Structural investigation of nucleic acids is usually carried out either in diluted buffered solutions or on crystals of these biomolecules. These environments markedly differ from the native one in which these molecules are found, where effects as macromolecular crowding or intermolecular interactions can play a significant role on the conformation.
Aim of the present study was to investigate whether the structures of short RNA duplexes would be different when placed in a buffered solution vs inside Xenopus lævis oocytes. The structural information was obtained as a set of distance constraints between paramagnetic tags covalently linked to the biomolecule under study; these constraints were obtained from 4-pulse PELDOR measurements.
In this work two different labelling strategies were used to label a uridine with an isoindoline-derived spin label, differing both by the flexibility of the linker and by the point of attachment. In the first case, the spin label was post-synthetically conjugated to the ribose sugar ring via a thiourea linkage. In the second case, the spin label was attached to the uracil base through a C-C bond; an intramolecular hydrogen bond between the label and the uracil restricts the rotation around the C-C bond, resulting in a semi-rigid label. For both strategies, the paramagnetic centre was protected against the reducing environment of the cell by replacing the normal gem-dimethyl groups, flanking the nitroxide moiety, with gem-diethyl groups.
The results showed a clear, reproducible shift of the mean inter-spin distance to shorter values upon internalisation inside cells. In order to understand these findings, the duplexes were further investigated under different in-vitro conditions, such as in cytoplasmic extract from the Xenopus lævis oocytes as well as in the presence of protein crowders (BSA and lysozyme). The results of these experiments indicate an involvement of electrostatic interactions in the observed conformational changes.