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Despite its clinical significance in conditions like acute myeloid leukemia and hepatocellular carcinoma (HCC), C\/EBP\u03b1 has remained undruggable due to its intrinsically disordered regions and lack of small-molecule binding pockets. In this study, we describe the development of a novel Oligonucleotide-based PROteolysis-TArgeting Chimera (Oligo-PROTAC or O'PROTAC) strategy designed to achieve targeted degradation of endogenous C\/EBP\u03b1. We designed and synthesized a 10-nucleotide double-stranded DNA decoy derived from a C\/EBP\u03b1-binding motif and conjugated it via variable linkers to the E3 ligase ligands pomalidomide or VH032. A total of six candidates were synthesized and evaluated in 3T3-J2 and Huh7 cell lines. We demonstrate that these O'PROTACs successfully localize to the nucleus and achieve dose-dependent degradation of C\/EBP\u03b1, with the most potent candidates exhibiting sub-micromolar DC50 via lipofection delivery. Notably, this degradation occurred without inducing significant cytotoxicity. We observe a transcriptional shift consistent with loss of C\/EBP\u03b1, including downregulation of cooperative factors involved in liver lineage specification and their downstream target genes. This work establishes O'PROTACs as a viable modality for targeting C\/EBP\u03b1, offering a novel strategy for mechanistic investigation and future therapeutic targeting.<\/jats:p>","DOI":"10.1021\/acschembio.6c00462","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T08:07:03Z","timestamp":1788336423000},"source":"Crossref","is-referenced-by-count":0,"title":["Development of Oligo-PROTACs That Target C\/EBP\u03b1"],"prefix":"10.1021","author":[{"given":"Junming","family":"Cai","sequence":"first","affiliation":[{"name":"Mayo Clinic College of Medicine and Science , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xianmei","family":"Qi","sequence":"additional","affiliation":[{"name":"Mayo Clinic College of Medicine and Science , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adam T.","family":"Smiley","sequence":"additional","affiliation":[{"name":"Mayo Clinic College of Medicine and Science , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4769-1518","authenticated-orcid":true,"given":"Douglas G.","family":"Brownfield","sequence":"additional","affiliation":[{"name":"Mayo Clinic College of Medicine and Science , , , ,","place":["Rochester, Minnesota, United States, 55905"]},{"name":"Mayo Clinic College of Medicine and Science , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,31]]},"reference":[{"issue":"5","key":"2026090204065677900_cit1","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1038\/nrc1363","article-title":"C\/EBPalpha mutations in acute myeloid leukaemias","volume":"4","author":"Nerlov","year":"2004","journal-title":"Nat. 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