Conformational and chemical selection by a trans-acting editing domain.

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain trans-editing proteins. ProXp-ala is a ubiquitous trans-editing enzyme that edits Ala-tRNAPro, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNAPro and mischarged Ala-tRNAAla is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of Caulobacter crescentus ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNAPro acceptor stem mimic, microhelixPro, or a nonhydrolyzable mischarged Ala-microhelixPro substrate analog identified residues important for binding and deacylation. Backbone 15N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a trans-editing domain to ensure acceptance of only mischarged Ala-tRNAPro, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.

Bioinspired titanium coatings: self-assembly of collagen-alginate films for enhanced osseointegration.

Achieving long term osseointegration is fundamental to the development of successful bone implants. A key aspect for improving long term osseointegration on titania surfaces is to gain control of nano- and microscale features. The so called biological approach is applied here to modify the surface of titania by coating it with a self-assembled and chemically crosslinked biopolymer film made of alginate and collagen. The biofilm coated titania closely mimics the bone extracellular matrix in bio-morphology and mechanical properties. Biofilms are prepared using the layer by layer technique combined with carbodiimide chemistry to achieve a stable and compact structure. Alginate-collagen coatings display fibrillar morphology with an apparent fiber diameter of ∼50 nm and lengths ranging from a few hundred nanometers to ∼3 µm, mimicking therefore the extracellular matrix of the bone in fiber length and extent. Osteoblast MC3T3-E1 cells showed enhanced adhesion on the coated surface compared to the bare titania and a superior biological activity of the alginate-terminated coating that interfaces the cells in biological fluids.

Polyelectrolytes Multilayers to Modulate Cell Adhesion: A Study of the Influence of Film Composition and Polyelectrolyte Interdigitation on the Adhesion of the A549 Cell Line.

Polyelectrolyte multilayers (PEMs) with different polycation/polyanion pairs are fabricated by the layer-by-layer technique employing synthetic, natural, and both types of polyelectrolytes. The impact of the chemical composition of PEMs on cell adhesion is assessed by studying cell shape, spreading area, focal contacts, and cell proliferation for the A549 cell line. Cells exhibit good adhesion on PEMs containing natural polycations and poly(sodium 4-styrenesulfonate) (PSS) as polyanion, but limited adhesion is observed on PEMs fabricated from both natural polyelectrolytes. PEMs are then assembled, depositing a block of natural polyelectrolytes on top of a stiffer block with PSS as polyanion. Cell adhesion is enhanced on top of the diblock PEMs compared to purely natural PEMs. This fact could be explained by the interdigitation between polyelectrolytes from the two blocks. Diblock PEM assembly provides a simple means to tune cell adhesion on biocompatible PEMs.

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 ŠX-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

Expanding the Scope of 2'-SCF3 Modified RNA.

The 2'-trifluoromethylthio (2'-SCF3 ) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2'-SCF3 adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc-protecting group strategy, are reported. RNA solid-phase synthesis provided site-specifically 2'-SCF3 -modified oligoribonucleotides that were investigated intensively. Their excellent behavior in (19) F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule-RNA interaction. Moreover, comparably to the 2'-SCF3 pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off-target effects and their performance in silencing of the BASP1 gene was demonstrated.

In-line alignment and Mg²âº coordination at the cleavage site of the env22 twister ribozyme.

Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. We report on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modelled 2'-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5' bond. Both an invariant guanosine and a Mg(2+) are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently reported twister ribozymes structures, which adopt similar global folds, but differ in conformational features around the cleavage site.

Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids.

The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2'-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2'-endo conformation of the 2'-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858-2865) that estimated the extent of destabilization caused by a single C2'-endo nucleotide within a native RNA duplex to amount to 6 kcal mol(-1) because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2'-endo conformation of 2'-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2'-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2'-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson-Crick base pair, adopted the same C3'-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2'-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2'-SCF3 nucleoside modification and correlate them to base pairing properties.