{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T16:12:30Z","timestamp":1790784750975,"version":"4.1.0"},"reference-count":57,"publisher":"Oxford University Press (OUP)","issue":"2","license":[{"start":{"date-parts":[[2023,1,9]],"date-time":"2023-01-09T00:00:00Z","timestamp":1673222400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000011","name":"Howard Hughes Medical Institute","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000011","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001797","name":"PhRMA Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100001797","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009559","name":"Life Sciences Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100009559","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Institutes of Health Chemical Biology","award":["T32 GM067543"],"award-info":[{"award-number":["T32 GM067543"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2023,1,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Over the past three decades, researchers have found that some engineered aptamers can be made to work well in test tubes but that these same aptamers might fail to function in cells. To help address this problem, we developed the \u2018Graftamer\u2019 approach, an experimental platform that exploits the architecture of a natural riboswitch to enhance in vitro aptamer selection and accelerate in vivo testing. Starting with combinatorial RNA pools that contain structural features of a guanine riboswitch aptamer interspersed with regions of random sequence, we performed multiplexed in vitro selection with a collection of small molecules. This effort yielded aptamers for quinine, guanine, and caffeine that appear to maintain structural features of the natural guanine riboswitch aptamer. Quinine and caffeine aptamers were each grafted onto a natural guanine riboswitch expression platform and reporter gene expression was monitored to determine that these aptamers function in cells. Additionally, we determined the secondary structure features and survival mechanism of a class of RNA sequences that evade the intended selection strategy, providing insight into improving this approach for future efforts. These results demonstrate that the Graftamer strategy described herein represents a convenient and straightforward approach to develop aptamers and validate their in vivo function.<\/jats:p>","DOI":"10.1093\/nar\/gkac1218","type":"journal-article","created":{"date-parts":[[2023,1,9]],"date-time":"2023-01-09T08:24:28Z","timestamp":1673252668000},"page":"966-981","source":"Crossref","is-referenced-by-count":58,"title":["Exploiting natural riboswitches for aptamer engineering and validation"],"prefix":"10.1093","volume":"51","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7400-9196","authenticated-orcid":false,"given":"Michael G","family":"Mohsen","sequence":"first","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven , CT 06511, USA"},{"name":"Howard Hughes Medical Institute, Yale University , New Haven , CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew K","family":"Midy","sequence":"additional","affiliation":[{"name":"Department of Molecular Biophysics and Biochemistry, Yale University , New Haven , CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aparaajita","family":"Balaji","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven , CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2165-536X","authenticated-orcid":false,"given":"Ronald\u00a0R","family":"Breaker","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven , CT 06511, USA"},{"name":"Howard Hughes Medical Institute, Yale University , New Haven , CT 06511, USA"},{"name":"Department of Molecular Biophysics and Biochemistry, Yale University , New Haven , CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2023,1,9]]},"reference":[{"key":"2023012711485411200_B1","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1038\/346818a0","article-title":"In vitro selection of RNA molecules that bind specific ligands","volume":"346","author":"Ellington","year":"1990","journal-title":"Nature"},{"key":"2023012711485411200_B2","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1038\/nrg2775","article-title":"Synthetic biology: applications come of age","volume":"11","author":"Khalil","year":"2010","journal-title":"Nat. Rev. Genet."},{"key":"2023012711485411200_B3","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1021\/acssynbio.9b00475","article-title":"The theophylline aptamer: 25 years as an important tool in cellular engineering research","volume":"9","author":"Wrist","year":"2020","journal-title":"ACS Synth. Biol."},{"key":"2023012711485411200_B4","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.trac.2007.12.004","article-title":"Aptamer-based biosensors","volume":"27","author":"Song","year":"2008","journal-title":"TrAC - Trends Anal. Chem."},{"key":"2023012711485411200_B5","doi-asserted-by":"crossref","first-page":"434","DOI":"10.3389\/fchem.2020.00434","article-title":"Aptamer-Based biosensors for environmental monitoring","volume":"8","author":"McConnell","year":"2020","journal-title":"Front. Chem."},{"key":"2023012711485411200_B6","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1016\/j.cbpa.2006.03.015","article-title":"Aptamer therapeutics advance","volume":"10","author":"Lee","year":"2006","journal-title":"Curr. Opin. Chem. Biol."},{"key":"2023012711485411200_B7","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1038\/nrd3141","article-title":"Aptamers as therapeutics","volume":"9","author":"Keefe","year":"2010","journal-title":"Nat. Rev. Drug Discov."},{"key":"2023012711485411200_B8","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1016\/S1074-5521(02)00224-7","article-title":"Genetic control by a metabolite binding mRNA","volume":"9","author":"Nahvi","year":"2002","journal-title":"Chem. Biol."},{"key":"2023012711485411200_B9","doi-asserted-by":"crossref","first-page":"995","DOI":"10.1261\/rna.061234.117","article-title":"Riboswitch diversity and distribution","volume":"23","author":"Mccown","year":"2017","journal-title":"RNA"},{"key":"2023012711485411200_B10","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/nrm1403","article-title":"Gene regulation by riboswitches","volume":"5","author":"Mandal","year":"2004","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"2023012711485411200_B11","doi-asserted-by":"crossref","first-page":"7054","DOI":"10.1073\/pnas.86.18.7054","article-title":"Modern metabolism as a palimpsest of the RNA world","volume":"86","author":"Benner","year":"1989","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023012711485411200_B12","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1002\/cbdv.200790055","article-title":"Ribozyme catalysis of metabolism in the RNA world","volume":"4","author":"Chen","year":"2007","journal-title":"Chem. Biodivers."},{"key":"2023012711485411200_B13","doi-asserted-by":"crossref","first-page":"a003566","DOI":"10.1101\/cshperspect.a003566","article-title":"Riboswitches and the RNA World","volume":"4","author":"Breaker","year":"2012","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"2023012711485411200_B14","doi-asserted-by":"crossref","first-page":"1277","DOI":"10.1016\/j.ymthe.2018.02.021","article-title":"Conditional regulation of gene expression by Ligand-Induced occlusion of a MicroRNA target sequence","volume":"26","author":"Mou","year":"2018","journal-title":"Mol. Ther."},{"key":"2023012711485411200_B15","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.copbio.2019.11.008","article-title":"Aptamers in RNA-based switches of gene expression","volume":"63","author":"Sp\u00f6ring","year":"2020","journal-title":"Curr. Opin. Biotechnol."},{"key":"2023012711485411200_B16","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1126\/science.2200121","article-title":"Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase","volume":"249","author":"Tuerk","year":"1990","journal-title":"Science"},{"key":"2023012711485411200_B17","doi-asserted-by":"crossref","first-page":"16299","DOI":"10.1021\/ja508478x","article-title":"Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution","volume":"136","author":"Filonov","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"2023012711485411200_B18","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1038\/nchembio.2278","article-title":"Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors","volume":"13","author":"Porter","year":"2017","journal-title":"Nat. Chem. Biol."},{"key":"2023012711485411200_B19","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1038\/s41589-021-00925-0","article-title":"Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag","volume":"18","author":"Dey","year":"2022","journal-title":"Nat. Chem. Biol."},{"key":"2023012711485411200_B20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/nar\/gkv1289","article-title":"Automated physics-based design of synthetic riboswitches from diverse RNA aptamers","volume":"44","author":"Espah\u00a0Borujeni","year":"2016","journal-title":"Nucleic Acids Res"},{"key":"2023012711485411200_B21","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/S0167-7799(99)01383-9","article-title":"Nucleic acid molecular switches","volume":"17","author":"Soukup","year":"1999","journal-title":"Trends Biotechnol"},{"key":"2023012711485411200_B22","doi-asserted-by":"crossref","first-page":"1194","DOI":"10.1126\/science.1218298","article-title":"Fluorescence imaging of cellular metabolites with RNA","volume":"335","author":"Paige","year":"2012","journal-title":"Science"},{"key":"2023012711485411200_B23","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1038\/14947","article-title":"Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP","volume":"6","author":"Koizumi","year":"1999","journal-title":"Nat. Struct. Biol."},{"key":"2023012711485411200_B24","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1002\/anie.200461848","article-title":"In vitro selection of structure-switching signaling aptamers","volume":"44","author":"Nutiu","year":"2005","journal-title":"Angew. Chem. Int. Ed."},{"key":"2023012711485411200_B25","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1007\/978-1-4939-7295-1_18","article-title":"A Capture-SELEX strategy for multiplexed selection of RNA aptamers against small molecules","volume":"1671","author":"Lauridsen","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"2023012711485411200_B26","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.ymeth.2016.04.032","article-title":"In vitro selection and amplification protocols for isolation of aptameric sensors for small molecules","volume":"106","author":"Yang","year":"2016","journal-title":"Methods"},{"key":"2023012711485411200_B27","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.ymeth.2019.04.004","article-title":"RNA-based Capture-SELEX for the selection of small molecule-binding aptamers","volume":"161","author":"Boussebayle","year":"2019","journal-title":"Methods"},{"key":"2023012711485411200_B28","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/S0092-8674(03)00391-X","article-title":"Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria","volume":"113","author":"Mandal","year":"2003","journal-title":"Cell"},{"key":"2023012711485411200_B29","doi-asserted-by":"crossref","first-page":"e2120246119","DOI":"10.1073\/pnas.2120246119","article-title":"Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2\u2032-deoxyguanosine","volume":"119","author":"Dhakal","year":"2022","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023012711485411200_B30","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1038\/nsmb710","article-title":"Adenine riboswitches and gene activation by disruption of a transcription terminator","volume":"11","author":"Mandal","year":"2004","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2023012711485411200_B31","doi-asserted-by":"crossref","first-page":"16092","DOI":"10.1073\/pnas.0705884104","article-title":"Guanine riboswitch variants from Mesoplasma florum selectively recognize 2\u2032-deoxyguanosine","volume":"104","author":"Kim","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023012711485411200_B32","doi-asserted-by":"crossref","first-page":"E2077","DOI":"10.1073\/pnas.1619581114","article-title":"Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity","volume":"114","author":"Weinberg","year":"2017","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023012711485411200_B33","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1038\/nature03037","article-title":"Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine","volume":"432","author":"Batey","year":"2004","journal-title":"Nature"},{"key":"2023012711485411200_B34","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1016\/j.chembiol.2004.11.018","article-title":"Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs","volume":"11","author":"Serganov","year":"2004","journal-title":"Chem. Biol."},{"key":"2023012711485411200_B35","doi-asserted-by":"crossref","first-page":"754","DOI":"10.1016\/j.jmb.2006.04.003","article-title":"Thermodynamic and kinetic characterization of Ligand binding to the Purine Riboswitch aptamer domain","volume":"359","author":"Gilbert","year":"2006","journal-title":"J. Mol. Biol."},{"key":"2023012711485411200_B36","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1042\/BC20070088","article-title":"Purine sensing by riboswitches","volume":"100","author":"Kim","year":"2008","journal-title":"Biol. Cell"},{"key":"2023012711485411200_B37","doi-asserted-by":"crossref","first-page":"13297","DOI":"10.1021\/bi700410g","article-title":"Mutational analysis of the purine riboswitch aptamer domain","volume":"46","author":"Gilbert","year":"2007","journal-title":"Biochemistry"},{"key":"2023012711485411200_B38","doi-asserted-by":"crossref","first-page":"8793","DOI":"10.1073\/pnas.92.19.8793","article-title":"Transcription termination at intrinsic terminators: the role of the RNA hairpin","volume":"92","author":"Wilson","year":"1995","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023012711485411200_B39","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1126\/science.284.5414.611","article-title":"Mechanism of intrinsic transcription termination and antitermination","volume":"284","author":"Yarnell","year":"1999","journal-title":"Science"},{"key":"2023012711485411200_B40","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1007\/978-1-59745-033-1_4","article-title":"In-line probing analysis of riboswitches","volume":"419","author":"Regulski","year":"2008","journal-title":"Methods Mol. Biol."},{"key":"2023012711485411200_B41","doi-asserted-by":"crossref","first-page":"e0185056","DOI":"10.1371\/journal.pone.0185056","article-title":"BBMerge \u2013 Accurate paired shotgun read merging via overlap","volume":"12","author":"Bushnell","year":"2017","journal-title":"PLoS One"},{"key":"2023012711485411200_B42","doi-asserted-by":"crossref","first-page":"e230","DOI":"10.1038\/mtna.2015.4","article-title":"FASTAptamer: a bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections","volume":"4","author":"Alam","year":"2015","journal-title":"Mol. Ther. - Nucleic Acids"},{"key":"2023012711485411200_B43","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1093\/bioinformatics\/btk008","article-title":"CMfinder - a covariance model based RNA motif finding algorithm","volume":"22","author":"Yao","year":"2006","journal-title":"Bioinformatics"},{"key":"2023012711485411200_B44","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/1471-2105-12-3","article-title":"R2R - software to speed the depiction of aesthetic consensus RNA secondary structures","volume":"12","author":"Weinberg","year":"2011","journal-title":"BMC Bioinformatics"},{"key":"2023012711485411200_B45","doi-asserted-by":"crossref","first-page":"1308","DOI":"10.1017\/S1355838299990891","article-title":"Relationship between internucleotide linkage geometry and the stability of RNA","volume":"5","author":"Soukup","year":"1999","journal-title":"RNA"},{"key":"2023012711485411200_B46","doi-asserted-by":"crossref","first-page":"012006","DOI":"10.1088\/1755-1315\/462\/1\/012006","article-title":"Antimicrobial activity of quinine derivatives against human pathogenic bacteria","volume":"462","author":"Antika","year":"2020","journal-title":"IOP Conf. Ser. Earth Environ. Sci."},{"key":"2023012711485411200_B47","doi-asserted-by":"crossref","first-page":"14","DOI":"10.3389\/fchem.2016.00014","article-title":"Small-molecule binding aptamers: selection strategies, characterization, and applications","volume":"4","author":"Ruscito","year":"2016","journal-title":"Front. Chem."},{"key":"2023012711485411200_B48","first-page":"145","article-title":"Selection in vitro of allosteric ribozymes","volume":"252","author":"Roth","year":"2004","journal-title":"Methods Mol. Biol."},{"key":"2023012711485411200_B49","doi-asserted-by":"crossref","first-page":"122274","DOI":"10.1016\/j.talanta.2021.122274","article-title":"Capture-SELEX for aptamer selection: a short review","volume":"229","author":"Lyu","year":"2021","journal-title":"Talanta"},{"key":"2023012711485411200_B50","doi-asserted-by":"crossref","first-page":"1611","DOI":"10.1021\/acssensors.8b00516","article-title":"Strategies for creating structure-Switching aptamers","volume":"3","author":"Feagin","year":"2018","journal-title":"ACS Sensors"},{"key":"2023012711485411200_B51","doi-asserted-by":"crossref","first-page":"11692","DOI":"10.1039\/D1SC02715H","article-title":"RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors","volume":"12","author":"Sanford","year":"2021","journal-title":"Chem. Sci."},{"key":"2023012711485411200_B52","doi-asserted-by":"crossref","first-page":"415697","DOI":"10.1155\/2012\/415697","article-title":"Capture-SELEX: selection of DNA aptamers for aminoglycoside antibiotics","volume":"2012","author":"Stoltenburg","year":"2012","journal-title":"J. Anal. Methods Chem."},{"key":"2023012711485411200_B53","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1016\/S1074-5521(97)90197-6","article-title":"Rational design of allosteric ribozymes","volume":"4","author":"Tang","year":"1997","journal-title":"Chem. Biol."},{"key":"2023012711485411200_B54","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1038\/ncomms12834","article-title":"Twister ribozymes as highly versatile expression platforms for artificial riboswitches","volume":"7","author":"Felletti","year":"2016","journal-title":"Nat. Commun."},{"key":"2023012711485411200_B55","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.1038\/gt.2009.81","article-title":"Engineering ligand-responsive gene-control elements: lessons learned from natural riboswitches","volume":"16","author":"Link","year":"2009","journal-title":"Gene Ther"},{"key":"2023012711485411200_B56","doi-asserted-by":"crossref","first-page":"E48","DOI":"10.1093\/nar\/gky062","article-title":"A small, portable RNA device for the control of exon skipping in mammalian cells","volume":"46","author":"Vogel","year":"2018","journal-title":"Nucleic Acids Res"},{"key":"2023012711485411200_B57","doi-asserted-by":"crossref","first-page":"E71","DOI":"10.1093\/nar\/gkab233","article-title":"Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans","volume":"49","author":"Finke","year":"2021","journal-title":"Nucleic Acids Res."}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/51\/2\/966\/48929514\/gkac1218.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/51\/2\/966\/48929514\/gkac1218.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,27]],"date-time":"2023-01-27T12:09:03Z","timestamp":1674821343000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/51\/2\/966\/6976067"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,9]]},"references-count":57,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,1,9]]},"published-print":{"date-parts":[[2023,1,25]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkac1218","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2023,1,25]]},"published":{"date-parts":[[2023,1,9]]}}}