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Here, we establish green fluorescent protein (GFP) variants as a tractable model system for studying protein\u2010binding aptamers using strategies commonly applied to small\u2010molecule aptamers. Independent SELEX experiments against mGreenLantern GFP (mGL\u2010GFP), superfolder GFP (sfGFP), and GFPuv yielded closely related aptamers with distinct specificity profiles. Sequence analysis, truncation studies, circular dichroism spectroscopy, and 2D nuclear magnetic resonance spectroscopy results are consistent with differences in epitopes among the GFP variants. Divalent metal ions strongly influence selection outcomes: aptamers isolated with Ca\n                    <jats:sup>2+<\/jats:sup>\n                    require Ca\n                    <jats:sup>2+<\/jats:sup>\n                    for binding, whereas Ca\n                    <jats:sup>2<\/jats:sup>\n                    <jats:sup>+<\/jats:sup>\n                    \u2010free selection produced a distinct family of Mg\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010dependent aptamers with potential intracellular utility. Together, these results establish GFP variants as a reliable platform for dissecting protein\u2013aptamer recognition and provide a roadmap for achieving small\u2010molecule\u2010level mechanistic insight into protein\u2010binding aptamers.\n                  <\/jats:p>","DOI":"10.1002\/anie.2160332","type":"journal-article","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T03:44:33Z","timestamp":1790739873000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Extending Small\u2010Molecule Aptamer Discovery Principles to Protein Targets: Selection and Characterization of Green Fluorescent Protein Aptamers"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2953-4730","authenticated-orcid":false,"given":"Stefen","family":"Stangherlin","sequence":"first","affiliation":[{"name":"Department of Chemistry Waterloo Institute for Nanotechnology University of Waterloo  Waterloo Ontario 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 Guelph Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iffat","family":"Tabassum","sequence":"additional","affiliation":[{"name":"Department of Chemistry Waterloo Institute for Nanotechnology University of Waterloo  Waterloo Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Songyang","family":"Miao","sequence":"additional","affiliation":[{"name":"Department of Chemistry Waterloo Institute for Nanotechnology University of Waterloo  Waterloo Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4064-6397","authenticated-orcid":false,"given":"Rui","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Guelph  Guelph Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5918-9336","authenticated-orcid":false,"given":"Juewen","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Chemistry Waterloo Institute for Nanotechnology University of Waterloo  Waterloo Ontario 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