Charlotte Förster

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.

Comparative crystallization and preliminary X-ray diffraction studies of locked nucleic acid and RNA stems of a tenascin C-binding aptamer

The pharmacokinetic properties of an aptamer against the tumour-marker protein tenascin-C have recently been successfully improved by the introduction of locked nucleic acids (LNAs) into the terminal stem of the aptamer. Since it is believed that this post-SELEX optimization is likely to provide a more general route to enhance the in vitro and in vivo stability of aptamers, elucidation of the structural basis of this improvement was embarked upon. Here, the crystallographic and X-ray diffraction data of the isolated aptamer stem encompassed in a six-base-pair duplex both with and without the LNA modification are presented. The obtained all-LNA crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 52.80, c = 62.83 angstroms; the all-RNA crystals belong to space group R32, with unit-cell parameters a = b = 45.21, c = 186.97 angstroms, gamma = 120.00 degrees.

Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex of Bacillus stearothermophilus initiation factor 2 C-­domain and fMet-tRNAfMet

Bacillus stearothermophilus translation initiation factor 2 (IF2) specifically binds initiator fMet-tRNAfMet and positions it into the ribosomal peptidyl site in the course of the initiation of protein biosynthesis. The isolated C-terminal domain of IF2 is capable of binding fMet-tRNAfMet, as shown by RNase A and hydrolysis protection experiments. In the presence of fMet-tRNAfMet, the IF2 C-domain yielded orthorhombic crystals of space group I222 (I212121) diffracting to 3.4 A resolution. The existence of equimolar amounts of tRNA and protein in the crystals was proven by Raman spectroscopy. The observed unit cell suggests the presence of two IF2 C- domain-fMet-tRNAfMet complexes per asymmetric unit of the crystal.

Human tRNAGly acceptor-stem microhelix: crystallization and preliminary X-ray diffraction analysis at 1.2 Å resolution

The major dissimilarities between the eukaryotic/archaebacterial-type and eubacterial-type glycyl-tRNA synthetase systems (GlyRS; class II aminoacyl-tRNA synthetases) represent an intriguing example of evolutionarily divergent solutions to similar biological functions. The differences in the identity elements of the respective tRNA(Gly) systems are located within the acceptor stem and include the discriminator base U73. In the present work, the human tRNA(Gly) acceptor-stem microhelix was crystallized in an attempt to analyze the structural features that govern the correct recognition of tRNA(Gly) by the eukaryotic/archaebacterial-type glycyl-tRNA synthetase. The crystals of the human tRNA(Gly) acceptor-stem helix belong to the monoclinic space group C2, with unit-cell parameters a = 37.12, b = 37.49, c = 30.38 A, alpha = gamma = 90, beta = 113.02 degrees, and contain one molecule per asymmetric unit. A high-resolution data set was acquired using synchrotron radiation and the data were processed to 1.2 A resolution.

Crystallization and preliminary X-ray diffraction analysis of an Escherichia coli tRNAGly acceptor-stem microhelix

The tRNA(Gly) and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNA(Gly). To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNA(Gly) acceptor-stem helix was crystallized. Data were collected to 2.0 A resolution using synchrotron radiation. Crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 35.35, c = 130.82 A, alpha = beta = 90, gamma = 120 degrees and two molecules in the asymmetric unit.

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 Seryl-tRNA synthetase/tRNASer acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA(Ser). The Seryl-tRNA-synthetase interacts with the tRNA(Ser) acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein-RNA complex. We solved the high resolution crystal structures of two tRNA(Ser) acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.

Superposition of two tRNASer acceptor stem crystal structures: comparison of structure, ligands and hydration.

We solved the X-ray structures of two Escherichia coli tRNA(Ser) acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNA(Ser) microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution.

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

The aminoacylation of tRNA is a crucial step in cellular protein biosynthesis. Recognition of the cognate tRNA by the correct aminoacyl-tRNA synthetase is ensured by tRNA identity elements. In tRNA(Arg), the identity elements consist of the anticodon, parts of the D-loop and the discriminator base. The minor groove of the aminoacyl stem interacts with the arginyl-tRNA synthetase. As a consequence of the redundancy of the genetic code, six tRNA(Arg) isoacceptors exist. In the present work, three different Escherichia coli tRNA(Arg) acceptor-stem helices were crystallized. Two of them, the tRNA(Arg) microhelices RR-1660 and RR-1662, were examined by X-ray diffraction analysis and diffracted to 1.7 and 1.8 A resolution, respectively. The tRNA(Arg) RR-1660 helix crystallized in space group P1, with unit-cell parameters a = 26.28, b = 28.92, c = 29.00 A, alpha = 105.74, beta = 99.01, gamma = 97.44 degrees , whereas the tRNA(Arg) RR-1662 helix crystallized in space group C2, with unit-cell parameters a = 33.18, b = 46.16, c = 26.04 A, beta = 101.50 degrees .

Crystallization and preliminary X-ray diffraction analysis of a tRNASer acceptor-stem microhelix

In order to understand elongator tRNA(Ser) and suppressor tRNA(Sec) identity elements, the respective acceptor-stem helices have been synthesized and crystallized in order to analyse and compare their structures in detail at high resolution. The synthesis, crystallization and preliminary X-ray diffraction results for a seven-base-pair tRNA(Ser) acceptor-stem helix are presented here. Diffraction data were collected to 1.8 A, applying synchrotron radiation and cryogenic cooling. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 36.14, b = 38.96, c = 30.81 A, beta = 110.69 degrees .