In-cell approaches in electron paramagnetic resonance spectroscopy to study conformations of DNA G-quadruplexes

Abstract

The current work deals with the development of EPR for biological applications. Based on existing in vitro techniques, in-cell approaches for determination of distance constraints by double electron-electron resonance (DEER) were established. After proof-of-principle experiments on rigid model systems, in-cell DEER has been applied to study formation of G-quadruplexes adopted by human telomeric DNA oligomers upon their microinjection into living cells.Distances between two spin labels are derived from a DEER experiment. Their incorporation into DNA oligomers was achieved by inserting nitroxide-modified 2′-deoxyuridines at places of thymidines during solid-phase synthesis. By means of CD spectroscopy it was shown that nitroxides incorporated in this way in loop regions of G-quadruplexes do not disturb their initial structures. In spite of the inherent flexibility of loops, measured interspin distances are characteristic: they vary significantly for different G-quadruplex topologies providing a criterion for identification of those topologies. The most probable interspin distance can be predicted from existing high-resolution structural data and compared with reference measurements of known structures allowing for allocation to a certain structure. Even several conformations can be identified within a single experiment if the difference between the most probable interspin distances exceeds 0.3 nm. In the ion-dependent folding of the d[AGGG(TTAGGG)3] the antiparallel-basket topology was found for Na+ (in agreement with high-resolution NMR structure) and for the first time a 1:1 mixture of the antiparallel-basket and the parallel-propeller in the presence of K+ ions was identified.G-quadruplexes may alter folding topology if they are formed one after another within a long DNA sequence. This influence of neighboring G-quadruplexes was studied in a sequence with three G-quadruplex-forming blocks where one of them – in the middle or at the side – was selectively labeled with nitroxides. Consequent distance measurements allowed for conclusion on topologies adopted solely by the middle or the terminal part of the DNA sequence and thus allowed for building models for the folded human telomeric DNA in solution.The intracellular environment is known to reduce nitroxides to EPR-silent hydroxylamines. Therefore, prior to novel in-cell distance measurements free nitroxides were deposited into cells via microinjection. Here, the reduction kinetics of two types of nitroxides was described within Michaelis-Menten formalism for enzymatic processes. Thus, an enzymatic origin of reduction in cellulo was proposed. It was found that inside X. laevis oocytes and in their extract the five member ring nitroxides (like PCA) are reduced much slower than their six member ring analogs. In principle, both six and five member ring nitroxides are suitable for in-cell DEER if no long incubation time is required. However, five member ring nitroxides are preferable due to longer half-life and thus easier handling.Conformations of the d[AGGG(TTAGGG)3] inside oocytes where nucleus-like conditions preside were studied after incubating the unfolded sequence for 15 min after injection. Two conformations at 1:1 ratio were found inside cells. These were the antiparallel-basket and the parallel-propeller. This result is the same as found in K+-containing buffer. Thus, a dominant role of K+ in in-cell folding of the human telomeric oligonucleotide was established. This result was also confirmed by in-extract measurement providing an alternative way to study conformations in cellulo. A time-dependent experiment allowed monitoring of the folding process.DEER was shown to be applicable for structural identification goals of biological objects under their native conditions. In-cell DEER can provide means to analyze conformations and folding of nucleic acid structures inside living cells which has hitherto been unachievable.