Biology subjects

Monari, L.

Publications and source records attributed to Monari, L..

3 recordsLinked to original sources

bioRxiv2025

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

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.

synthetic biology↗

bioRxiv2025

PyFuRNAce: An integrated design engine for RNA origami

To realize the full potential of RNA nanotechnology and RNA origami, user-friendly design tools are needed. Here, we present pyFuRNAce, an open-source, Python-based software package with a graphical user interface that enables the design of complex RNA nanostructures. PyFuRNAce integrates the entire RNA origami workflow--from motif definition and blueprint design to sequence generation and primer selection--into a single, user-friendly platform. Built around a motif-based assembly paradigm, the software enables users to create and modify custom nanostructures through an intuitive web interface with streamlined design steps and real-time 3D visualization. By consolidating multiple design stages into a unified environment, pyFuRNAce reduces the entry barrier for RNA nanotechnology and accelerates the development of functional RNA origami structures for applications in medicine, biotechnology, and synthetic biology.

bioengineering↗

bioRxiv2024

Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

The central dogma at the core of molecular biology states that information flows from DNA to RNA and then to protein. Our research seeks to introduce a conceptually novel approach towards synthetic life by leveraging RNA origami, as an alternative to proteins, requiring only a single copying step between genetic information and function. Here, we report the genetic encoding and expression of an RNA origami cytoskeleton-mimic within giant unilamellar lipid vesicles (GUVs). We design the first RNA origami tiles which fold co-transcriptionally from a DNA template and self-assemble into higher-order 3D RNA origami nanotubes at constant 37{whitebullet} C in GUVs, where they reach several micrometers in length. Unlike pre-formed and encapsulated DNA cytoskeletons, these GUVs produce their own molecular hardware in an out-of-equilibrium process fuelled by nucleotide feeding. To establish genotype-phenotype correlations, we investigate how sequence mutations govern the contour and persistence length of the RNA origami nanotubes with experiments and coarse-grained molecular-dynamics simulations, realizing a phenotypic transition to closed rings. Finally, we achieve RNA origami cortex formation and GUV deformation without chemical functionalization by introducing RNA aptamers into the tile design.Altogether, this work pioneers the expression of RNA origami-based hardware in vesicles as a new approach towards active, evolvable and RNA-based synthetic cells.

synthetic biology↗