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What are the Physical Properties - Branching Characteristics and 3D sizes - of viral-length RNA molecules?

The figure to the left is a cryo-electron microscopy image that we obtained [RNA 2012] from a purified solution of the RNA molecules (circled in red) corresponding to one of the genes of CCMV. Each of the molecules in the micrograph is chemically identical to the others – the same 3200 nucleotide (nt)-long sequence of RNA. But they appear different because they have different secondary and tertiary structures and because they have different orientations in the vitreous water in which they are trapped at low temperature; accordingly, they have different 2D projections in the transmission micrograph. Indeed, an RNA molecule this long must be regarded as a “statistical object” that must be represented by an ensemble of configurations, much like a long semi-flexible polymer.

How can in vitro packaged self-amplifying RNA genes be used for in situ expression of proteins


In our general introduction of "In Vivo Self Amplifying RNA research projects", we emphasized how important it is to mimic the natural use of RNA replicons by a wide range of positive-strand RNA viruses, for purposes of high-level protein expression. We featured the particular case of Nodamura virus, with its two-molecule genome consisting of RNA1 coding for the RNA replicase (RdRp) and RNA2 coding for the capsid protein. One way to use this system for delivery of genes of interest (GOIs) is to simply insert the GOI into the end of RNA1, immediately following a self-cleaving proteolytic sequence, so that the GOI RNA is replicated along with RNA1 and so that its gene product – the desired therapeutic or reporter protein – will be cleaved in functional form from the RdRp. We have done this using EYFP as the reporter gene.

In Vivo Self-Amplifying RNA Research Projects

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By “in vivo” experiments we mean ones performed in host cells (rather than in host animals, which is how the term “in vivo” is more generally used, in virology and medical contexts). And by “host cells” we mean controlled monolayers of cells in petri dishes. In this classical form the cells can easily be transfected by RNA or VLPs, or infected by virus, and the transformed cells can easily be assayed in a large number of ways. Ultimately, we would like to transform and assay cells that have been targeted in animals, but we need first to demonstrate and understand how changes of interest can be effected under the controlled conditions of cell culture.

IN VITRO/PHYSICAL CHEMISTRY PROJECTS

 

Virus particles can be as simple as a molecule of RNA or DNA inside a spherical shell – the “capsid” – made up of multiple copies of a single protein. Further, because viruses only “become alive” when they are inside their hosts, it is possible to study them as physical objects, i.e., to do the same controlled experiments (and theory) on them that one routinely does with more familiar polymer and colloidal systems.

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