As the number and complexity of RNA and DNA structures continue to expand, there is a growing need for robust yet accessible tools that support their accurate interpretation, validation, and refinement. We present DNATCO v5.0 (dnatco.datmos.org), an interactive web application for comprehensive structural analysis of nucleic acids. DNATCO integrates the NtC dinucleotide conformational classes and the CANA structural alphabet to provide an intuitive, geometrically complete description of local backbone and base orientations, complemented by interactive visualization of base pairing. The platform performs quantitative validation of conformational similarity and covalent bond lengths and angles, using newly established nucleic-acid valence-geometry standards. Quantitative validation encompasses the confal score and scattergrams mapping the fit between experimental electron density and geometry similarity to the closest NtC class. All outputs are downloadable. Integrated diagnostic tools help users identify unusual or problematic regions, explore alternative conformations, and generate torsion-restraint files for downstream. DNATCO v5.0 is implemented entirely client-side via WebAssembly, ensuring fast performance and preserving data privacy, and supports both PDB and user-provided structural models. By combining a rigorous geometric framework with an approachable interface, DNATCO enables both non-experts and specialists to evaluate nucleic-acid structures with greater confidence and to improve models in ways that support accurate biological interpretation.

Lincosamides are an important class of clinically used antibiotics that inhibit translation by binding to the peptidyl transferase center of the ribosome. However, their efficacy is impaired by the widespread acquisition of genes coding for erythromycin resistance methyltransferase (Erm), which confer resistance by dimethylating adenine A2058 in the 23S ribosomal RNA. Here, we report clincelin, a novel chimeric lincosamide that retains conserved lincosamide thiooctose core and integrates structural features from two natural antibiotics—a propylated proline from lincomycin and salicylate from celesticetin—together with additional clinically validated C7 chlorination. Biochemical and cellular analyses revealed that the presence of salicylate and chlorination synergistically enhances intracellular accumulation, giving clincelin greater antibacterial activity than the parent compounds and the clinically used clindamycin. Notably, clincelin partially retains its efficacy against erm -mediated resistant pathogens, which is explained by cryo-electron microscopy showing that the salicylate moiety adopts distinct binding modes on drug-sensitive versus A2058-dimethylated Staphylococcus aureus ribosomes. Altogether, a comprehensive functional understanding of lincosamide structural modifications will enable the development of novel agents designed to overcome existing resistance.

Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following the start codon can significantly influence protein expression. However, the impact of these sequences is construct-specific and is not universally applicable to all proteins. Most of the previous research has been limited to selecting from a few rationally designed sequences. In contrast, we used a directed evolution-based methodology, screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increased expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.

Nine new crystal structures of CG-rich DNA 18-mers with the sequence 5'-GGTGGGGGC-XZ-GCCCCACC-3', which are related to the bacterial repetitive extragenic palindromes, are reported. 18-mer oligonucleotides with the central XZ dinucleotide systematically mutated to all 16 sequences show complex behavior in solution, but all ten so far successfully crystallized 18-mers crystallized as A-form duplexes. The refinement protocol benefited from the recurrent use of geometries of the dinucleotide conformer (NtC) classes as refinement restraints in regions of poor electron density. The restraints are automatically generated at the dnatco.datmos.org web service and are available for download. This NtC-driven protocol significantly helped to stabilize the structure refinement. The NtC-driven refinement protocol can be adapted to other low-resolution data such as cryo-EM maps. To test the quality of the final structural models, a novel validation method based on comparison of the electron density and conformational similarity to the NtC classes was employed.

Solution and crystal data are reported for DNA 18-mers with sequences related to those of bacterial noncoding single-stranded DNA segments called repetitive extragenic palindromes (REPs). Solution CD and melting data showed that the CG-rich, near-palindromic REPs from various bacterial species exhibit dynamic temperature-dependent and concentration-dependent equilibria, including architectures compatible with not only hairpins, which are expected to be biologically relevant, but also antiparallel duplexes and bimolecular tetraplexes. Three 18-mer oligonucleotides named Hpar-18 (PDB entry 6rou), Chom-18 (PDB entry 6ros) and its brominated variant Chom-18Br (PDB entry 6ror) crystallized as isomorphic right-handed A-like duplexes. The low-resolution crystal structures were solved with the help of experimental phases for Chom-18Br. The center of the duplexes is formed by two successive T-T noncanonical base pairs (mismatches). They do not deform the double-helical geometry. The presence of T-T mismatches prompted an analysis of the geometries of these and other noncanonical pairs in other DNA crystals in terms of their fit to the experimental electron densities (RSCC) and their geometric fit to the NtC (dinucleotide conformational) classes (https://dnatco.datmos.org/). Throughout this work, knowledge of the NtC classes was used to refine and validate the crystal structures, and to analyze the mismatches.

By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them observed for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical analysis of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased analysis of nucleic acid structures by structural and molecular biologists.