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

Cai, Bo; Kim, Dong-Wook; Akhand, Saeed Salehin; Sun, Yubo; Cassell, Robert J.; Alpsoy, Aktan; Dykhuizen, Emily C.; Rijn, Richard M. van; Wendt, Michael K.; Krusemark, Casey J.

doi:10.1021/jacs.9b08085

Cited by 93 publications

(101 citation statements)

References 38 publications

(29 reference statements)

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“…Encouragingly,the recent report on intracellular screening of DNA-encoded chemical library (DEL) showed cell-penetrating peptides could enable intracellular operation of DNAconjugated small molecules. [30] Second, this study used pan-HDAC inhibitors to engage multiple HDACsa nd identify more proteins,b ut additional steps were necessary to deconvolute the specific interactions.U sing isoform-specific ligands would provide more specific information on individual targets.W ew ill perform more in-depth studies and explore the biological applications of this method in future studies.…”

Section: Resultsmentioning

confidence: 99%

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

et al. 2020

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Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions.

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Section: Resultsmentioning

confidence: 99%

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

et al. 2020

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“…The Krusemark group recently presented a sophisticated solution to the problem of on-and in-cell DEL selections. [43] A DEL was utilized that included an appended cyclic cell penetrating peptide (cCPP) and a reactive crosslinking agent. cCPP allows the DEL to access the cytosolic space and the crosslinking moiety allows the covalent crosslinking upon formation of a transient interaction between a library member and a HaloTag (a widely used self-labeling protein tag derived from a bacterial haloalkane dehalogenase) [44] fusion protein of interest.…”

Section: Selection Strategiesmentioning

confidence: 99%

“…The Krusemark group recently presented a sophisticated solution to the problem of on‐ and in‐cell DEL selections . A DEL was utilized that included an appended cyclic cell penetrating peptide (cCPP) and a reactive crosslinking agent.…”

Section: Selection Strategiesmentioning

confidence: 99%

DNA Encoded Libraries: A Visitor's Guide

Flood

Kingston

Vantourout

et al. 2020

Israel Journal of Chemistry

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In 1992, Brenner and Lerner hypothesized that individual chemical transformations could be encoded in DNA, allowing the rapid synthesis and screening of large collections of small molecules. Since their report, huge investments into the development of the DNA encoded library (DEL) technology have enabled the acceleration of the drug discovery process especially early phase discovery undertakings such as target validation and hit identification. As DEL lies at the nexus between chemistry and biology, there is an increasing need to expand the toolboxes of both organic transformations and biological methods. However, the myriad of techniques and reactions already reported can be difficult to digest for practitioners whose expertise resides outside the realm of DEL. This review therefore focuses on a stepwise presentation of DEL from the basic concepts to newest developments. The presentation includes the history, fundamentals, and successes of DEL, different methods for DEL synthesis and affinity selection, the conventional transformations, and finally the latest developments from a synthetic organic perspective.

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“…Covalent conjugation of target proteins and small-molecular ligands was recently actively studied in the field of chemical biology, bioimaging, and drug discovery [19][20][21][22]. Chemical and photoaffinity labeling of DNA-encoded compounds [23,24] and DNA aptamers [25] to target proteins were reported to identify unknown targets or screen novel binders. Furthermore, Liu et al reported a method called "interaction determination using unpurified proteins" to selectively amplify DNA sequences encoding the information of ligand-target pairs from a heterogenous mixture of DNA-encoded chemical library and proteins [26].…”

Section: Introductionmentioning

confidence: 99%

Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection

2020

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Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems.

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

Cited by 93 publications

References 38 publications

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

DNA Encoded Libraries: A Visitor's Guide

Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection

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