Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.

Current interest in lone‐pair⋅⋅⋅π (lp⋅⋅⋅π) interactions is gaining momentum in biochemistry and (supramolecular) chemistry. However, the physicochemical origin of the exceptionally short (ca. 2.8 Å) oxygen‐to‐nucleobase plane distances observed in prototypical Z‐DNA CpG steps remains unclear. High‐level quantum mechanical calculations, including SAPT2+3 interaction energy decompositions, demonstrate that lp⋅⋅⋅π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatic interactions combined with stereochemical effects imposed by the locally strained structural context. They also suggest that the carbon van der Waals (vdW) radii, originally derived for sp3 carbons, should not be used for smaller sp2 carbons attached to electron‐withdrawing groups. Using a more adapted carbon vdW radius results in these lp⋅⋅⋅π contacts being no longer of the sub‐vdW type. These findings challenge the whole lp⋅⋅⋅π concept that refers to elusive orbital interactions that fail to explain short interatomic contact distances.

When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its "natural" fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen-π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for "GNRA-type" folds) and a newly described Z-turn (for "UNCG-type" folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict.