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. 2019 Oct 30;141(43):17057-17061.
doi: 10.1021/jacs.9b08085. Epub 2019 Oct 22.

Selection of DNA-Encoded Libraries to Protein Targets within and on Living Cells

Bo Cai  1 , Dongwook Kim  1 , Saeed Akhand  1 , Yixing Sun  1 , Robert J Cassell  1 , Aktan Alpsoy  1 , Emily C Dykhuizen  1 , Richard M Van Rijn  1 , Michael K Wendt  1 , Casey J Krusemark  1
Affiliations

Selection of DNA-Encoded Libraries to Protein Targets within and on Living Cells

Bo Cai et al. J Am Chem Soc. .

Abstract

We report the selection of DNA-encoded small molecule libraries against protein targets within the cytosol and on the surface of live cells. The approach relies on generation of a covalent linkage of the DNA to protein targets by affinity labeling. This cross-linking event enables subsequent copurification by a tag on the recombinant protein. To access targets within cells, a cyclic cell-penetrating peptide is appended to DNA-encoded libraries for delivery across the cell membrane. As this approach assesses binding of DELs to targets in live cells, it provides a strategy for selection of DELs against challenging targets that cannot be expressed and purified as active.

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Figures

Figure 1.
Figure 1.
Cell penetration of DNA is enabled by cCPP12. (A) Live cell entry of a dsDNA-cCPP12 conjugate. 20-base dsDNAs containing FAM labels and a cCPP12-FAM conjugate were incubated with HMLE cells at 2 μM for 4.5 h and imaged. (B) qPCR analysis of DNA cell entry. (C) CAPA performed with 5 μM 20-base dsDNA. P-values were calculated using unpaired Student’s two-tailed t test: ***P < 0.001.
Figure 2.
Figure 2.
Assessing DNA-linked ligand-target interactions inside live cells. Affinity labeling of CBX7-ChD (A) and eDHFR (B) with DNA-linked ligands analyzed by SDS-PAGE.
Figure 3.
Figure 3.
DEL selection against Halotag-CBX7-ChD within living cells. (A) Recovery and enrichment of tested DNA constructs in a mock selection at 1000-fold dilution. (molar ratios P1/N1/N2 = 1:500:500; [P1] = 1 nM). (B) Correlation plot of live cell and cell lysate selections. (C) The parental peptide for a DNA-encoded PSL and a heat map of live cell enrichments. The color scale represents the log enrichment relative to the nonligand control. See Figures S6 and S7 for structures. (D) IC50 values of off-DNA hits in binding assay to Halotag-CBX7-ChD. Line and symbol colors correspond to live cell enrichment values from the selection, as indicated on the color scale.
Figure 4.
Figure 4.
DEL selection against SNAPtag-DOR on live cell surfaces. (A) Cross-linking approach to DEL selections on live cell surfaces. BG-biotin = O-(6)-benzylguanine-biotin. (B) Parental peptides for DNA-encoded PSLs and heat maps of live cell enrichments. The color scale represents the log enrichment relative to the nonligand control. See Figures S8 and S9 for structures. (C) Ki values of off-DNA hits binding to SNAPtag-DOR. Line colors correspond to live cell enrichment values from the selection, as indicated on the color scale. *Ki of HA-P(2)-#5 was determined on Prolink-tagged DOR (DiscoverX).
Scheme 1.
Scheme 1.
Cross-linking Approach to Live Cell DEL Selections
See this image and copyright information in PMC

Comment in

  • DELs Inside Cells.
    Roy A, Kodadek T. Roy A, et al. Trends Pharmacol Sci. 2020 Apr;41(4):225-227. doi: 10.1016/j.tips.2020.01.007. Epub 2020 Feb 4. Trends Pharmacol Sci. 2020. PMID: 32033772

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