Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells

Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells | 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 Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells Koki Kamiya, Sumin Lee, Kotaro Baba This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7138498/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Mar, 2026 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Dynamic regulation of lipid membrane composition is fundamental to living cells; however, synthetic analogs capable of such regulation remain scarce. Here, we present an artificial cell platform in which riboswitch-mediated expression of phospholipase D (PLD) enables stimulus-responsive lipid remodeling within lipid vesicles. In this system, chemically induced activation of a fluoride-responsive riboswitch triggers cell-free synthesis of PLD, which hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) in the inner leaflet of the vesicles. This enzymatic reaction generates a negatively charged asymmetric membrane, enabling functionalization with mechanosensitive channels such as the mechanosensitive channel of large conductance (MscL). We characterized the kinetics of asymmetry generation by varying plasmid DNA and fluoride concentrations, and evaluated membrane behavior using lipid compositions with or without cholesterol. Our platform demonstrates a novel strategy for coupling gene expression to dynamic membrane remodeling and underscores the potential of riboswitch-regulated lipid transitions in building environment-responsive artificial cells with programmable functions. Biological sciences/Systems biology/Synthetic biology Biological sciences/Chemical biology/Biophysical chemistry Biological sciences/Biophysics Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SUPPLEMENTARYINFORMATION.pdf Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells Cite Share Download PDF Status: Published Journal Publication published 19 Mar, 2026 Read the published version in Communications Biology → 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. 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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-7138498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":487498232,"identity":"bc9979ae-3191-4fd5-8060-91b27311ecf5","order_by":0,"name":"Koki Kamiya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBACCSA+wMBggyKYQIyWNBK1AMFhEhwm2X468TBPzXm7Dbe7Ez8wtjHI8zcwPHuAT4s0T+6GwzzHbidvuHN2swRQi+GMAwzpBvi0yDGAtLDdTja4kbtB+m8bA+MGoM8k8GrhfwvU8u8cSMvmH0Bb7AlqkZYA2sLbdsAOqGUbyGGJBLVIzni74eDcvuQESaAWC4ZzEskzDhPwi8T53M0f3nyzs+cDOuwGQ5mNbX97T9oDfFpAgImHgSGxAWoEAwMzTxpe5SDA+IOBwR6Jz36MoJZRMApGwSgYUQAApZhMfyw8JiUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9143-9841","institution":"Gunma University","correspondingAuthor":true,"prefix":"","firstName":"Koki","middleName":"","lastName":"Kamiya","suffix":""},{"id":487498233,"identity":"8c42b559-db66-4eb8-b1ef-c1985fc335e3","order_by":1,"name":"Sumin Lee","email":"","orcid":"","institution":"Gunma University","correspondingAuthor":false,"prefix":"","firstName":"Sumin","middleName":"","lastName":"Lee","suffix":""},{"id":487498234,"identity":"fb4e605f-3227-47e9-b0db-a1c03d66062c","order_by":2,"name":"Kotaro Baba","email":"","orcid":"","institution":"Gunma University","correspondingAuthor":false,"prefix":"","firstName":"Kotaro","middleName":"","lastName":"Baba","suffix":""}],"badges":[],"createdAt":"2025-07-16 09:50:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7138498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7138498/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-026-09890-7","type":"published","date":"2026-03-19T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":107868014,"identity":"38b1d510-c03e-4bab-97d7-c38241bb13df","added_by":"auto","created_at":"2026-04-27 07:06:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1630858,"visible":true,"origin":"","legend":"Article File","description":"","filename":"PLDpaper250714.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7138498/v1_covered_0e4bf8d8-bcc8-4d3c-96b1-9db44749b585.pdf"},{"id":88391829,"identity":"1ad97c2c-eced-4d2e-9e74-2f4d08b95804","added_by":"auto","created_at":"2025-08-06 04:39:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":675164,"visible":true,"origin":"","legend":"Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells","description":"","filename":"SUPPLEMENTARYINFORMATION.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7138498/v1/8a20661e0e820e1df54fa6e5.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells","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-7138498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7138498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Dynamic regulation of lipid membrane composition is fundamental to living cells; however, synthetic analogs capable of such regulation remain scarce. 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