Biology subjects

Nuccio, S. P.

Publications and source records attributed to Nuccio, S. P..

4 recordsLinked to original sources

bioRxiv2025

Expression of nano-engineered RNA organelles in bacteria

Designing synthetic biomolecular condensates, or membrane-less organelles, offers insights on the functions of their natural counterparts, and is equally valuable for cellular and metabolic engineering. Choosing E. coli for its biotechnological relevance, we deploy RNA nanotechnology to design and express non-natural membrane-less organelles in vivo. The designer condensates assemble co-transcriptionally from branched RNA motifs interacting via base-pairing. Exploiting binding selectivity we express orthogonal, non-mixing condensates, and by embedding a protein-binding aptamer we achieve selective protein recruitment. Condensates can be made to dissolve and re-assemble upon thermal cycling, thereby reversibly releasing and re-capturing protein clients. The synthetic organelles are expressed robustly across the cell population and remain stable despite enzymatic RNA processing. Compared with existing solutions based on peptide building blocks or repetitive RNA sequences, these nanostructured RNA motifs enable algorithmic control over interactions, affinity for clients, and condensate microstructure, opening new directions in synthetic biology and biotechnology.

synthetic biology↗

bioRxiv2024

Individual G-quadruplex targeting with ligand-functionalized CRISPR-Cas9 uncovers transcriptional-dependent functional responses

The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, severely limiting their scope. Herein, we describe a new Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrated that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA revealed that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4-site. We further demonstrated that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides an innovative platform to investigate G4- and iM-biology with high precision and unveils the therapeutic relevance of individual DNA structures with unprecedented selectivity.

cell biology↗

bioRxiv2024

Programmable RNA writing with trans-splicing

RNA editing offers the opportunity to introduce either stable or transient modifications to nucleic acid sequence without permanent off-target effects, but installation of arbitrary edits into the transcriptome is currently infeasible. Here, we describe Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5' or 3' trans-splicing. In trans-splicing, an exogenous template is introduced to compete with the endogenous pre-mRNA, allowing for replacement of upstream or downstream exon sequence. Using Cas7-11 cleavage of pre-mRNAs to bias towards editing outcomes, we boost the efficiency of RNA trans-splicing by 10-100 fold, achieving editing rates between 5-50% and 85% on endogenous and reporter transcripts, respectively, while maintaining high-fidelity. We demonstrate PRECISE editing across 11 distinct endogenous transcripts of widely varying expression levels, showcasing more than 50 types of edits, including all 12 possible transversions and transitions, insertions ranging from 1 to 1,863 nucleotides, and deletions. We show high efficiency replacement of exon 4 of MECP2, addressing most mutations that drive the Rett Syndrome; editing of SHANK3 transcripts, a gene involved in Autism; and replacement of exon 1 of HTT, removing the hallmark repeat expansions of Huntington's disease. Whole transcriptome sequencing reveals the high precision of PRECISE editing and lack of off-target trans-splicing activity. Furthermore, we combine payload engineering and ribozymes for protein-free, high-efficiency trans-splicing, with demonstrated efficiency in editing HTT exon 1 via AAV delivery. We show that the high activity of PRECISE editing enables editing in non-dividing neurons and patient-derived Huntingtons disease fibroblasts. PRECISE editing markedly broadens the scope of genetic editing, is straightforward to deliver over existing gene editing tools like prime editing, lacks permanent off-targets, and can enable any type of genetic edit large or small, including edits not otherwise possible with existing RNA base editors, widening the spectrum of addressable diseases.

molecular biology↗

bioRxiv2023

Co-transcriptional production of programmable RNA condensates and synthetic organelles

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate guest molecules. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with controlled number, size, morphology and composition, and that display the ability to selectively capture proteins. The in situ expression of programmable RNA condensates could underpin spatial organisation of functionalities in both biological and synthetic cells.

synthetic biology↗