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1. The Nonmonotonic Dose Dependence of Protein Expression in Cells Transfected with Self-Amplifying RNA

   https://doi.org/10.1128/jvi.01858-21  

   Tanimoto, C. R.; Thurm, A. R.; Brandt, D. S.; Knobler, C. M.; Gelbart, W. M. (April 2022, Journal of virology)

   Self-amplifying (sa) RNA molecules—“replicons”—derived from the genomes of positive-sense RNA viruses are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a nonmonotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-saRNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a “sweet spot” at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of Lipofectamine, the replicons were mixed with a “carrier” RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as 1/10 of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements. 

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2. Intracellular bottlenecking permits no more than three tomato yellow leaf curl virus genomes to initiate replication in a single cell

   https://doi.org/10.1371/journal.ppat.1011365  

   Ren, Ruifan; Zheng, Limin; Han, Junping; Perdoncini Carvalho, Camila; Miyashita, Shuhei; Zhang, Deyong; Qu, Feng (May 2023, PLOS Pathogens)

   Wang, Aiming (Ed.)

   Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. Nevertheless, it is unclear if similarly stringent reproductive bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication. 

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3. Replication and single-cycle delivery of SARS-CoV-2 replicons

   https://doi.org/10.1126/science.abj8430  

   Ricardo-Lax, Inna; Luna, Joseph M.; Thao, Tran Thi; Le Pen, Jérémie; Yu, Yingpu; Hoffmann, H.-Heinrich; Schneider, William M.; Razooky, Brandon S.; Fernandez-Martinez, Javier; Schmidt, Fabian; et al (November 2021, Science)

   Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization. 

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4. RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds

   https://doi.org/10.1038/s41557-021-00679-1  

   Cody Geary, Guido Grossi (May 2021, Nature chemistry)

   null (Ed.)

   RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology. 

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5. Nanomechanics and co-transcriptional folding of Spinach and Mango

   https://doi.org/10.1038/s41467-019-12299-y  

   Mitra, Jaba; Ha, Taekjip (September 2019, Nature Communications)

   Abstract Recent advances in fluorogen-binding “light-up” RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2,iMangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding iniMangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications. 

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