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

Institute of Chemistry and Biochemistry, Free University Berlin, 14195 Berlin, Germany.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.53). 09/2009; 65(Pt 8):809-12. DOI: 10.1107/S1744309109026281
Source: PubMed


Modified nucleic acids are of great interest with respect to their nuclease resistance and enhanced thermostability. In therapeutical and diagnostic applications, such molecules can substitute for labile natural nucleic acids that are targeted against particular diseases or applied in gene therapy. The so-called 'locked nucleic acids' contain modified sugar moieties such as 2'-O,4'-C-methylene-bridged beta-D-ribofuranose and are known to be very stable nucleic acid derivatives. The structure of locked nucleic acids in single or multiple LNA-substituted natural nucleic acids and in LNA-DNA or LNA-RNA heteroduplexes has been well investigated, but the X-ray structure of an ;all-locked' nucleic acid double helix has not been described to date. Here, the crystallization and X-ray diffraction data analysis of an 'all-locked' nucleic acid helix, which was designed as an LNA originating from a tRNA(Ser) microhelix RNA structure, is presented. The crystals belonged to space group C2, with unit-cell parameters a = 77.91, b = 40.74, c = 30.06 A, beta = 91.02 degrees . A high-resolution and a low-resolution data set were recorded, with the high-resolution data showing diffraction to 1.9 A resolution. The crystals contained two double helices per asymmetric unit, with a Matthews coefficient of 2.48 A(3) Da(-1) and a solvent content of 66.49% for the merged data.

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    • "The chemically synthesized single strands with the sequences 5′-(G-G-T-G-A-G-G)L-3′ and 5′-(m5C-m5C-T-m5C-A-m5C-m5C)L-3′ were purchased from IBA (Göttingen, Germany) with HPLC purification grade. Crystals were grown within 3-4 days using 40 mM sodium cacodylate, pH 5.5, 20 mM cobalt hexamine, 80 mM sodium chloride, 20 mM magnesium chloride, and 10% (v/v) MPD with equilibration against 1 mL 33–41% (v/v) MPD at 21°C using the hanging drop vapour diffusion technique [16]. "
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    ABSTRACT: “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.
    Full-text · Article · May 2012 · Journal of nucleic acids
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    • "In summary, the crystal packing of the LNA duplexes is favored by hydrophobic interactions of the ‘tail-to-tail’ guanosine stacking and is further facilitated by interactions with the cobalt hexamine, which acts like a bridge between the two helices. A comparison of the crystallographic parameters between the previously solved RNA structure (24) and the here presented LNA structure demonstrates significant differences in crystal packing, although the RNA and LNA crystallize under identical conditions (24,26). The tRNASer microhelix crystallizes with one RNA helix per asymmetric unit, whereas the molecule packing consists of two molecules per asymmetric unit for the LNA duplex. "
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    ABSTRACT: ‘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.
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