How does hydroxyl introduction influence the double helical structure: the stabilization of an altritol nucleic acid:ribonucleic acid duplex

Margriet Ovaere 2 Jiri Sponer 0 1 Judit E. Sponer 0 1 Piet Herdewijn 3 Luc Van Meervelt 2 0 CEITEC - Central European Institute of Technology, Masaryk University , Campus Bohunice, Kamenice 5, 625 00 Brno, Czech Republic 1 Institute of Biophysics, Academy of Sciences of the Czech Republic , Kra lovopolska 135, CZ-61265, Brno, Czech Republic 2 Department of Chemistry, Katholieke Universiteit Leuven, Biomolecular Architecture and BioMacS , Celestijnenlaan 200F, B-3001 Leuven, Belgium 3 Laboratory of Medicinal Chemistry, Katholieke Universiteit Leuven, Rega Institute for Medical Research and BioMacS , Minderbroedersstraat 10, B-3000 Leuven, Belgium Altritol nucleic acids (ANAs) are a promising new tool in the development of artificial small interfering ribonucleic acids (siRNAs) for therapeutical applications. To mimic the siRNA:messenger RNA (mRNA) interactions, the crystal structure of the ANA:RNA construct a(CCGUAAUGCC-P):r(GGCAUUACGG) was determined to 1.96 A resolution which revealed the hybrid to form an A-type helix. As this A-form is a major requirement in the RNAi process, this crystal structure confirms the potential of altritol-modified siRNAs. Moreover, in the ANA strands, a new type of intrastrand interactions was found between the O20 hydroxyl group of one residue and the sugar ring O40 atom of the next residue. These interactions were further investigated by quantum chemical methods. Besides hydration effects, these intrastrand hydrogen bonds may also contribute to the stability of ANA:RNA duplexes. - In antisense technology, the antisense oligonucleotides have to hybridize strongly and selectively with their messenger ribonucleic acid (mRNA) complement. A variety of nucleic acid modifications have been synthesized for these purposes. Insertion of a methylene group between the ring oxygen atom and the anomeric carbon atom of the furanose ring of RNA gives altritol nucleic acid (ANA, Figure 1a). This chemical insertion has a profound effect on the physicochemistry and the biology of these nucleic acids. The nucleic acid becomes chemically and enzymatically more stable than RNA (1), while keeping very selective and strong hybridization properties following Watson-Crick rules (2). As a result of this, ANA has scored very well in a small interfering RNA (siRNA) screening assay (3). When carrying out the same insertion in DNA, hexitol nucleic acid (HNA) is obtained. Likewise, HNA-modified RNAs show strong siRNA effects (4). The sugar rings of DNA and RNA (having a furanose sugar moiety) are more flexible than the sugar rings of HNA and ANA (having a reduced pyranose sugar moiety) and hybrids between ANA and DNA or RNA are more stable than hybrids between HNA and DNA or RNA, which points to the importance of the presence of the OH group for duplex stabilization. Herein, we report on the structural and physiochemical reasons for the duplex stabilization effect of this OH group in an ANA:RNA hybrid, which is not observed in regular dsRNAs. MATERIALS AND METHODS Oligonucleotide synthesis Synthesis and assembly of the ANA strand were performed according to Ovaere et al. (5). At the 30-end of the ANA strand, an extra phosphate group was added because of solid support choice. The RNA sequence was purchased from Eurogentec. Hybridization was accomplished by titration and monitored by NMR. Crystallization conditions Crystals were formed after about 1 year by the vapour diffusion hanging drop method at 289 K using Crystal Screen II (Hampton Research) in a condition which includes 0.01 M nickel chloride hexahydrate, 1.0 M lithium sulphate monohydrate and 0.1 M Tris (tris(hydroxyl-methyl)aminomethane) pH 8.5 as buffer. For the crystallization drop, 1 ml of 0.5 M ANA:RNA decamer (duplex concentration) was combined with 1 ml screening condition and equilibrated against 500 ml crystallization screen. One rod-form crystal (dimensions 0.3 mm 0.10 mm 0.06 mm) was obtained and cryoprotected with 40% propylene glycol. Data collection and processing Diffraction data were recorded at the Swiss Light Source PXIII beamline (Paul Scherrer Institute, Villigen, Switzerland) on a MAR225 CCD detector (100 K, wavelength 1.000 A , crystal to detector distance 200 mm). In total, 200 frames ( increment 0.5 ) covering a 100 -data range were collected to a resolution of 1.96 A. Two crystals with similar unit cell could be found in the reflection data, and these data were integrated separately by MOSFLM (6). Nevertheless, the diffraction data originate mainly from one crystal and only these data could be used for structure solution. The data were scaled by Scala (7), and processing statistics is listed in Table 1. The crystal belongs to the orthorhombic crystal class 222 with a = 26.07, b = 42.51 and c = 157.79 A . Systematic absences analysed by Pointless (7) suggested P212121 as space group. Structure solution and refinement As the crystal structure of the ANA:RNA decamer was already partly solved in another unit cell but suffering from lattice translocation defects (unpublished results), a decamer of this crystal structure was used as molecular replacement model without the extra phosphate group and without the last base pair at the ANA 30-side. Molecular replacement by Phaser (8) resulted in a helix oriented almost parallel to the c-axis. As the Matthews coefficient (2.22 A3/Dalton) suggested three decamers in the asymmetric unit, two more duplexes were searched for. The three decamers are stacked on each other in a head-to-tail manner. Structure refinement was performed by Refmac (9) using the restraints based on the crystal structure of the altritol adenosine building block (Supplementary Data) for the ANA sugar rings and standard dictionary restraints for the ANA bases and the RNA strand. The Fo Fc and 2Fo Fc electron density maps were carefully studied for any inconsistency, and bond distances, angles and chiral volumes were monitored. The Find Waters option of the program Coot was used to localize 239 water molecules (10). Disordered solvent regions are modelled according to Babinets principle (11). Finally, a R1 value of 22.44% was obtained (Rfree value: 23.42%). Refinement statistics is listed in Table 1. Final coordinates and structure factor amplitudes have been deposited with the Protein Data Bank (3OK2) and Nucleic Acid Data Bank (NA0770). Quantum chemical calculations Geometry optimizations were performed at B3LYP/ 6-311++G(2d,2p) level of theory. Initial positions of the C, O and P atoms were taken from the X-ray geometry. Selection of the starting geometries is described in the Results section. Starting H-atom positions were generated Values in parentheses are for the outermost shell. arbitrarily and later fully refined in the course of the geometry optimizations. The model systems carried a total charge of 1 and comprised two altritol moieties as well as a phosphate group linker. For simplicity, the hydroxymethyl group of the altritol unit phosphorylated at 3 (...truncated)