Rational Development of a Ligand for Theophylline Riboswitch Aptamer Expands Its Regulatory Dynamic Range in Prokaryotic and Eukaryotic Systems

Rational Development of a Ligand for Theophylline Riboswitch Aptamer Expands Its Regulatory Dynamic Range in Prokaryotic and Eukaryotic Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rational Development of a Ligand for Theophylline Riboswitch Aptamer Expands Its Regulatory Dynamic Range in Prokaryotic and Eukaryotic Systems Ambadas Rode, Rushikesh Khadake, Krushna Shinde This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7535131/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The theophylline riboswitch has served as a foundational tool in synthetic biology for three decades, but efforts to optimize its regulatory performance are limited. Here, we report the development of 4-quinazolinone derivatives, a new class of synthetic ligands designed using a structure-based approach. The biophysical techniques, including Fluorescence assay, ITC, SPR, RNase footprinting, and inline probing, were used to investigate aptamer-ligand interaction; developed ligands interact with the theophylline aptamer with 30-fold higher affinity (KD = 31-96 nM) than the native theophylline ligand (KD = 919 nM). The higher association rate (kon) and lower rate of dissociation (koff) resulted in the enhancement of binding affinity. The synthetic compounds also showed up to 3-fold increase in cellular penetration. These enhancements in binding affinity and superior cellular penetration (~3-fold) of the developed ligand resulted in expanding the riboswitch's regulatory capabilities. We validated the riboswitch activity using fluorescence imaging, spectrofluorometry, flow cytometry, western blotting, and qRT-PCR. In bacterial systems, our 4-quinazolinone ligands enhanced "ON" gene expression by up to 157-fold (compared to 47-fold with theophylline), while dramatically improving "OFF" switch suppression to 81% (relative to 28% with theophylline) at the same ligand concentration. In mycobacteria, 4-quinazolinone derivatives demonstrated an activation ratio of 20.45-fold (versus 6.09-fold with theophylline), further demonstrating the superior performance of our synthetic ligands across diverse bacterial systems. In eukaryotes, these ligands increased gene expression up to 11.27-fold, compared to 2.93-fold with theophylline. Furthermore, Docking and inline-probing data confirmed that the 4-quinazolinone derivatives bind to the same pocket as the native theophylline ligand. Our findings expanded the utility and dynamic range of the theophylline riboswitch, providing a more robust platform for diverse biomedical applications. Biological sciences/Systems biology/Synthetic biology Biological sciences/Chemical biology/RNA Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupportingNBT04092025.pdf Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7535131","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":513004894,"identity":"5cfbd0d8-9bec-4bca-811c-b2677151a218","order_by":0,"name":"Ambadas Rode","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACCRDBxiDHwMADEWAjVosx6VoSG2BaCAL+2Q1sHz6U2aRvbz97gLmioo6BT7qBgCV3DjDPnHEuLXfOmbwExjNnDjOwyRwgYM2NBGZm3rbDuTMkeAwYG9sOMLBJJODXIQ/R8j9dAqzlXx1hLQYQLQcSIFoamAlrMbxzsJlxxrlkwxk8eQkHG44d5iGoRe5282GGD2V28hLsZw8+bKipk5OfQUALAwNjA5x5gIGB6NgZBaNgFIyCUYAPAAArijnnMbC2RgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7252-0385","institution":"Regional Centre for Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Ambadas","middleName":"","lastName":"Rode","suffix":""},{"id":513004895,"identity":"fe1e3020-aea0-431a-8dc6-5b978acf7535","order_by":1,"name":"Rushikesh Khadake","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Rushikesh","middleName":"","lastName":"Khadake","suffix":""},{"id":513004896,"identity":"8b3603b2-45f0-452b-8544-8d55e7ccade2","order_by":2,"name":"Krushna Shinde","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Krushna","middleName":"","lastName":"Shinde","suffix":""}],"badges":[],"createdAt":"2025-09-04 10:37:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7535131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7535131/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91069588,"identity":"5eb4287b-42e6-4405-9a96-30dbce590f60","added_by":"auto","created_at":"2025-09-11 10:32:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1757244,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ManuscriptNBT04092025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7535131/v1_covered_c6864974-a177-42aa-9fbd-66510a827b2d.pdf"},{"id":91069185,"identity":"a49901dd-5bc5-4a53-a47c-3fb663c210be","added_by":"auto","created_at":"2025-09-11 10:24:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2251477,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupportingNBT04092025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7535131/v1/23cdcfff53c0f36564cbd2c2.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Rational Development of a Ligand for Theophylline Riboswitch Aptamer Expands Its Regulatory Dynamic Range in Prokaryotic and Eukaryotic Systems","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7535131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7535131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The theophylline riboswitch has served as a foundational tool in synthetic biology for three decades, but efforts to optimize its regulatory performance are limited. Here, we report the development of 4-quinazolinone derivatives, a new class of synthetic ligands designed using a structure-based approach. The biophysical techniques, including Fluorescence assay, ITC, SPR, RNase footprinting, and inline probing, were used to investigate aptamer-ligand interaction; developed ligands interact with the theophylline aptamer with 30-fold higher affinity (KD = 31-96 nM) than the native theophylline ligand (KD = 919 nM). The higher association rate (kon) and lower rate of dissociation (koff) resulted in the enhancement of binding affinity. The synthetic compounds also showed up to 3-fold increase in cellular penetration. These enhancements in binding affinity and superior cellular penetration (~3-fold) of the developed ligand resulted in expanding the riboswitch's regulatory capabilities. We validated the riboswitch activity using fluorescence imaging, spectrofluorometry, flow cytometry, western blotting, and qRT-PCR. In bacterial systems, our 4-quinazolinone ligands enhanced \"ON\" gene expression by up to 157-fold (compared to 47-fold with theophylline), while dramatically improving \"OFF\" switch suppression to 81% (relative to 28% with theophylline) at the same ligand concentration. In mycobacteria, 4-quinazolinone derivatives demonstrated an activation ratio of 20.45-fold (versus 6.09-fold with theophylline), further demonstrating the superior performance of our synthetic ligands across diverse bacterial systems. In eukaryotes, these ligands increased gene expression up to 11.27-fold, compared to 2.93-fold with theophylline. Furthermore, Docking and inline-probing data confirmed that the 4-quinazolinone derivatives bind to the same pocket as the native theophylline ligand. Our findings expanded the utility and dynamic range of the theophylline riboswitch, providing a more robust platform for diverse biomedical applications.","manuscriptTitle":"Rational Development of a Ligand for Theophylline Riboswitch Aptamer Expands Its Regulatory Dynamic Range in Prokaryotic and Eukaryotic Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 10:24:30","doi":"10.21203/rs.3.rs-7535131/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"06e7cf7c-0528-4c9c-afd9-3cfce53d3be1","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54494031,"name":"Biological sciences/Systems biology/Synthetic biology"},{"id":54494032,"name":"Biological sciences/Chemical biology/RNA"}],"tags":[],"updatedAt":"2025-09-23T12:31:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 10:24:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7535131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7535131","identity":"rs-7535131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}