While standard biochemistry and molecular biological techniques suffice for much of our work, several of our studies require sophisticated state-of-the-art equipment that is made available to us at the California NanoSystems Institute (CNSI) here at UCLA or in the laboratories of our collaborators.
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.
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.