Spatial\nControl over Reactions via Localized Transcription\nwithin Membraneless DNA Nanostar Droplets

Biomolecular condensates control\nwhere and how fast many chemical\nreactions occur in cells by partitioning reactants and catalysts,\nenabling simultaneous reactions in different spatial locations of\na cell. Even without a membrane or physical barrier, the partitioning\nof the reactants can affect the rates of downstream reaction cascades\nin ways that depend on reaction location. Such effects can enable\nsystems of biomolecular condensates to spatiotemporally orchestrate\nchemical reaction networks in cells to facilitate complex behaviors\nsuch as ribosome assembly. Here, we develop a system for developing\nsuch control in synthetic systems. We localize different transcription\ntemplates within different phase-separated, membraneless DNA nanostar\n(NS) dropletsprogrammable, in vitro liquid–liquid phase\nseparation systems for partitioning of substrates and localization\nof reactions to membraneless droplets. When RNA produced within such\ndroplets is also degraded in the bulk, droplet-localized transcription\ncreates RNA concentration gradients. Consistent with the formation\nof these gradients, toehold-mediated strand displacement reactions\ninvolving transcripts are 2-fold slower far from the site of transcription\nthan when nearby. We then demonstrate how multiple such gradients\ncan form and be maintained independently by simultaneous transcription\nreactions occurring in tandem, each localized to different NS droplet\ntypes. Our results provide a means for constructing reaction systems\nin which different reactions are spatially localized and controlled\nwithout the need for physical membranes. This system also provides\na means for generally studying how localized reactions and the exchange\nof reaction products might occur between protocells.