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It also preferentially interacts with oligomeric tau and is required for tau-mediated neurodegeneration in a mouse model of Alzheimer\u2019s disease. To study endogenous hnRNPA2\/B1 and tau, we generated nanobodies that specifically recognize these proteins. We screened yeast surface display nanobody libraries using an avidity-enhanced screening strategy that enabled selection of binders against short peptide ligands. This led to isolation of anti-hnRNPA2\/B1 and anti-tau nanobodies with defined epitopes. Directed evolution of the anti-hnRNPA2\/B1 nanobody improved binding affinity by over 20-fold but caused cytoplasmic aggregation, demonstrating a tradeoff between affinity and intracellular behavior. Although the final nanobodies retained modest affinities, they showed robust intracellular colocalization with their targets. Furthermore, fusion to ubiquitin ligase adaptor domains significantly decreased hnRNPA2\/B1 and tau levels. Collectively, these nanobodies provide valuable tools for studying hnRNPA2\/B1 and tau dynamics in their native cellular context.<\/jats:p>","DOI":"10.1101\/2025.11.21.689763","type":"posted-content","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T22:05:43Z","timestamp":1764021943000},"source":"Crossref","is-referenced-by-count":0,"title":["Nanobodies targeting hnRNPA2\/B1 and tau"],"prefix":"10.64898","author":[{"given":"Azady","family":"Pirhanov","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristian","family":"Rodriguez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatemeh","family":"Tashakori-Asfestani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raven","family":"Gonsoulin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rayne","family":"Santiago","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarnut","family":"Tobunluepop","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmanuella","family":"Erhunmwunsee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sambhavi","family":"Puri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Arbaciauskaite","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wu","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Wolozin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5621-521X","authenticated-orcid":false,"given":"Yongku","family":"Cho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025112604303525000_2025.11.21.689763v1.1","first-page":"458","article-title":"and RNA-Binding Proteins: Complex Interactions in Health and Neurodegenerative Diseases","volume":"30","year":"2023","journal-title":"The Neuroscientist"},{"key":"2025112604303525000_2025.11.21.689763v1.2","doi-asserted-by":"crossref","unstructured":"J. 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