked to the binders will be PCR-amplified and enriched from the selection. Alternatively, gel purification can also separate the binders from the non-binders by polymerase-extending the PC-DNA [‌Scheme 2‌, part (b)].[‌9‌] Finally, the library itself may be designed in a hairpin format with a built-in photo-crosslinker [‌Scheme 2‌, part (c)].[‌10‌] To prepare the hairpin-shaped DEL, a universal non-coding DNA sequence complementary to the primer binding site is ligated to the library DNA. After library–target protein incubation, the selection mixture is subjected to UV irradiation and gel purification. The covalent binder DNA–protein complex can be easily isolated from the non-binders.

‌Meeeee 8‌ Meeee MMM Meeeeeeee Meeeeee Meeee ee MMMM[‌8‌–‌88‌]

Meeee eeee eeeeeeeeeeee e eeeeeeee eeeeeeeeee eeeeeee eee eeeeee eee eee eeeeeeeee eee eeeeeeeee, eee eeeee-eeeeeeeeeee eee eeee eeee eeee ee eee eeeeeeeee ee MMM-eeeeeee eeeeeee eeeeeeeee (MMMMe) eee eeeee-eeeeee MMM eeeeeeeeeeee. Me eeeee ee ‌Meeeee 8‌, eeee (e), eeee eee eeeeeeee ee eee eeeeee eeeeeee, eee eeeeeeee eeeeeeeeeee eeeee eee-eeeeeeeee eeeee eeeee eee eeeeeeeee ee eee eeeeeeee eeeee ee eeee eeeeeee eeeeeeee, eee eeeee-eeeeeeeeeeee eeeeeeeeee eeeeeee eee eee MMM eeeeeee eee eeee eeee eee eeeeeee eeeeeeeeeee eee eeeeeeee eee MMM eeeeeeee. Me eeeee ee eeeeee MMM eeee e eeeee eeeeee, eeeeeeeeeee eeeeeeeee eee ee eeeeeeeeee eeeeeee eee eeeeee eeeeeee eee eeeeee eeeeeeeeeeee [‌Meeeee 8‌, eeee (e)],[‌88‌] eeeee eeee-eeeeeee MMMM eee eeeeeeee eeeee eeeeee eeeee eeeeeee eeeee eeeeeee [‌Meeeee 8‌, eeee (e)].[‌88‌]

‌Meeeee 8‌ MMMM Meeeeeeeee Meeee Meeee-eeeeeeeeeeee[‌88‌,‌88‌]

Meeeee, MMMM ee e eeeeeeeee eeeeeeee ee MMM eeeeeeeeee. MMMM eee ee eeeeeeeeeee ee eee eeeeeeeee eeeeeee eeeeeeee eeeeeeee ee eeee eeeee. Meeeee eeeeeeeee ee MMMM, eee eeeeeeee-eeeeeee eeeeee ee eeeeeee eeee e MMM eee, eeeee eeee eeee ee e eeeeee eeeeee ee eeee ee eee eeeeeee, eeeeeee eeeeeeeee eeee eeee eeeeee eeeeeeeeeeeee eee eeeeee eeeeeeeeeee (‌Meeeee 8‌).[‌88‌]

‌Meeeee 8‌ Meee-Meee Meeeeeeee Meeeeeee Meeeeee Meeeeeee Meeeeeee eeee MMMM[‌88‌]

Meeeeeeeeeee Meeeeeeee

Meeeeee Meeeeeeeeeeee; Meeeeee Meeeeeeee:[‌88‌]

8 M MeMMe (8.8 eeeeee; eM 8.8) eee eeeee ee eee eeeeeee MMM eeee (eeeeeee eee eeeeee eeeeee ee eeee eeee 888 µM) ee eeeeee eee eeeeee eM ee ~8.8. Meee, 88 ee/eM eeeeeeee (8.888 eeeeee) eee eee MeMM (8.8 eeeeee) eeee eeeee. Mee eeee eee eeeeeeeeee ee −88 °M eee ee eeeee 8 e, eee eeee eeeeeeeeeee ee 88 888 e eee 88 eee ee 8 °M. Mee eeeeeeeeeee eee eeeeeeeee, eee eee eeeeee eee eeeeeeeee eeeeee eeee eeee eeee 88% MeMM. Mee eeeeee eee eeeeeeeeeee ee 88 888 e eee eeeeeee 8 eee ee 8 °M, eee eeeeeeeeeee eee eeeeeeeee, eee eee eeeeee eee eeeee eeeee e MeeeeMee. Mee eeeeeeeee eeeeee eee eeeeeeeee ee ee eeeeeeeeeee eeeeee eee eeeeeeeeee eeeeeeee ee eeeeeeeeeee.

Meeeeeee Meeeeeeee MMM Meeeeeeee [‌Meeeee 8‌, Meee (e)]:[‌8‌]

Mee MMM, eee MM-MMM (8 ee eeeeeeeeeeeee ee MMM), eee eee eeeeeee eeeeee (8:8:8 eeeee) eeee eeeeeee eeee 8M MMM ee e eeeee eeeeeeeeeeeee ee 8.88 µM eee eeee eee eeeeeee eee eee eeeeeee. Mee eeeeeee eee eeeeeeeee ee 8 °M eee 8 e, eee eeee eeeeeeeeee (888 ee) ee eee eee 88 e ee e Meeee MM MMM eeeee eeeee eeeeee. M eeeeee (88 µM) ee eee eeeeeee eee eeeeeeeeeee ee e eee eeeeeeeeee eeee, eee 88e Meeeeeeeeee M eeeeee (8.8 µM), MeeM (88 eeeee/µM; 8 µM, 88 eeeee), eee M8M (88.8 µM) eeee eeeee ee eeeee e eeeee eeeeee ee 88 µM. Mee eeeeeee eee eeeeeeeee ee 88 °M eee 8 e. Meeee eee eeeeeeeee, eee eeeeeeee eeee eeee-eeeeeeeee ee 88 °M eee 88 eee ee e eee eeee; eee eeeeee eeeeeee eeeeee ee eeeeeee ee eee eeeee ee 8:8888 eeeeee MMM.

MMM Meeeee-eeee-Meeeee MMM Meeeeeeee [‌Meeeee 8‌, Meee (e)]:[‌8‌]

Mee MMM, eee MM-MMM (88 ee eeeeeeeeeeeee ee eee MMM), eee eee eeeeeee eeeeee (8:8:8 eeeee) eeee eeeeeee eeee 8M MMM eee 8.8 M MeMe ee e eeeee eeeeeeeeeeeee ee 8.8 µM eee eeee eee eeeeeee eee eee eeeeeee. Mee eeeeeee eee eeeeeeeee ee 8 °M eee 8 e, eee eeee eeeeeeeeee (888 ee) ee eee eee 88 e ee e Meeee MM MMM eeeee eeeee eeeeee. 88e M8 MMM eeeeeeeeee eeeeee (8.8 eeeeee), 88 eM Meeeeeeeeeeeeee Meeeeeee Mee, eee 88 eM MMM eeee eeee eeeee ee eee eeeeeeee eeee, eeeeeeee ee M8 MMM eeeeeeeeee [8 µM (8 eeeee) eee 888 µM ee eeeeeeee eeee] ee 8 °M. Mee eeeeeeeee eeeeeeeee eeee eeeeeee eee ee 88 °M eee 8 e. Mee eeeeee eee eeeeeeee ee MeMM eeeeeeeeeeeee (eee eee eeeeeee eeeeeeeee eeeee), eee eee eeeeee eee eeeeeeeee eeee M8M (88 µM) eee 8e eeeeeee eeeeee (8 µM), eee eeee-eeeeeeeee ee 88 °M eee 88 eee eeee e eee eeee. Mee eeeeeeee eeee eeeeeeeee ee MMM-MMMM eeeeeeee. Mee eeeeeeeee eee eeeee eeee eeeeeee eeee 8 × 8 ee eeeeeee eee eeeeeeeeeee ee e eeeeeeeeeeeeeee eeee. M eeeee eeee eee eeeeeee eeee eee eee ee eee eeee eeee e eeeee eeeeee, eee eee eeeeeeeeeeeeeee eeee eee eeeeee ee e eeeeee eeee eee eeeeeeeeeee. Meeee eeeeeeeeeeeeee, eee eee eeeeeee eeeeee eeee eeeeeeeee. Mee eee eeeeeeeee ee eee eeeeee ee eee eeeee eeee eeee eeeeeeeee, eeeeeeeee ee 88 °M eee 88 eee ee 8M MMM eeeeee, eee eeee eeeeeeee eee 88 e ee 88 °M. Mee eeeeee eee eeeeeeee ee eeeeeeee eee 8 eee ee 8888 eee. Mee eeeeeeee eee eeeeeeeee eeee eeeeeeeee ee 88 °M eee 88 eee, eee eeee eeeeeeeeeee eeeeee ee eee eee eeeeeeee eeeee; eeee eeeeeee–eeeeeee eeeeeee eee eeeeeeee eeeee. Mee eeee-eeeeeee eeeee eeee eeeeeeee, eee eee MMMe eeee eeeeeeeee ee MeMM eeeeeeeeeeeee. Mee eeeeee eee eeeeeeeee ee M8M (88 µM) eeeeee MMM.

Meeeee-eeee-Meeeee MMM Meeeeeeee [‌Meeeee 8‌, Meee (e)]:[‌88‌]

Mee MMM, eee MM-MMM, eee eeeeee MMM (8:8:8 eeeee) eeee eeeeeee eeee M8M (888 µM) ee e eeeee eeeeeeeeeeeee ee 8 µM. Mee eeeeeee eee eeeeee ee 88 °M eee 88 e, eee eeee eeeeee eeeeee ee ee. 88e M8 MMM eeeeee eeeeee (88 µM) eee M8 MMM eeeeee (8 µM, 888 eeeee) eeee eeeee ee eee eeee ee 8 °M, eee eee eeeeeeee eeeeeeee eee eeeeeee eee ee 88 °M eee 88 e. Mee eeeeeee eee eeeeeeeee ee MeMM eeeeeeeeeeeee (eee eee eeeeeee eeeeeeeee eeeee) eee eeeeeeeeee MMMM eeeeeeee. Mee eeeeeee eeeee eeee eeeeeee eee eee eeeeeeee eeee eeeeeeeee eeee 8e MM eeeeee eeeeeeeee eee eeeeeeeee eeeeeeeee eeeee, eee eeee eeeeeeee ee eeeeeee eeeeeeeeeeeee. Mee eeeeeee eeeeeee (888 eeee) eee eee eeeeeee eeeeee (888 eeee) eeee eeeeeee eeee 8M MMM eee 8.8 M MeMe ee e eeeee eeeeeeeeeeeee ee 8.8 µM eee eeee eee eeeeeee eee eee eeeeeee. Mee eeeeeee eee eeeeeeeee ee 8 °M eee 8 e, eee eeee eeeeeeeeee (888 ee) ee eee eee 88 e ee e Meeee MM MMM eeeee eeeee eeeeee. Mee eeeeeee eee eeeeeeeeeee eee eee eeeeee eee eeeeeeeee eeee M8M (88 µM) eee 8e eeeeeee eeeeee (8 µM), eee eeee-eeeeeeeee ee 88 °M eee 88 eee eeee e eee eeee. Mee eeeeeeee eeee eeeeeeeee ee MMM-MMMM eeeeeeee. Mee eeeeeee eeeee eeee eeeeeee eee eee eeeeeeee eeee eeeeeeeee eeee 8e MM eeeeee eeeeeeeee eee eeeeeeeee eeeeeeeee eeeee, eee eee MMM eee eeeeeeee ee MeMM eeeeeeeeeeeee. Mee eeeeee eee eeeeeeeee ee M8M (88 µM) eeeeee MMM.

Meeeee-MMM Meeeeee MMMM Meeeeeeee [‌Meeeee 8‌, Meee (e)]:[‌88‌]

Mee eeeeeeeeee MMM eee eeeeee-MMM (8 ee eeeeeeeeeeeee ee eeeeeeeeee MMM) eeee eeeee ee e eeeeee ee 8.8 M MMMM/8.8 M MeMe (eM 8.8) ee e eeeee eeeeeeeeeeeee ee 888 eM eee eeeeee MMM eee 888 eM eee eeeeeeeeee MMM. Mee eeeeeee eeee eeeeeee eeeeee ee 88 °M eee eeeeee eeeeee ee ee. Me eeeee eeeeee ee eee eeeeee eeeeeee eee eeeee eeee eee eeeeee eee eee eeeeeeeeee eeeeeee eee eeeeeee ee e eeeee eeeeee ee 888 µM. Mee eeeeeee eeee eeeeeeeee ee 88 °M eee 8 e, eee eeee eeeeeeeeee (888 ee) ee eee eee 88 e eeeee e Meeee MM MMM eeeee eeeee eeeeee. 8% Meeeee eeeeeee eeeeeee (MMM; 88 µM) eee eeeee eee eee eeeeeeee eeee eeeeeeeee ee 88 °M eee 88 eee eeee e eee eeee. Mee eeeeeee eee eeeeeeee ee eeeeeee eeeeeeeeeeeee (eee eee eeeeeee eeeeeeeee eeeee) eee eee eeeeeeeee eeeeeee eeee eeeeeeee eeeee 88% MMM-Meee eeeeeeeeee MMMM eee. Mee eeeeeee eeeee eeee eeeeeee, eee eee eeeeeeee eeee eeeeeeeee eeee 8e MM eeeeee ee eee eeeeeeeee eeeeeeeee eeeee eee eeeeeeee ee eeeeeee eeeeeeeeeeeee. Mee eeeeee eee eeeeeeeee ee M8M (88 µM) eeeeee MMM.

Meee-Meeeeee MMMM Meeeeeeee [‌Meeeee 8‌, Meee (e)]:[‌88‌]

Mee eeee eeeeeeeeee MMM (8 ee eeeeeeeeeeeee ee eeee eeeee; 8:8 eeeee) eee eeeee eeee 8M MMM. Me eeeee eeeeee ee eee eeeeeee eeeeee eee eeeee ee eee eeee, eeeee eee eeeeeee ee e eeeee eeeeee ee 888 µM. Mee eeeeeee eee eeeeeeeee ee 88 °M eee 8 e, eee eeee eeeeeeeeee (888 ee) ee eee eee 88 e eeeee e Meeee MM MMM eeeee eeeee eeeeee. 8% eeeeee eeeeeee eeeeeee (MMM; 88 µM) eee eeeee eee eee eeeeeeee eeee eeeeeeeee ee 88 °M eee 88 eee eeee e eee eeee. Mee eeeeeee eee eeeeeeee ee MeMM eeeeeeeeeeeee eee eee eeeeeeeee eeeeeee eeee eeeeeeee ee 88% MMM-Meee eeeeeeeeee MMMM (eeeeeeeeee 88% eeeeeeeee). Mee eeeee ee eeeeeeeeeee MMM eeeeeeee eeee eeeeeee eee eee eeeeeeee eeee eeeeeeeee eeee 8e MM eeeeee eeeee eee eeeeeeeee eeeeeeeee eeeee, eee eeee eeeeeeee ee MeMM eeeeeeeeeeeee (eee eee eeeeeee eeeeeeeee eeeee). Mee eeeeeeeee eeeeeee eeee eeeeeeeee ee M8M (8 µM), eee M8 MMM eeeeee eeeeee (8 µM), 88 µM eeeee eeeeee (8 µM), eee 88 µM eeeeeeee eeeeee (8 µM) eeee eeeee. Mee eeeeeee eee eeeeeee eeeeee ee 88 °M eeee e eee eeee eee eeee eeeeee eeeeee ee 88 °M. M8 MMM eeeeeeeeee (8 M/μM; 8 µM) eee 8.8 eM eMMM (88 µM) eeee eeeee ee eee eeee, eee eee eeeeeee eee eeeeeeeee ee 88 °M eee 88 eee. M8 MMM eeeeee (88 M/μM; 8 µM) eee 88 eM MMM (8 µM) eeee eeeee ee eee eeee, eee eee eeeeeee eee eeeeeeeee ee 88 °M eee 8 e eee eeee eeeeeeee ee MeMM eeeeeeeeeeeee. Mee eeeeeeeee eeeeeee eeee eeeeeeeee eeeeee MMM eeeeeeeeeeeee.

Me-Meee MMM Meeeeeeee (‌Meeeee 8‌):[‌88‌]

Mee eeeee eeee eeeeeeeee eee eeeeee eeeee eeeee eeee 8M MMM eeeeee ee eeeeee eeeeeeeeeeee eee eeeeeeeeeeeeee ee 888 e eee 8 eee. Mee eeeeee-eeeeeeeeee eeeeeee eeeee eee eeeeeeeee eeeee (8 µM eeee, 88 ee eeeeeeeeeeeee ee eeee eeeee) eeee eeeee ee 8M MMM eeeeee; eee eeeeeee eee eeeeee ee 88 °M eee 8 eee, eee eeee eeeeee eeeeee ee ee. Mee eeeee eeee eeeeeeeeeee ee eee eeeeeeee eeeee eeeeeee, eeeee eee eeeeeeeee ee 8 °M eee 8.8 e eee eeee eeeeeeeeee (888 ee) ee eee eee 88 e eeeee e Meeee MM MMM eeeee eeeee eeeeee. Mee eeeee eeee eeeeee eeeee eeeee eeee 8M MMM eeeeee ee eeeeee eeeeeeeee eeeeee eee eeee eeeeeeeeeee ee e 8 µM eeee ee eeeeeeeeeeee eeeee (88 ee eeeeeeeeeeeee ee eeeeeee eeeee) ee 8M MMM eeeeee ee ee eee 88 eee. Mee eeeee eeee eeeeee eeeee eeeee eeee 8M MMM eeeeee ee eeeeee eee eeeeeee eeeee–eeeeeeeeeeee eeeee eeeeeee. Mee eeeee eeee eeeeeeeeeee ee 8M MMM eeeeee (888 µM) eeeeeeeeee 8.8 µM MMM, eee eeeeeeeee ee 88 °M eee 8 e. Mee eeeee eeee eeeeee 88 eeeee eeee 8M MMM eeeeee ee eeeeee eeeeeee eeeeeee, eee eeee eeeeee ee 8M MMM ee 88 °M eee 88 eee. Mee eeeeeee eee eeeeeeeeeee ee 88 888 eee eee 88 eee, eee eee eeeeeeeeeee eee eeeeeeeee. Mee MMM eee eeeeeeee ee MeMM eeeeeeeeeeeee (eee eee eeeeeee eeeeeeeee eeeee), eee eee eeeeeeeee eeeeeee eeee eeeeeeeee eeeee ee MMM eeeeeeeeeeeee.

References