Enzymatic and Chemoenzymatic Three-Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines.

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet-Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol.

5SRNAdb: an information resource for 5S ribosomal RNAs.

Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.

Enantioselective, continuous (R)- and (S)-2-butanol synthesis: achieving high space-time yields with recombinant E. coli cells in a micro-aqueous, solvent-free reaction system.

The stereoselective production of (R)- or (S)-2-butanol is highly challenging. A potent synthesis strategy is the biocatalytic asymmetric reduction of 2-butanone applying alcohol dehydrogenases. However, due to a time-dependent racemisation process, high stereoselectivity is only obtained at incomplete conversion after short reaction times.

Spiegelzymes® mirror-image hammerhead ribozymes and mirror-image DNAzymes, an alternative to siRNAs and microRNAs to cleave mRNAs in vivo?

With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited.

Spiegelzymes: sequence specific hydrolysis of L-RNA with mirror image hammerhead ribozymes and DNAzymes.

In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera.

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.

Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells.

Probing the SELEX process with next-generation sequencing.

Background: SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process.

Pushing the detection limits: the evanescent field in surface plasmon resonance and analyte-induced folding observation of long human telomeric repeats.

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte interaction results in a large-scale contraction, then the refractive index changes due to the increasing amount of macromolecules close to the surface.

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.

The crystal structure of a Thermus thermophilus tRNA(Gly) acceptor stem microhelix at 1.6 Å resolution.

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem.

A streamlined protocol for emulsion polymerase chain reaction and subsequent purification.

Compartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complex libraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-free manner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion (ePCR) including sample recovery by DNA purification.

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.

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.

A calibrated diversity assay for nucleic acid libraries using DiStRO--a Diversity Standard of Random Oligonucleotides.

We have determined diversities exceeding 10(12) different sequences in an annealing and melting assay using synthetic randomized oligonucleotides as a standard. For such high diversities, the annealing kinetics differ from those observed for low diversities, favouring the remelting curve after annealing as the best indicator of complexity. Direct comparisons of nucleic acid pools obtained from an aptamer selection demonstrate that even highly complex populations can be evaluated by using DiStRO, without the need of complicated calculations.

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

Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as beta-D-2'-O,4'-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The beta-D-2'-O,4'-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability.

Crystallization and X-ray diffraction analysis of an 'all-locked' nucleic acid duplex derived from a tRNA(Ser) microhelix.

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.

Combination of soluble coxsackievirus-adenovirus receptor and anti-coxsackievirus siRNAs exerts synergistic antiviral activity against coxsackievirus B3.

Coxsackievirus B3 (CVB-3) is a major causative agent of chronic heart muscle infections. The present study describes a cell culture system with an ongoing virus infection to evaluate two novel inhibitory strategies, either individually or combined: (1) RNA interference (RNAi) to degrade cytoplasmatic CVB-3 RNA and (2) a vector-delivered soluble variant of the coxsackievirus-adenovirus receptor fused to a human immunoglobulin (sCAR-Fc), which inhibits cellular uptake of CVB-3. Both approaches were capable of inhibiting CVB-3 in persistently infected human myocardial fibroblasts.

The 1.2A crystal structure of an E. coli tRNASer)acceptor stem microhelix reveals two magnesium binding sites.

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNA(Ser) belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to 'class I', where tRNA determinants are more complex and are located within different regions of the tRNA. The structure of an Escherichia coli tRNA(Ser) acceptor stem microhelix was solved by high resolution X-ray structure analysis.

Crystal structure of the E. coli tRNA(Arg) aminoacyl stem isoacceptor RR-1660 at 2.0 A resolution.

Due to the redundancy of the genetic code there exist six mRNA codons for arginine and several tRNA(Arg) isoacceptors which translate these triplets to protein within the context of the mRNA. The tRNA identity elements assure the correct aminoacylation of the tRNA with the cognate amino acid by the aminoacyl-tRNA-synthetases. In tRNA(Arg), the identity elements consist of the anticodon, parts of the D-loop and the discriminator base.

Locked nucleic acid aptamers.

The aptamer technology has been introduced in the early 1990s. With this technique ligands for organic dyes and proteins have been identified in many research field, providing various inhibitory molecules that allow functional interference in biological systems. Aptamers can therefore be employed for various applications ranging from diagnostic to therapeutic assay formats.

Crystal structure of the human tRNAGly microhelix isoacceptor G9990 at 1.18A resolution.

The tRNA(Gly)/Glycyl-tRNA synthetase system belongs to the so called 'class II' in which tRNA identity elements consist of relative few and simple motifs, as compared to 'class I' where the tRNA determinants are more complicated and spread over different parts of the tRNA, mostly including the anticodon. The determinants from 'class II' although, are located in the aminoacyl stem and sometimes include the discriminator base. There exist predominant structure differences for the Glycyl-tRNA-synthetases and for the tRNA(Gly) identity elements comparing eucaryotic/archaebacterial and eubacterial systems.

Long-term cardiac-targeted RNA interference for the treatment of heart failure restores cardiac function and reduces pathological hypertrophy.

Background: RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca(2+) homeostasis.