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To improve their stability and circulation time, oligonucleotides are often conjugated to hydrophilic polymers, such as polyethylene glycol (PEG). However, current bulk techniques fail to resolve PEG heterogeneity, especially in complex biological environments. Here, we use nanopore sensing to quantify the PEG conjugation efficiency of the FDA-approved RNA aptamer pegaptanib. We assembled DNA nanostructures that bind pegaptanib, and then we used solid-state nanopores to quantify pegaptanib PEGylation. We further assessed pegaptanib PEGylation and stability in a serum background and demonstrated that nanopore sensing resolves PEG moieties of distinct molecular weights within the oligonucleotide conjugates. Single-molecule profiling of polymer-RNA conjugates enables iterative improvements in oligonucleotide design and provides a direct means to assess their stability in complex biological environments, thereby advancing the development of more effective NATs.<\/jats:p>","DOI":"10.1021\/jacs.6c12398","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:10:02Z","timestamp":1785849002000},"page":"33980-33989","source":"Crossref","is-referenced-by-count":0,"title":["Nanopore-Based Profiling\nof PEGylation in Nucleic\nAcid Therapeutics"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-5742-7601","authenticated-orcid":true,"given":"Gerardo","family":"Patin\u0303o Guille\u0301n","sequence":"first","affiliation":[{"name":"University of Cambridge , , ,","place":["Cambridge, U.K., CB3 0HE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6802-3036","authenticated-orcid":true,"given":"Thieme T.","family":"Schmidt","sequence":"additional","affiliation":[{"name":"University of Cambridge , , ,","place":["Cambridge, U.K., CB3 0HE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9606-9488","authenticated-orcid":true,"given":"Jeremy J.","family":"Baumberg","sequence":"additional","affiliation":[{"name":"University of Cambridge , , ,","place":["Cambridge, U.K., CB3 0HE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4131-1203","authenticated-orcid":true,"given":"Jack W.","family":"Szostak","sequence":"additional","affiliation":[{"name":"The University of Chicago , , , ,","place":["Chicago, Illinois, United States, 60637"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulrich F.","family":"Keyser","sequence":"additional","affiliation":[{"name":"University of Cambridge , , ,","place":["Cambridge, U.K., CB3 0HE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7663-2408","authenticated-orcid":true,"given":"Filip","family":"Bos\u030ckovic\u0301","sequence":"additional","affiliation":[{"name":"The University of Chicago , , , ,","place":["Chicago, Illinois, United States, 60637"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"issue":"6","key":"2026081210065136600_cit1","doi-asserted-by":"publisher","first-page":"630","DOI":"10.1038\/s41565-021-00898-0","article-title":"The Current Landscape of Nucleic\nAcid Therapeutics","volume":"16","author":"Kulkarni","year":"2021","journal-title":"Nat. Nanotechnol."},{"issue":"8","key":"2026081210065136600_cit2","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1038\/s41568-023-00586-2","article-title":"MRNA-Based\nCancer Therapeutics","volume":"23","author":"Liu","year":"2023","journal-title":"Nat. Rev. Cancer"},{"issue":"4","key":"2026081210065136600_cit3","doi-asserted-by":"publisher","first-page":"1493","DOI":"10.1021\/jacs.1c09574","article-title":"Aptamers Entirely\nBuilt from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy","volume":"144","author":"Zhu","year":"2022","journal-title":"J. Am. Chem. Soc."},{"issue":"9","key":"2026081210065136600_cit4","doi-asserted-by":"publisher","first-page":"3416","DOI":"10.1021\/jacs.0c12043","article-title":"Glucagon like Peptide 1 Receptor Agonists for Targeted\nDelivery of Antisense Oligonucleotides to Pancreatic Beta Cell","volume":"143","author":"Knerr","year":"2021","journal-title":"J. Am. Chem. Soc."},{"issue":"36","key":"2026081210065136600_cit5","doi-asserted-by":"publisher","first-page":"19800","DOI":"10.1021\/jacs.3c05574","article-title":"A Unified Strategy to Improve Lipid Nanoparticle Mediated\nMRNA Delivery Using Adenosine Triphosphate","volume":"145","author":"Ma","year":"2023","journal-title":"J. Am. Chem. Soc."},{"issue":"7","key":"2026081210065136600_cit6","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41419-022-05075-2","article-title":"RNA-Based Therapeutics: An Overview\nand Prospectus","volume":"13","author":"Zhu","year":"2022","journal-title":"Cell Death & Dis"},{"issue":"10","key":"2026081210065136600_cit7","doi-asserted-by":"publisher","first-page":"673","DOI":"10.1038\/s41573-020-0075-7","article-title":"Advances\nin Oligonucleotide Drug\nDelivery","volume":"19","author":"Roberts","year":"2020","journal-title":"Nat. Rev. Drug Discovery"},{"issue":"1","key":"2026081210065136600_cit8","doi-asserted-by":"publisher","DOI":"10.1002\/btm2.10600","article-title":"PEGylated\nTherapeutics in the Clinic","volume":"9","author":"Gao","year":"2024","journal-title":"Bioeng. Transl.\nMed."},{"issue":"9","key":"2026081210065136600_cit9","doi-asserted-by":"publisher","first-page":"app.40293","DOI":"10.1002\/app.40293","article-title":"Impacts of PEGylation\non the Gene and Oligonucleotide\nDelivery System","volume":"131","author":"Ikeda","year":"2014","journal-title":"J. Appl. Polym. Sci."},{"issue":"6","key":"2026081210065136600_cit10","doi-asserted-by":"publisher","first-page":"800","DOI":"10.3390\/pharmaceutics13060800","article-title":"PEGylation of MRNA by Hybridization\nof Complementary PEG-RNA Oligonucleotides Stabilizes MRNA without\nUsing Cationic Materials","volume":"13","author":"Yoshinaga","year":"2021","journal-title":"Pharmaceutics"},{"issue":"10","key":"2026081210065136600_cit11","doi-asserted-by":"publisher","first-page":"5519","DOI":"10.1007\/s12274-018-2131-8","article-title":"PEGylation of Therapeutic\nOligonucletides: From Linear to Highly\nBranched PEG Architectures","volume":"11","author":"Lu","year":"2018","journal-title":"Nano Res."},{"issue":"3","key":"2026081210065136600_cit12","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1078\/0171-9335-00363","article-title":"PEGylation\nSignificantly Affects\nCellular Uptake and Intracellular Trafficking of Non-Viral Gene Delivery\nParticles","volume":"83","author":"Mishra","year":"2004","journal-title":"Eur. J. Cell Biol."},{"issue":"11","key":"2026081210065136600_cit13","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0050028","article-title":"PEGylation\nExtends Circulation Half-Life\nWhile Preserving In Vitro and In Vivo Activity of Tissue Inhibitor\nof Metalloproteinases-1 (TIMP-1)","volume":"7","author":"Batra","year":"2012","journal-title":"PLoS One"},{"issue":"9","key":"2026081210065136600_cit14","doi-asserted-by":"publisher","first-page":"14022","DOI":"10.1021\/acsnano.1c05922","article-title":"Polyethylene\nGlycol Immunogenicity:\nTheoretical, Clinical, and Practical Aspects of Anti-Polyethylene\nGlycol Antibodies","volume":"15","author":"Chen","year":"2021","journal-title":"ACS Nano"},{"issue":"7","key":"2026081210065136600_cit15","doi-asserted-by":"publisher","first-page":"6996","DOI":"10.1021\/acsnano.5b01326","article-title":"Surface Functionalization of Nanoparticles\nwith Polyethylene Glycol: Effects on Protein Adsorption and Cellular\nUptake","volume":"9","author":"Pelaz","year":"2015","journal-title":"ACS Nano"},{"issue":"24","key":"2026081210065136600_cit16","doi-asserted-by":"publisher","first-page":"5758","DOI":"10.1016\/j.bmcl.2014.10.045","article-title":"Oligonucleotides Conjugated\nwith Short Chemically Defined Polyethylene Glycol Chains Are Efficient\nAntisense Agents","volume":"24","author":"Shokrzadeh","year":"2014","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"2026081210065136600_cit17","doi-asserted-by":"publisher","first-page":"101219","DOI":"10.1016\/j.preteyeres.2023.101219","article-title":"From Randomised\nControlled Trials\nto Real-World Data: Clinical Evidence to Guide Management of Diabetic\nMacular Oedema","volume":"97","author":"Gabrielle","year":"2023","journal-title":"Prog. Retin. Eye Res."},{"issue":"7","key":"2026081210065136600_cit18","doi-asserted-by":"publisher","first-page":"1264","DOI":"10.1021\/bc100067t","article-title":"Sensitive\nQuantification of PEGylated Compounds by Second-Generation Anti-Poly(Ethylene\nGlycol) Monoclonal Antibodies","volume":"21","author":"Su","year":"2010","journal-title":"Bioconjugate\nChem."},{"issue":"15","key":"2026081210065136600_cit19","doi-asserted-by":"publisher","first-page":"7642","DOI":"10.1021\/ac501507g","article-title":"Quantitative Analysis\nof Polyethylene Glycol (PEG) and PEGylated Proteins in Animal Tissues\nby LC-MS\/MS Coupled with in-Source CID","volume":"86","author":"Gong","year":"2014","journal-title":"Anal.\nChem."},{"issue":"5","key":"2026081210065136600_cit20","doi-asserted-by":"publisher","first-page":"881","DOI":"10.1021\/bc200478w","article-title":"Analytical Measurement\nof Pegylated Molecules","volume":"23","author":"Cheng","year":"2012","journal-title":"Bioconjugate Chem."},{"issue":"3\u20134","key":"2026081210065136600_cit21","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1002\/ansa.202100066","article-title":"Advancements in\nthe Characterisation of Oligonucleotides\nby High Performance Liquid Chromatography-Mass Spectrometry in 2021:\nA Short Review","volume":"3","author":"Hannauer","year":"2022","journal-title":"Anal. Sci. Adv."},{"issue":"4","key":"2026081210065136600_cit22","doi-asserted-by":"publisher","first-page":"860","DOI":"10.1021\/acs.oprd.3c00428","article-title":"Striving for\nUniformity: A Review on Advances and Challenges To Achieve Uniform\nPolyethylene Glycol","volume":"28","author":"Bento","year":"2024","journal-title":"Org. Process Res. Dev."},{"key":"2026081210065136600_cit23","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1038\/s41565-022-01287-x","article-title":"Simultaneous\nIdentification of Viruses and SARS-CoV-2 Variants with Programmable\nDNA Nanobait","volume":"18","author":"Bos\u030ckovic\u0301","year":"2023","journal-title":"Nat. Nanotechnol."},{"issue":"1","key":"2026081210065136600_cit24","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1089\/nat.2024.0065","article-title":"Bioanalytical\nAssays\nfor Oligonucleotide Therapeutics: Adding Antibody-Based Immunoassays\nto the Toolbox as an Orthogonal Approach to LC-MS\/MS and Ligand Binding\nAssays","volume":"35","author":"Chimento","year":"2025","journal-title":"Nucleic Acid Ther."},{"issue":"11","key":"2026081210065136600_cit25","doi-asserted-by":"publisher","first-page":"18590","DOI":"10.1021\/acsnano.1c09275","article-title":"Correction\nto Delivery of Oligonucleotide\nTherapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular\nVesicles","volume":"15","author":"Bost","year":"2021","journal-title":"ACS Nano"},{"issue":"11","key":"2026081210065136600_cit26","doi-asserted-by":"publisher","first-page":"2705","DOI":"10.1093\/nar\/gkg393","article-title":"Structural\nVariations and Stabilising Modifications of Synthetic SiRNAs in Mammalian\nCells","volume":"31","author":"Czauderna","year":"2003","journal-title":"Nucleic Acids Res."},{"issue":"3","key":"2026081210065136600_cit27","doi-asserted-by":"publisher","first-page":"1359","DOI":"10.1093\/nar\/gkad1134","article-title":"Internal RNA\n2\u2032-O-Methylation on the HIV-1 Genome Impairs Reverse Transcription","volume":"52","author":"Decombe","year":"2024","journal-title":"Nucleic Acids Res."},{"issue":"6","key":"2026081210065136600_cit28","doi-asserted-by":"publisher","first-page":"6634","DOI":"10.1021\/acs.chemrev.1c00795","article-title":"Recent Developments for the Deuterium and Tritium Labeling\nof Organic Molecules","volume":"122","author":"Kopf","year":"2022","journal-title":"Chem. Rev."},{"issue":"5","key":"2026081210065136600_cit29","doi-asserted-by":"publisher","first-page":"287","DOI":"10.1089\/nat.2023.0011","article-title":"OSWG Recommended\nApproaches to the\nNonclinical Pharmacokinetic (ADME) Characterization of Therapeutic\nOligonucleotides","volume":"33","author":"Berman","year":"2023","journal-title":"Nucleic Acid Ther."},{"key":"2026081210065136600_cit30","doi-asserted-by":"publisher","DOI":"10.1002\/advs.75319","article-title":"DNA Nanoarray\nfor Multiplexed RNA Detection With Single-Molecule Readout","volume":"13","author":"Li","year":"2026","journal-title":"Adv. Sci."},{"issue":"7","key":"2026081210065136600_cit31","doi-asserted-by":"publisher","first-page":"645","DOI":"10.1038\/nnano.2016.50","article-title":"Digitally Encoded\nDNA Nanostructures for Multiplexed,\nSingle-Molecule Protein Sensing with Nanopores","volume":"11","author":"Bell","year":"2016","journal-title":"Nat. Nanotechnol."},{"issue":"23","key":"2026081210065136600_cit32","doi-asserted-by":"publisher","first-page":"15013","DOI":"10.1021\/acsnano.4c01466","article-title":"Nanopore Translocation\nReveals Electrophoretic Force on Noncanonical RNA:DNA Double Helix","volume":"18","author":"Bos\u030ckovic\u0301","year":"2024","journal-title":"ACS Nano"},{"key":"2026081210065136600_cit33","doi-asserted-by":"publisher","first-page":"4094","DOI":"10.1021\/acs.macromol.5c00119","article-title":"Folding Dynamics\nof Linear SsDNA-DsDNA Heterostructures through Solid-State Nanopores","volume":"58","author":"Mensing","year":"2025","journal-title":"Macromolecules"},{"issue":"1","key":"2026081210065136600_cit34","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41467-018-07924-1","article-title":"Detecting Topological Variations of DNA at Single-Molecule\nLevel","volume":"10","author":"Liu","year":"2019","journal-title":"Nat. Commun."},{"issue":"21","key":"2026081210065136600_cit35","doi-asserted-by":"publisher","first-page":"8719","DOI":"10.1021\/acs.nanolett.2c03546","article-title":"Ultrafast\nPolymer Dynamics through a Nanopore","volume":"22","author":"Lin","year":"2022","journal-title":"Nano Lett."},{"issue":"23","key":"2026081210065136600_cit36","doi-asserted-by":"publisher","first-page":"14380","DOI":"10.1039\/C4NR05001K","article-title":"Probing the\nSize of Proteins with\nGlass Nanopores","volume":"6","author":"Steinbock","year":"2014","journal-title":"Nanoscale"},{"issue":"12","key":"2026081210065136600_cit37","doi-asserted-by":"publisher","first-page":"20075","DOI":"10.1021\/acsnano.2c08312","article-title":"Probing RNA Conformations\nUsing a Polymer-Electrolyte Solid-State Nanopore","volume":"16","author":"Chau","year":"2022","journal-title":"ACS Nano"},{"issue":"95","key":"2026081210065136600_cit38","doi-asserted-by":"publisher","first-page":"14097","DOI":"10.1039\/D4CC05344C","article-title":"Improving Macromolecule Crowding\nConfigurations in Nanopores for Protein Sensing","volume":"60","author":"Zheng","year":"2024","journal-title":"Chem. Commun."},{"issue":"1","key":"2026081210065136600_cit39","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/srep38675","article-title":"High Temperature Extends the Range\nof Size Discrimination of Nonionic Polymers by a Biological Nanopore","volume":"6","author":"Piguet","year":"2016","journal-title":"Sci. Rep."},{"issue":"20","key":"2026081210065136600_cit40","doi-asserted-by":"publisher","first-page":"8207","DOI":"10.1073\/pnas.0611085104","article-title":"Single-Molecule Mass Spectrometry in Solution Using\na Solitary Nanopore","volume":"104","author":"Robertson","year":"2007","journal-title":"Proc. Natl. Acad. Sci.\nU. S. A."},{"issue":"1","key":"2026081210065136600_cit41","doi-asserted-by":"publisher","first-page":"684","DOI":"10.1038\/srep00684","article-title":"PEG-Labeled Nucleotides and Nanopore\nDetection for Single Molecule DNASequencing by Synthesis","volume":"2","author":"Kumar","year":"2012","journal-title":"Sci. Rep."},{"issue":"18","key":"2026081210065136600_cit42","doi-asserted-by":"publisher","first-page":"689134","DOI":"10.1101\/2025.11.18.689134","article-title":"A Nucleic\nAcid Labeling Chemistry Reveals Surface DNA on Exosomes","volume":"11","author":"Boskovic","year":"2025","journal-title":"bioRxiv"},{"key":"2026081210065136600_cit43","doi-asserted-by":"publisher","first-page":"6219","DOI":"10.1038\/s41467-026-72819-5","article-title":"Quantification\nof Disease-Associated RNA Tandem Repeats by Nanopore Sensing","volume":"17","author":"Patin\u0303o-Guille\u0301n","year":"2026","journal-title":"Nat. Commun."},{"issue":"12","key":"2026081210065136600_cit44","doi-asserted-by":"publisher","first-page":"931","DOI":"10.1038\/s41578-020-0229-6","article-title":"Solid-State Nanopore\nSensors","volume":"5","author":"Xue","year":"2020","journal-title":"Nat. Rev. Mater."},{"issue":"1","key":"2026081210065136600_cit45","doi-asserted-by":"publisher","first-page":"4473","DOI":"10.1038\/s41467-019-12358-4","article-title":"Complex DNA Knots Detected with a Nanopore Sensor","volume":"10","author":"Kumar Sharma","year":"2019","journal-title":"Nat. Commun."},{"issue":"8","key":"2026081210065136600_cit46","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0290559","article-title":"Synthesis\nof Length-Tunable DNA Carriers for Nanopore\nSensing","volume":"18","author":"Roelen","year":"2023","journal-title":"PLoS One"},{"issue":"24","key":"2026081210065136600_cit47","doi-asserted-by":"publisher","first-page":"244902","DOI":"10.1063\/5.0094221","article-title":"How Capture\nAffects Polymer Translocation in a Solitary\nNanopore","volume":"156","author":"Seth","year":"2022","journal-title":"J. Chem. Phys."},{"issue":"15","key":"2026081210065136600_cit48","doi-asserted-by":"publisher","first-page":"7054","DOI":"10.1021\/acs.nanolett.3c01823","article-title":"DNA Volume, Topology,\nand Flexibility Dictate Nanopore Current Signals","volume":"23","author":"Li","year":"2023","journal-title":"Nano Lett."},{"issue":"34","key":"2026081210065136600_cit49","doi-asserted-by":"publisher","first-page":"10527","DOI":"10.1021\/acs.nanolett.4c02538","article-title":"Improved Conductance\nBlockage Modeling of Cylindrical Nanopores, from 2D to Thick Membranes","volume":"24","author":"Charron","year":"2024","journal-title":"Nano Lett."},{"issue":"12","key":"2026081210065136600_cit50","doi-asserted-by":"publisher","first-page":"1093","DOI":"10.1038\/nnano.2016.153","article-title":"Direct Observation\nof DNA Knots Using\na Solid-State Nanopore","volume":"11","author":"Plesa","year":"2016","journal-title":"Nat. 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