2025 Hyper Recent •CC0 1.0 Universal

This work is dedicated to the public domain. No rights reserved.

Access Preprint From Server
August 29th, 2025
Version: 1
Heidelberg University
synthetic biology
bioRxiv

Growth, dissolution and segregation of genetically encoded RNA droplets by ribozyme catalysis

Giessler, F.Open in Google Scholar•Verstraeten, W.Open in Google Scholar•Abele, T.Open in Google Scholar•Maurer, S. J.Open in Google Scholar•Monari, L.Open in Google Scholar•Göpfrich, K.Open in Google Scholar

Active droplets, membraneless compartments driven by internal chemical reactions, are compelling models for protocells and synthetic life. A central challenge is to program their dynamic behaviors using heritable genetic information, which would grant them the capacity to evolve. Here, we create transiently active RNA droplets by integrating sites for ribozyme catalysis directly into the sequence of self-assembling, four-arm RNA nanostars. To enable perfusion and observe the resulting dynamics over time, we develop a method for trapping individual droplets in hydrogel cages by targeted in situ photopolymerization. This enables us to quantify the sequence-programmable droplet dissolution and to control the degradation kinetics by choosing between fast (hammerhead) and slow (hairpin) ribozymes. Furthermore, we trigger the segregation of mixed droplet populations via the sequence-specific cleavage of a chimeric linker RNA. The droplet-encapsulated DNA templates code for the regrowth of new droplets, establishing the proof-of-principle for a minimal, genetically encoded cycle of dissolution and regrowth. By directly linking RNA sequence to droplet stability, composition, and life-cycle dynamics, our work provides a robust platform for engineering evolvable materials and advancing the bottom-up construction of synthetic cells.

Similar Papers

bioRxiv
Tue Sep 02 2025
Tunable Low-Rate Genomic Recombination with Cre-lox in Escherichia coli: A Versatile Tool for Environmental Biosensing and Synthetic Biology
The ability to induce heritable genomic changes in response to environmental cues is valuable for environmental biosensing, for experimentally probing microbial ecology and evolution, and for synthetic biology applications. Site-specific recombinases provide a route to genetic memory via targeted DNA modifications, but their high specificity and efficiency are offset by leaky expression and limite...
Garabello, E.
•
Yoon, H.
•
Reid, M. C.
•
Giometto, A.
bioRxiv
Tue Sep 02 2025
A cytokine receptor-targeting chimera (kineTAC) toolbox for expanding extracellular targeted protein degradation.
Extracellular targeted protein degradation (eTPD) is as an important new modality for manipulating the extracellular proteome. However, most eTPD receptors are expressed broadly or are restricted to the liver. Cytokine receptor targeting chimeras (kineTACs) are genetically encoded bispecifics for eTPD that fuse a natural ligand like CXCL12 to an antibody, directing soluble or membrane proteins for...
Kumru, K.
•
Yao, Z.
•
Holmes, B. B.
•
Zhao, F.
...•
Wells, J. A.
bioRxiv
Tue Sep 02 2025
In Vivo Pathway Optimization in Yeast via LoxPsym-Mediated Shuffling of Upstream Activating Sequences
The budding yeast Saccharomyces cerevisiae plays an integral role in the bioeconomy as a powerful host for industrial bio-manufacturing, driving the production of diverse bio-based products. Achieving optimal product yields requires precise fine-tuning of the expression levels of multiple pathway genes, which often relies on cloning-intensive methods. Here, we present PULSE, an in vivo promoter en...
Ruehmkorff, C.
•
Tafazoli Yazdi, A.
•
Hochrein, L.
bioRxiv
Mon Sep 01 2025
Synthetic ratio computation for programming population composition and multicellular morphology
Recent advancements in genetic engineering have provided diverse tools for artificially synthesizing population diversity in both prokaryotic and eukaryotic systems. However, achieving precise control over the ratios of multiple cell types within a population derived from a single founder remains a significant challenge. In this study, we introduce a suite of recombinase-mediated genetic devices d...
An, B.
•
Tang, T.-C.
•
Zhang, Q.
•
Wang, T.
...•
Zhong, C.
bioRxiv
Mon Sep 01 2025
Software System Design to Support Scale in Mammalian Cell Line Engineering.
Cell line engineering (CLE) is the process of gene editing living cell lines for various purposes such as protein production, fluorescent tagging, compound screening tools, reference standards or diagnostics. Manufacturing processes of the line were typically low throughput and we aimed to increase the capacity of the platform to meet market demand. Software is required to match any throughput and...
McClymont, D. W.
•
McIlwraith, B.
•
Green, A.
•
Coulson, S.
•
Scott, E.
bioRxiv
Sun Aug 31 2025
Phage-mediated just-in-time circuit amplification and delivery for recombinant expression of toxic proteins
In order to address outstanding challenges in bacterial bioreactor bioprocessing, we have developed an approach that alleviates the need for chemical inducers and selective agents (such as antibiotics), and mitigates leaky expression of the production circuit. More specifically, temperate bacteriophage Lambda ({lambda}) was engineered as a chassis to just-in-time amplify and deliver a production c...
Baecker, L. E.
•
Baert, F.
•
Broux, K.
•
Oome, D.
...•
Aertsen, A.
bioRxiv
Sun Aug 31 2025
Synthetic yeast genome SCRaMbLEing uncovers a new role for ribosomal proteins in genetic code expansion
The translation of proteins with non-canonical amino acids (ncAAs) has emerged as a powerful technology for embedding new functional elements into proteins, enabling the development of novel enzymes, materials, and biopharmaceuticals. However, the utility of this approach has been hindered by weak translation efficiencies. To address this challenge, we sought to substantially improve orthogonal tr...
Sanders, J. E. J.
•
Hoffmann, S. A.
•
Moody, E. R.
•
McCullough, M.
...•
Cai, Y.
bioRxiv
Thu Aug 28 2025
Exploration of mRNA-sized RNA import into Saccharomyces cerevisiae mitochondria by a combined synthetic biology and adaptive laboratory evolution approach
Efficient gene integration using RNA-guided endonucleases has not yet been achieved in the mitochondrial genome. Import of nucleic acids into mitochondria, a controversial feature, is essential for implementation of Cas9-mediated genome engineering of mitochondria. Import of short RNAs naturally occurs in mitochondria, and several putative import mechanisms and determinants have been proposed. How...
Koster, C.
•
Kohabir, K.
•
den Ridder, M.
•
Luttik, M.
...•
Daran-Lapujade, P.
bioRxiv
Wed Aug 27 2025
Efficient biosynthesis of 3-hydroxypropionic acid in recombinant Escherichia coli by metabolic engineering
3-Hydroxypropionic acid (3-HP) is a platform compound that can produce many chemical commodities. This study focuses on establishing and optimizing the production of 3-HP in E. coli.To increase the metabolic flux of acetyl-CoA, a precursor for the synthesis of 3-HP, CRISPR/Cas9-based DNA editing technique was used to knock out the genes encoding pyruvate oxidase (poxB), lactate dehydrogenase (ldhA...
Wang, Y.
•
Suo, C.
•
Yang, J.
•
Cui, Y.
...•
Liu, C.