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.

Single-particle studies of the effects of RNA-protein interactions on the self-assembly of RNA virus particles.

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.