Polymerase-Extension-Based Selection Method for DNA-Encoded

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Letter

Polymerase-extension-based Selection Method for DNA-encoded Chemical Libraries Against Non-immobilized Protein Targets Bingbing Shi, Yuqing Deng, and Xiaoyu Li ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.9b00011 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Combinatorial Science

Polymerase-extension-based Selection Method for DNA-encoded Chemical Libraries Against Non-immobilized Protein Targets Bingbing Shi†, Yuqing Deng‡, and Xiaoyu Li*,‡ †Department

of Food Science, Tibet Agriculture and Animal Husbandry University, 100 Yucai Road West, Nyingchi, China 860000 ‡Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Email: [email protected] ABSTRACT: DNA-encoded chemical libraries (DELs) have become an important ligand discovery technology in biomedical research and drug discovery. DELs can be comprised of hundreds of millions to billions of candidate molecules and provide outstanding chemical diversity for discovering novel ligands and inhibitors for a large variety of biological targets. However, in most cases, DELs are selected against purified and immobilized proteins based on binding affinity. The development and application of DELs to more complex biological targets requires selection methods compatible with non-immobilized and unpurified proteins. Here, we describe an approach using polymerase-based extension and target-directed photo-crosslinking and its application to the interrogation of a solution-phase protein target, carbonic anhydrase II.

Screening large-scale libraries against biological targets is one of the most important approaches for ligand discovery in biomedical research and drug discovery. Various technology platforms of encoded libraries, such as phage display,1-2 mRNA display,3-4 yeast surface display,5 ribosome display,6 SELEX,7-8 have been developed. Recently, DNA-encoded chemical library (DEL) has become an increasingly important ultra-highthroughput screening platform for small-molecule-based ligand discovery.9-13 In a DEL, each small molecule is covalently conjugated with a unique DNA tag, serving as the identifier for the chemical structure of the compound, and the entire library can be prepared and screened against the target simultaneously at minute scale (fmol-pmol). Since the original concept was proposed by Brenner and Lerner in 1992,14 this field has seen significant advancements in both technological developments and real-world applications. Today, DEL has been widely adopted by pharmaceutical companies in drug discovery programs.9, 15-16 In most DEL selections, the library is incubated with an immobilized protein target. Bound ligands are separated from non-binders by washing before they are eluted under denaturing conditions. Eluted binders are then subjected to PCR amplification and DNA sequencing. Although being straightforward, the affinity-based selection method is not applicable to proteins that are not compatible to purification and/or immobilization, such as unpurified endogenous proteins, protein complexes, membrane proteins, and non-adherent cells. Moreover, with selections using purified protein targets, certain desirable biological features of the target are missing, such as the post-translation protein modifications and endogenous binding partners.17 In the early works by Brenner, Janda, Gallop and their respective co-workers, DELs were prepared on solid support so that soluble proteins could be directly used as the targets.18-19 More recently, Paegel, Kodadek and their respective coworkers have built large-scale DELs on solid phase and selected them against soluble proteins.20-22 Moreover, many strategies

compatible with soluble targets have also been reported, including the interaction-dependent PCR,17, 23 water-oil emulsion,24 covalent crosslinking,25-29 and biophysical methods.30 Recently, we reported a DEL selection method compatible with non-immobilized targets using a “ligate-crosslink-purify” approach (Figure 1a).31 In this method, a short DNA bearing a photo-crosslinker is tethered to the original library. The library DNA could form a hairpin structure; therefore, upon ligandtarget binding, UV irradiation ligates the encoding DNA strand with the target protein and forms a covalent protein-DNA conjugate that can be easily purified for hit deconvolution. However, this method requires additional steps for library modification (ligation with PC-DNA, photo-crosslinking DNA) and purification, which may be detrimental to the integrity of the library (e.g. compound stability) and result in low material recovery after purification. Here we report a simplified method that circumvents these issues and is also compatible with nonimmobilized targets. Previously, Neri and co-workers have employed polymerase-mediated extension as an encoding method in the DEL synthesis.32-35 We propose to use polymerase extension as a selection strategy. In our previous work, a short DNA strand conjugated with a photo-crosslinker is ligated to the distal end of the library DNA, which can loop back and form a hairpin structure to bring the crosslinker close to the compound (Figure 1a).31 Here, we directly hybridize a photo-crosslinking DNA (PC-DNA) at the primer-binding site (PBS), which has a common sequence for all library members. After target incubation and UV irradiation, for binders, the PC-DNA will covalently capture the target protein and form a DNA-target conjugate (Figure 1b). Next, DNA polymerase is added to copy the encoding DNA sequences. The extended DNA-target conjugate can be gel-purified and subjected to hit deconvolution with PCR amplification and DNA sequencing. For non-binders, although polymerase extension also occurs, the PC-DNA is not crosslinked to the target and will be removed during gel

ACS Paragon Plus Environment

ACS Combinatorial Science 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

purification due to the small size.31 Recently, Krusemark and co-workers

Figure 1. a) Our previous approach for DEL selections against non-immobilized targets.31 Prior to selection, library is tethered with a photocrosslinker, which captures the protein target upon ligand binding. 2) The proposed polymerase-extension-based selection method. A PCDNA (photo-crosslinker DNA) hybridizes at the primer binding site (PBS) of the library. After binding and UV irradiation, PC-DNA is crosslinked to the target; DNA polymerase then copies the encoding sequences. The DNA-target conjugates are purified for hit identification.

have developed an elegant selection method that also employs a photo-crosslinking DNA to capture the target; however, the method did not generate a covalent DNA-protein conjugate and affinity pulldown is necessary to isolate the binders.36 First, we prepared a 49-nt DNA (1; Figure 2a), modified with desthiobiotin at the 3’-end, which is a high affinity ligand for avidin (Kd ~2.0 nM). The desthiobiotin is encoded by a “TTT” codon. A 10-nt DNA with a 5’-phenylazide crosslinker was prepared as the PC-DNA.37 1 and PC-DNA were mixed and incubated with avidin (2.5 µM each), irradiated under 365 nm for 30 seconds, and then extended with T4 DNA polymerase before denaturing electrophoresis. As shown in Figure 2b, two slower moving bands were observed (marked by arrows) and their intensities increased when higher concentration of polymerases were used. Presumably, the lower band is the extended single-stranded PC-DNA-avidin conjugate and the upper band is the corresponding duplex with the library DNA.37 No DNA-avidin conjugate was observed without UV (Figure 2b). The bands were excised, extracted, PCR-amplified, and analyzed by Sanger sequencing. The results clearly show “TTT” at the coding region and confirmed that the band is the extended PC-DNA-avidin conjugate (Figure 2c). Next, we proceeded to validate that the DNA strands encoding high-affinity binders can be enriched from an excess of non-binding background DNAs in a model library. First, the same desthiobiotin-DNA (1) was mixed with a negative control DNA without desthiobiotin (2) at 1:10 and 1:100 ratios (Figure 3a). The negative control DNA 2 has a mixed sequence (DDD; D = A, C or G) at the encoding site. After adding PC-DNA, the mixtures of 1 and 2 were subjected to the same selection with avidin, photo-crosslinking, polymerase extension, and gel purification procedures and conditions as in Figure 2. Similarly, two gel bands corresponding to the PC-DNA-avidin conjugate and the renatured DNA duplexes were observed (Figure 3b; lane 3-8). Again, these bands were gel-purified, amplified, and analyzed by Sanger sequencing. The sequencing results were compared with the samples of the pre-selection mixtures. As shown in Figure 3c, since the percentage of 1 was low in the original mixtures, the “TTT” sequence could not be observed

at the encoding site; however, after selection, in both the 1:10 and 1:100 Figure 2. a) A desthiobiotin-conjugated DNA (1) is selected against avidin. 1, 2.5 µM; PC-DNA, 2.5 µM; avidin: 2.5 µM; buffer: 0.1 M NaCl with 1x PBS; UV: 365 nm, 30 sec., 0 °C; T4 DNA polymerase extension: 1 h at 16 °C. b) Electrophoresis analysis after extension. Lane 1: positive control (a mixture of 1 and the PC-DNA-avidin standard); lane 2: no UV; lane 3-5: with increasing T4 DNA ligase concentration. The weak band in lane 2 (*) is presumed to be the DNA duplex/avidin complex. c) The bands indicated by the arrows were excised, PCR-amplified and analyzed with Sanger sequencing. See Supporting Information for details.

experiments, the “TTT” codon became significantly more distinct, indicating that the desthiobiotin-conjugated DNA 1 has been enriched from the mixture. These results have demonstrated the capability of this method in selecting specific


Page 2 of 6

Page 3 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Combinatorial Science binders from a large excess of non-binding background. Finally, in order to further test the applicability of our method, we prepared another library and selected it against nonimmobilized carbonic anhydrase II (CA-II). First, this library was spiked-in with two known CA-II ligands, GLCBS and CBS (Figure 4a; DNAs

Figure 3. a) The mixtures of 1 and 2 (1:10 and 1:100) were selected against avidin following the same procedure as in Figure 2. D denotes either A, C or G. b) Electrophoresis analysis after polymerase extension. M: marker; lane 1: positive control (the extended PC-DNA-avidin standard sample);37 lane 2: 1 only in the selection; lane 3-5: the 1:10 mixture; lane 6-8: the 1:100 mixture. Extension products that were gel-purified are marked by arrows. b) Sanger sequencing results before and after the selection. Encoding sites are highlighted with brackets.

are encoded with “CAGTAC” and “GGATCC”, respectively). Second, a mix of DNAs with 16,384 different sequences (at equal ratio; see the SI for details on library sequence and preparation) was used in the library as the non-binding background (Figure 4a). After adding the PC-DNA (1 eq.), this 16,386-member library (all at equal ratio) was directly selected against soluble CA-II, following the same conditions and procedure as in Figure 2. After polymerase extension and gel purification of the selected PC-DNA-CA-II conjugates, Next generation sequencing (NGS) was employed for hit deconvolution. The sequencing data were processed with a custom script to quantitatively tally the codons for each compound and calculate the enrichment fold (post-selection % / pre-selection %). The results are shown in scatter plots (x-axis: post-sequencing counts; y-axis: enrichment fold).25, 28 In each plot, compounds with high enrichment fold and high postselection sequencing counts are considered as potential hits. As shown in Figure 4b, the sequences encoding the ligands GLCBS and CBS have been enriched with large enrichment folds, while none of the background sequences have been significantly enriched (Figure 4c). The enrichment folds are

comparable to the previous methods25, 31 and is lower than a crosslinking/solid-phase-based method,26 but it indicates that the selection may cover the typical nM to low µM affinity range for small molecules. Previously we have optimized the experimental conditions to minimize non-specific crosslinking31, 37-38 and the same conditions were employed here. In addition, the CA-II selection

Figure 4. a) A 16,386-member model library containing two positive controls GLCBS (3) and CBS (4) was selected against non-immobilized CA-II (2.5 µM) with the same procedure as in Figure 2, except high throughput sequencing was used. b) Selection result is shown in a scatter plot: enrichment folds (y-axis) vs. postselection sequence counts (x-axis). Enrichment fold = (postselection fraction)/(pre-selection fraction).25, 39 c) The left lower portion of b) is enlarged. See the Supplementary Information for details.

was also performed with a lower protein concentration (0.2 µM); the results showed similar enrichment folds (Figure S5). Future work with large diverse libraries will be able to further refine the method to suppress background interference. The model selection results have demonstrated that this method may be used to enrich the stereotypical binders from DELs. In conclusion, we have developed a new method for selecting DNA-encoded libraries against non-immobilized protein targets. Compared with other crosslinking-based methods previously reported,25-26, 31 it establishes a direct and stable link between the target protein and the encoding sequence of the binding ligand and also avoids the tedious and often detrimental steps of library modification and purification as in our previous report.31 Instead, this method uses a simple PCDNA-primed extension to copy the encoding DNA sequences, which has been proven to be an efficient and reliable encoding technique in the DEL synthesis.32-35 However, it is worth noting that polymerase extends single-stranded DNA (ssDNA) in the 5’- to 3’- direction; therefore, the current scheme is limited to DELs encoded with ssDNA and with the compound at the 3’end. For libraries with 5’-small molecules, an extension from



the distal 5’-end coupled with a nick-sealing ligation with the PC-DNA may be used. For libraries encoded with doublestranded DNAs (dsDNAs), converting the dsDNAs to singlestranded DNAs using methods such as biotin-tagging, affinity separation, strand displacement, or Lambda exonuclease, may be explored. Finally, this study is still preliminary. The two proteins, avidin and CA-II, are well-validated, proof-ofprinciple targets; and only model libraries have been used. The effectiveness and the capability in enriching specific binders of this approach will be further pursued with larger, chemically diverse libraries and non-model targets, especially the ones that are intractable to immobilization.

ASSOCIATED CONTENT Supporting Information. DNA synthesis, characterization, library information, DNA sequences, selection protocols and data analysis methods, and other experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author * Email: [email protected] Notes The corresponding author is a shareholder of Y-gene Biotech, a company operating in the field of DNA-encoded library.

ACKNOWLEDGMENT This work was supported by grants from the start-up fund from Tibet Agriculture and Animal Husbandry University, Research Grants Council of Hong Kong, China (AoE/P-705/16, 17321916, 17302817, 17301118), and from National Natural Science Foundation of China (21572014, 21877093).

REFERENCES 1. McCafferty, J.; Griffiths, A. D.; Winter, G.; Chiswell, D. J., Phage antibodies: filamentous phage displaying antibody variable domains. Nature 1990, 348 (6301), 552-4. 2. Kang, A. S.; Barbas, C. F.; Janda, K. D.; Benkovic, S. J.; Lerner, R. A., Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces. Proc. Nat. Acad. Sci. USA 1991, 88 (10), 4363-4366. 3. Wilson, D. S.; Keefe, A. D.; Szostak, J. W., The use of mRNA display to select high-affinity protein-binding peptides. Proc. Nat. Acad. Sci. USA 2001, 98 (7), 3750-3755. 4. Huang, Y.; Wiedmann, M. M.; Suga, H., RNA Display Methods for the Discovery of Bioactive Macrocycles. Chem. Rev. 2018, 10.1021/acs.chemrev.8b00430. 5. Boder, E. T.; Wittrup, K. D., Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 1997, 15 (6), 5537. 6. Hanes, J.; Pluckthun, A., In vitro selection and evolution of functional proteins by using ribosome display. Proc. Nat. Acad. Sci. USA 1997, 94 (10), 4937-4942. 7. Ellington, A. D.; Szostak, J. W., In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346 (6287), 818-22. 8. Morris, K. N.; Jensen, K. B.; Julin, C. M.; Weil, M.; Gold, L., High affinity ligands from in vitro selection: complex targets. Proc. Nat. Acad. Sci. USA 1998, 95 (6), 2902-7. 9. Goodnow, R. A., Jr.; Dumelin, C. E.; Keefe, A. D., DNA-encoded chemistry: enabling the deeper sampling of chemical space. Nat. Rev. Drug Discov. 2017, 16 (2), 131-147. 10. Shi, B.; Zhou, Y.; Huang, Y.; Zhang, J.; Li, X., Recent advances on the encoding and selection methods of DNA-encoded chemical library. Bioorg. Med. Chem. Lett. 2017, 27 (3), 361-369.

11. Yuen, L. H.; Franzini, R. M., Achievements, Challenges, and Opportunities in DNA-Encoded Library Research: An Academic Point of View. ChemBioChem 2017, 18 (9), 829-836. 12. Neri, D.; Lerner, R. A., DNA-Encoded Chemical Libraries: A Selection System Based On Endowing Organic Compounds with Amplifiable Information. Annu. Rev. Biochem. 2018, 87, 479-502. 13. Kunig, V.; Potowski, M.; Gohla, A.; Brunschweiger, A., DNAencoded libraries - an efficient small molecule discovery technology for the biomedical sciences. Biol. Chem. 2018, 399 (7), 691-710. 14. Brenner, S.; Lerner, R. A., Encoded Combinatorial Chemistry. Proc. Nat. Acad. Sci. USA 1992, 89 (12), 5381-5383. 15. Arico-Muendel, C. C., From haystack to needle: finding value with DNA encoded library technology at GSK. Medchemcomm 2016, 7 (10), 1898-1909. 16. Halford, B., Breakthroughs with bar codes DNA-encoded libraries help pharma find drug leads. Chem. Eng. News 2017, 95 (25), 28-33. 17. McGregor, L. M.; Jain, T.; Liu, D. R., Identification of ligandtarget pairs from combined libraries of small molecules and unpurified protein targets in cell lysates. J. Am. Chem. Soc. 2014, 136 (8), 326470. 18. Needels, M. C.; Jones, D. G.; Tate, E. H.; Heinkel, G. L.; Kochersperger, L. M.; Dower, W. J.; Barrett, R. W.; Gallop, M. A., Generation and screening of an oligonucleotide-encoded synthetic peptide library. Proc. Nat. Acad. Sci. USA 1993, 90 (22), 10700-4. 19. Nielsen, J., Brenner, S., Janda, K.D., Synthetic Methods for the Implementation of Encoded Combinatorial Chemistry. J. Am. Chem. Soc. 1993, 115, 9812-9813. 20. MacConnell, A. B.; McEnaney, P. J.; Cavett, V. J.; Paegel, B. M., DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis. ACS Comb. Sci. 2015, 17 (9), 518-34. 21. Mendes, K. R.; Malone, M. L.; Ndungu, J. M.; SuponitskyKroyter, I.; Cavett, V. J.; McEnaney, P. J.; MacConnell, A. B.; Doran, T. M.; Ronacher, K.; Stanley, K.; Utset, O.; Walzl, G.; Paegel, B. M.; Kodadek, T., High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections. ACS Chem. Biol. 2017, 12 (1), 234-243. 22. Erharuyi, O.; Simanski, S.; McEnaney, P. J.; Kodadek, T., Screening one bead one compound libraries against serum using a flow cytometer: Determination of the minimum antibody concentration required for ligand discovery. Bioorg. Med. Chem. Lett. 2018, 28 (16), 2773-2778. 23. Chan, A. I.; McGregor, L. M.; Jain, T.; Liu, D. R., Discovery of a covalent kinase inhibitor from a DNA-encoded smallmolecule library X protein library selection. J. Am. Chem. Soc. 2017, 139 (30), 1019210195. 24. Blakskjaer, P.; Heitner, T.; Hansen, N. J., Fidelity by design: Yoctoreactor and binder trap enrichment for small-molecule DNAencoded libraries and drug discovery. Curr. Opin. Chem. Biol. 2015, 26, 62-71. 25. Zhao, P.; Chen, Z.; Li, Y.; Sun, D.; Gao, Y.; Huang, Y.; Li, X., Selection of DNA-encoded small molecule libraries against unmodified and non-immobilized protein targets. Angew. Chem. Int. Ed. 2014, 53 (38), 10056-9. 26. Denton, K. E.; Krusemark, C. J., Crosslinking of DNA-linked ligands to target proteins for enrichment from DNA-encoded libraries. Medchemcomm 2016, 7 (10), 2020-2027. 27. Jetson, R. R.; Krusemark, C. J., Sensing Enzymatic Activity by Exposure and Selection of DNA-Encoded Probes. Angew. Chem. Int. Ed. 2016, 55 (33), 9562-6. 28. Li, G.; Zheng, W.; Chen, Z.; Zhou, Y.; Liu, Y.; Yang, J.; Huang, Y.; Li, X., Design, preparation, and selection of DNA-encoded dynamic libraries. Chem.Sci. 2015, 6, 7097-7104. 29. Zhou, Y.; Li, C.; Peng, J.; Xie, L.; Meng, L.; Li, Q.; Zhang, J.; Li, X. D.; Li, X.; Huang, X.; Li, X., DNA-Encoded Dynamic Chemical Library and Its Applications in Ligand Discovery. J. Am. Chem. Soc. 2018, 140 (46), 15859-15867. 30. Kochmann, S.; Le, A. T. H.; Hili, R.; Krylov, S. N., Predicting efficiency of NECEEM-based partitioning of protein binders from nonbinders in DNA-encoded libraries. Electrophoresis 2018, 39 (23), 2991-2996.


Page 4 of 6

Page 5 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Combinatorial Science 31. Shi, B.; Deng, Y.; Zhao, P.; Li, X., Selecting a DNA-Encoded Chemical Library against Non-immobilized Proteins Using a "LigateCross-Link-Purify" Strategy. Bioconjug. Chem. 2017, 28 (9), 22932301. 32. Buller, F.; Mannocci, L.; Zhang, Y.; Dumelin, C. E.; Scheuermann, J.; Neri, D., Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions. Bioorg. Med. Chem. Lett. 2008, 18 (22), 5926-31. 33. Wichert, M.; Krall, N.; Decurtins, W.; Franzini, R. M.; Pretto, F.; Schneider, P.; Neri, D.; Scheuermann, J., Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation. Nat. Chem. 2015, 7 (3), 241-9. 34. Mannocci, L.; Melkko, S.; Buller, F.; Molnar, I.; Bianke, J. P.; Dumelin, C. E.; Scheuermann, J.; Neri, D., Isolation of potent and specific trypsin inhibitors from a DNA-encoded chemical library. Bioconjug. Chem. 2010, 21 (10), 1836-41. 35. Buller, F.; Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.; Kalisch, M.; Buhlmann, P.; Supuran, C. T.; Neri, D., Selection of Carbonic Anhydrase IX Inhibitors from One Million DNA-Encoded Compounds. ACS Chem. Biol. 2011, 6 (4), 336-44. 36. Kim, D.; Jetson, R. R.; Krusemark, C. J., A DNA-assisted immunoassay for enzyme activity via a DNA-linked, activity-based probe. Chem. Commun. 2017, 53 (68), 9474-9477. 37. Li, G.; Liu, Y.; Chen, L.; Wu, S.; Li, X., Photoaffinity Labeling of Small-Molecule-Binding Proteins by DNA-Templated Chemistry. Angew. Chem. Int. Ed. 2013, 52 (36), 9544-9549. 38. Li, G.; Liu, Y.; Yu, X.; Li, X., Multivalent photoaffinity probe for labeling small molecule binding proteins. Bioconjug. Chem. 2014, 25 (6), 1172-80. 39. Kleiner, R. E.; Dumelin, C. E.; Tiu, G. C.; Sakurai, K.; Liu, D. R., In vitro selection of a DNA-templated small-molecule library reveals a class of macrocyclic kinase inhibitors. J. Am. Chem. Soc. 2010, 132 (33), 11779-91.



TOC graphics


Page 6 of 6

Polymerase-Extension-Based Selection Method for DNA-Encoded

Recommend Documents