Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

“Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery

The crystal structure of an ‘All Locked’ nucleic acid duplex

‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern

Diffraction techniques and vibrational spectroscopy opportunities to characterise bones

From a histological point of view, bones that allow body mobility and protection of internal organs consist not only of different organic and inorganic tissues but include vascular and nervous elements as well. Moreover, due to its ability to host different ions and cations, its mineral part represents an important reservoir, playing a key role in the metabolic activity of the organism. From a structural point of view, bones can be considered as a composite material displaying a hierarchical structure at different scales. At the nanometre scale, an organic part, i.e. collagen fibrils and an inorganic part, i.e. calcium phosphate nanocrystals are intimately mixed to assure particular mechanical properties

Comparative X-ray structure analyses of nucleic acids

Hochaufgelöste Strukturen von Nukleinsäuren geben Aufschluss über die Konformation, Hydratation und Ligandenbindung der Moleküle. Als Modellsysteme für natürliche Nukleinsäuren wurden zwei tRNA-Akzeptorstämme und zum Vergleich eine davon abgeleitete modifizierte „locked“ Nukleinsäure untersucht. Um eine maximale Auflösung der Strukturen zu erzielen, wurden Kristallisationsansätze unter Mikrogravitationsbedingungen durchgeführt und gleichzeitig die Länge der untersuchten Moleküle auf sieben Basenpaare begrenzt. Anhand der Struktur des tRNAArg-Akzeptorstammes konnte beispielhaft die Bindung von kleinen Molekülen an einfache Nukleinsäurefaltungsmotive gezeigt werden. Die Ergebnisse stehen im Einklang mit Untersuchungen zur basen-spezifischen Bindung von kleinen Molekülen an Aptamere und Riboswitches. Die Struktur des tRNASer- Akzeptorstammes weist durch ihre hohe Auflösung von 1,2 Å eine nahezu vollständige Hydratation aller Basenpaare auf. Da der Akzeptorstamm von tRNAs wesentliche Identitätsmerkmale für die Erkennung durch die korrespondierenden Aminoacyl-tRNA-Synthetasen trägt, konnte durch einen Vergleich mit einem Isoakzeptor der Einfluss des Wassernetzwerkes auf die Interaktion mit natürlichen Bindungspartnern untersucht werden. Dies wurde durch eine Superpositionierung der beiden Isoakzeptoren mit der Seryl-tRNA-Synthetase bestätigt und ausgeweitet. Die Sequenz und Struktur des tRNASer- Akzeptorstammes stellten die Grundlage für die Untersuchung der davon abgeleiteten Struktur einer „locked“ Nukleinsäure hinsichtlich Konformation, Hydratation und Ligandenbindung dar. Dies ermöglichte einen sequenzunabhängigen Vergleich der LNA mit der korrespondierenden RNA. Dabei zeigte sich, dass die LNA eine vorher nicht beschriebene Doppelhelixstruktur einnimmt, aus der heraus Gründe für die hohe thermische Stabilität dieser Nukleinsäureklasse abgeleitet werden konnten. Die Duplex ist im Vergleich zur RNA durch eine Streckung bei gleichzeitiger Öffnung charakterisiert, was konformationell eher anderen modifizierten als der natürlichen Nukleinsäure ähnelt. Trotz der strukturellen Unterschiede gleichen das Hydratationsmuster und das Ligandenbindungsverhalten in weiten Bereichen dem der natürlichen Nukleinsäure.High-resolution X-ray crystallography of nucleic acids is a powerful method to investigate conformation, hydration and ligand-binding of these molecules. Two tRNA-acceptorstem-microhelices where employed as model systems for natural nucleic acids and compared to a modified “locked” nucleic acid, which sequence was derived from one of those acceptorstems. Oligonucleotides were restricted to a length of seven base pairs and crystallization was even performed under microgravity conditions to maximize resolution of the structures. The structure of the tRNAArg acceptorstem can be seen as a model for the binding of small molecules to simple structural motifs of nucleic acids. The results are consistent with investigations on base-specific interactions of small molecules with aptamers and riboswitches. Due to its high resolution of 1.2 Å, the structure of the tRNASer acceptorstem reveals a nearly complete hydration of all base pairs. Since tRNA acceptorstems carry important identity elements for the interaction with the cognate aminoacyl-tRNA-synthetase, a comparison with an isoacceptor yielded insight into the impact of the hydration network for the interaction of tRNAs with other molecules. These findings were confirmed and extended by a superposition of the two isoacceptors with the seryl-tRNA-synthetase. The highly-resolved structure of the tRNASer acceptorstem was the fundament for the investigation of a “locked” nucleic acid. The sequence of the LNA was derived from the acceptorstem. This allowed a sequence independent comparison of the modified nucleic acid with its corresponding natural counterpart. The structure was investigated with focus on conformation, hydration and ligand-binding. It could be demonstrated, that the LNA shows a double helix geometry, which differs significantly from natural nucleic acids and can be rather brought in vicinity to other modified nucleic acids. The structure of the LNA duplex can be described as a stretched helix with an enlarged helical pitch. Despite these structural differences the hydration and ligand-binding ability of the modified nucleic acid are similar to those observed in RNA

Crystallization and X-ray diffraction analysis of an ‘all-locked’ nucleic acid duplex derived from a tRNASer microhelix

A completely ‘all-locked’ nucleic acid duplex was designed from an E. coli tRNASer microhelix. The helix consists exclusively of LNA building blocks and was crystallized. The crystals diffracted to 1.9 Å resolution

Escherichia coli tRNAArg acceptor-stem isoacceptors: comparative crystallization and preliminary X-ray diffraction analysis

Various E. coli tRNAArg acceptor-stem microhelix isoacceptors have been crystallized and investigated by high-resolution X-ray diffraction analysis

Crystallization and preliminary X-ray diffraction data of an LNA 7-mer duplex derived from a ricin aptamer

An all-LNA duplex was designed from the stem region of an RNA aptamer which has been generated against ricin. The LNA duplex was crystallized and preliminary X-ray diffraction analysis revealed diffraction to a resolution of up to 2.8 Å