Biology subjects

Villarreal, D. A.

Publications and source records attributed to Villarreal, D. A..

3 recordsLinked to original sources

bioRxiv2026

Screening metatranscriptomes for ultrastable RNA secondary structures reveals hidden bacteriophages and novel capsid nanomaterials

Metatranscriptomics has transformed our view of RNA bacteriophage diversity, revealing vast numbers of single-stranded RNA (ssRNA) phages whose protein capsids can be engineered for biotechnology applications. However, many ssRNA phages remain hidden from current detection methods, which require protein-level similarity to known phages. Here we show that RNA structure provides an additional signal for the detection of ssRNA phages in metatranscriptomes, including hidden phages missed by prior protein-based methods. By computationally folding each contig and screening for exceptionally stable RNA secondary structures, we find evidence of thousands of previously unrecognized phages encoding novel coat proteins. We express a library of 12,000 such coat proteins in E. coli and find that most assemble into nuclease-resistant capsids. We determine the 3D structure of one such capsid by cryo-electron microscopy and demonstrate that it can be disassembled and reassembled in vitro to package heterologous RNA--a key step toward repurposing these particles as RNA delivery vehicles. We compile the newly discovered ssRNA phages with previously known ones into a database that contains sequence and structural information for over 460,000 unique RNA molecules and over 100,000 distinct coat proteins, providing a comprehensive resource for microbiology and nanomaterials research.

biochemistry↗

bioRxiv2025

Measuring the selective packaging of RNA molecules by viral coat proteins in cells

Some RNA viruses package their genomes with extraordinary selectivity, assembling protein capsids around their own viral RNA while excluding nearly all host RNA. How the assembling proteins distinguish viral RNA from host RNA is not fully understood, but RNA structure is thought to play a key role. To test this idea, we perform in-cellulo packaging experiments using bacteriophage MS2 coat proteins and a variety of RNA molecules in E. coli. In each experiment, plasmid-derived RNA molecules with a specified sequence compete against the cellular transcriptome for packaging by plasmid-derived coat proteins. Following this competition, we quantify the total amount and relative composition of the packaged RNA using electron microscopy, interferometric scattering microscopy, and high-throughput sequencing. By systematically varying the input RNA sequence and measuring changes in packaging outcomes, we are able to directly test competing models of selective packaging. Our results rule out a longstanding model in which selective packaging requires the well-known TR stem-loop, and instead support more recent models in which selectivity emerges from the collective interactions of multiple coat proteins and multiple stem-loops distributed across the RNA molecule. These findings establish a framework for understanding selective packaging in a range of natural viruses and virus-like particles, and lay the groundwork for engineering synthetic systems that package specific RNA cargoes. Significance StatementBacteriophage MS2 packages its RNA genome into protective protein shells called capsids while excluding nearly all host-cell RNA. Engineering synthetic capsids with similar selectivity could enable a broad range of RNA-based technologies, including CRISPR gene editing systems, mRNA vaccines, and other emerging RNA-based therapeutics. Our study shows that selective packaging in MS2 is not dictated by a single, high-affinity RNA-protein interaction but instead emerges from the collective interactions of multiple coat proteins and an ensemble of stem-loops distributed across the RNA molecule. By establishing these collective interactions as the basis of selectivity, our findings provide a foundation for engineering synthetic capsids capable of selectively packaging target RNAs for next-generation RNA-based technologies.

biophysics↗

bioRxiv2023

Measuring intramolecular connectivity in long RNA molecules using two-dimensional DNA patch-probe arrays

We describe a simple method to infer intramolecular connections in a population of long RNA molecules in vitro. First we add DNA oligonucleotide "patches" that perturb the RNA connections, then we use a microarray containing a complete set of DNA oligonucleotide "probes" to record where perturbations occur. The pattern of perturbations reveals couplings between different regions of the RNA sequence, from which we infer connections as well as their prevalences in the population. We validate this patch-probe method using the 1,058-nucleotide RNA genome of satellite tobacco mosaic virus (STMV), which has previously been shown to have multiple long-range connections. Our results not only indicate long duplexes that agree with previous structures but also reveal the prevalence of competing connections. Together, these results suggest that globally-folded and locally-folded structures coexist in solution. We show that the prevalence of connections changes when pseudouridine, an important component of natural and synthetic RNA molecules, is substituted for uridine in STMV RNA.

biophysics↗