{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T11:19:36Z","timestamp":1768216776316,"version":"3.49.0"},"reference-count":33,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T00:00:00Z","timestamp":1768176000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>With the primary aim of generating antibody tools useful for diagnostic and therapeutic applications relevant to oligonucleotide-templated reactions, an scFv filamentous phage library was screened using a specific 2\u2032-O-methyl RNA template:oligonucleotide click reaction complex as the target structure. Such an antibody would not be expected to recognize any naturally occurring biological molecules. Two promising candidate scFv clones were converted into human IgG1 antibodies for further characterization. Surprisingly, although the best antibody (IgG1-DS5) was bound to the original selection complex, its recognition preference was shown to be directed toward short 2\u2032-O-methyl RNA duplexes without click modifications. The IgG1-DS5 antibody showed no binding to the separate single strands comprising the recognized duplex, nor to the corresponding duplexes with RNA or DNA template strands. Versions of the target 2\u2032-O-methyl RNA duplex with divergent sequences were also not recognized by IgG1-DS5. The unexpected binding properties of the IgG1-DS5 antibody were directed toward potential applications based on the molecular proximity of tethered single strands. Initially, SKBr3 cells were coated with biotins by means of surface azide metabolic labeling with peracetylated N-azidoacetylmannosamine, followed by treatment with a click-reactive DBCO-PEG4-biotin compound. Subsequent cell treatment with the tetravalent biotin-binding protein neutravidin (NAV), carrying subsaturating levels of biotinylated 2\u2032-O-methyl RNA target duplexes, showed strong IgG1-DS5 staining on cell surfaces. These observations were extended with biotinylated anti-EGFR antibody linked with biotinylated 2\u2032-O-methyl RNA single strands, also by means of the NAV protein as an adaptor. Flow cytometry analysis showed that DS5 antibody binding was only obtained when combinations of separate preparations of antibodies carrying top and bottom target strands were applied sequentially to EGFR-positive cells. These results show that proximity-based surface annealing of the IgG1-DS5 antibody target single strands can act to define cell populations with a surface marker of sufficient density. Where IgG1-DS5 is derivatized with either a fluorescent moiety or a cytotoxic drug, this antibody may find application in diagnostic or therapeutic tumor targeting.<\/jats:p>","DOI":"10.3389\/fimmu.2025.1699400","type":"journal-article","created":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T08:19:34Z","timestamp":1768205974000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["An antibody uniquely binding short 2\u2032-O-methyl RNA oligonucleotide duplexes: formation and recognition of target duplexes on cell surfaces"],"prefix":"10.3389","volume":"16","author":[{"given":"Ian S.","family":"Dunn","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew M.","family":"Lawler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leah","family":"Fagundes","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Milto","family":"Simoes Junior","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"April","family":"Mangold","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Domenic","family":"Rinaldi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lenora B.","family":"Rose","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James T.","family":"Kurnick","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,1,12]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1002\/1521-3773(20021104)41:21<4104::AID-ANIE4104>3.0.CO;2-O","article-title":"Directing otherwise incompatible reactions in a single solution by using DNA-templated organic synthesis","volume":"41","author":"Calderone","year":"2002","journal-title":"Angew Chem Int Ed Engl"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1102629","article-title":"DNA-templated organic synthesis and selection of a library of macrocycles","volume":"305","author":"Gartner","year":"2004","journal-title":"Science"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200400656","article-title":"DNA-templated organic synthesis: nature\u2019s strategy for controlling chemical reactivity applied to synthetic molecules","volume":"43","author":"Li","year":"2004","journal-title":"Angew Chem Int Ed Engl"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1021\/acs.nanolett.2c03928","article-title":"DNA-templated anchoring of proteins for programmable cell functionalization and immunological response","volume":"23","author":"Xiong","year":"2023","journal-title":"Nano Lett"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1101\/2024.09.18.613743","article-title":"DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4\/6 complex protein degradation","author":"Zheng","year":"2024","journal-title":"bioRxiv"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.3390\/molecules27206831","article-title":"Assembly of biologically functional structures by nucleic acid templating: implementation of a strategy to overcome inhibition by template excess","volume":"27","author":"Lawler","year":"2022","journal-title":"Molecules"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1007\/s12033-019-00207-0","article-title":"Advances in the production and batch reformatting of phage antibody libraries","volume":"61","author":"Reader","year":"2019","journal-title":"Mol Biotechnol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1126\/science.aat0271","article-title":"Programming self-organizing multicellular structures with synthetic cell-cell signaling","volume":"361","author":"Toda","year":"2018","journal-title":"Science"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1126\/science.add9665","article-title":"The emerging era of cell engineering: Harnessing the modularity of cells to program complex biological function","volume":"378","author":"Lim","year":"2022","journal-title":"Science"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1158\/2159-8290.CD-23-0101","article-title":"Programming CAR T cell tumor recognition: tuned antigen sensing and logic gating","volume":"13","author":"Hamieh","year":"2023","journal-title":"Cancer Discov"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0502-473","article-title":"Protein detection using proximity-dependent DNA ligation assays","volume":"20","author":"Fredriksson","year":"2002","journal-title":"Nat Biotechnol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2017.05.003","article-title":"Close encounters - probing proximal proteins in live or fixed cells","volume":"42","author":"L\u00f6nn","year":"2017","journal-title":"Trends Biochem Sci"},{"key":"B13","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1186\/s12868-020-0554-0","article-title":"Proximity ligation assay reveals both pre- and postsynaptic localization of the APP-processing enzymes ADAM10 and BACE1 in rat and human adult brain","volume":"21","author":"Lundgren","year":"2020","journal-title":"BMC Neurosci"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3862-0_17","article-title":"Proximity ligation assay to detect the proximity between host proteins and viral proteins of HIV-1","volume":"2807","author":"Chakraborty","year":"2024","journal-title":"Methods Mol Biol"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot108502","article-title":"Using the proximity ligation assay to visualize colocalization of proteins at the drosophila larval neuromuscular junction","volume":"7","author":"Ashley","year":"2024","journal-title":"Cold Spring Harb Protoc"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-4188-0_12","article-title":"Use of proximity ligation assay to study lymphoid Malignancies","volume":"2865","author":"Rinaldi","year":"2025","journal-title":"Methods Mol Biol"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1126\/science.287.5460.2007","article-title":"Cell surface engineering by a modified Staudinger reaction","volume":"287","author":"Saxon","year":"2000","journal-title":"Science"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(03)01018-8","article-title":"Constructing azide-labeled cell surfaces using polysaccharide biosynthetic pathways","volume":"362","author":"Luchansky","year":"2003","journal-title":"Methods Enzymol"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3381-6_18","article-title":"Antibody isolation from human synthetic libraries of single-chain antibodies and analysis using NGS","volume":"2702","author":"Amir","year":"2023","journal-title":"Methods Mol Biol"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.3390\/pharmaceutics15020600","article-title":"A review of protein- and peptide-based chemical conjugates: past, present, and future","volume":"15","author":"Holz","year":"2023","journal-title":"Pharmaceutics"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1021\/bc500296p","article-title":"Plug-and-go\u201d strategy to manipulate streptavidin valencies","volume":"25","author":"Sun","year":"2014","journal-title":"Bioconjug Chem"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1134\/S0006297913020028","article-title":"Natural antibodies to nucleic acids","volume":"78","author":"Buneva","year":"2013","journal-title":"Biochem (Mosc)"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.2c00584","article-title":"Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies","volume":"94","author":"Al-Amin","year":"2022","journal-title":"Anal Chem"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202000037","article-title":"Visualizing and manipulating biological processes by using haloTag and SNAP-tag technologies","volume":"21","author":"Hoelzel","year":"2020","journal-title":"Chembiochem"},{"key":"B25","doi-asserted-by":"publisher","first-page":"107118","DOI":"10.1016\/j.bioorg.2024.107118","article-title":"HUH endonuclease: A sequence-specific fusion protein tag for precise DNA-protein conjugation","volume":"144","author":"Du","year":"2024","journal-title":"Bioorg Chem"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1101\/2024.08.13.607811","article-title":"Sequence-directed covalent protein-RNA linkages in a single step using engineered HUH-tags","author":"Smiley","year":"2024","journal-title":"bioRxiv"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1093\/annonc\/12.suppl_1.S43","article-title":"Pharmacologic insights into the future of trastuzumab","author":"Leyland-Jones","year":"2001","journal-title":"Ann Oncol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-024-03021-7","article-title":"Trastuzumab deruxtecan versus trastuzumab emtansine in HER2-positive metastatic breast cancer: long-term survival analysis of the DESTINY-Breast03 trial","volume":"30","author":"Cort\u00e9s","year":"2024","journal-title":"Nat Med"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1111\/cas.16234","article-title":"Trastuzumab deruxtecan versus trastuzumab emtansine in Asian patients with HER2-positive metastatic breast cancer","volume":"115","author":"Iwata","year":"2024","journal-title":"Cancer Sci"},{"key":"B30","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s10549-013-2732-6","article-title":"Trastuzumab-related cardiac events in the treatment of early breast cancer","volume":"142","author":"Fried","year":"2013","journal-title":"Breast Cancer Res Treat"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.3390\/cancers14143353","article-title":"Biomarkers of trastuzumab-induced cardiac toxicity in HER2- positive breast cancer patient population","volume":"14","author":"Grela-Wojewoda","year":"2022","journal-title":"Cancers (Basel)"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1914510","article-title":"Trastuzumab deruxtecan in previously treated HER2-positive breast cancer","volume":"382","author":"Modi","year":"2020","journal-title":"N Engl J Med"},{"key":"B33","doi-asserted-by":"publisher","first-page":"oyaf057","DOI":"10.1093\/oncolo\/oyaf057","article-title":"Correlation between tumor growth rate and survival in patients with metastatic breast cancer treated with trastuzumab deruxtecan","volume":"30","author":"He","year":"2025","journal-title":"Oncologist"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1699400\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T08:19:39Z","timestamp":1768205979000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1699400\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,12]]},"references-count":33,"alternative-id":["10.3389\/fimmu.2025.1699400"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1699400","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,12]]},"article-number":"1699400"}}