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.
An analytical model for the free energy change during collapse of an RNA molecule from an extended to a compact conformation is proposed. It considers explicit binding of water and ion molecules to the RNA and the exchange of these molecules with the aqueous solution. Microscopic states of the system are captured on a two-dimensional square lattice and evaluated using contact energies. We compute the free energy as a function of a collapse variable and the number of ions bound to the RNA. The major driving force to the collapse of the RNA chain is the gain in water entropy once expelled from the surface of the RNA molecule illustrated by decomposing the free energy into species contributions and their energy and entropy components. The sensitivity of the conclusions of the model to variations in parameters is computed and appears to be weak.
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