Search

RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pKa values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pKa for A nucleotides in single-stranded RNA. This is the largest pKa shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pKa shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pKa shift.

RNA-Tertiarstrukturmotive werden durch viele verschiedene Wasserstoffbrucken stabilisiert. Protonierte A- und C-Nukleotide werden normalerweise nicht als geeignete Bausteine fur solche Motive betrachtet, da ihre pKa-Werte weit vom physiologischen pH-Wert entfernt sind. Hier beschreiben wir die NMR-Struktur eines in vitro selektierten GTP-bindenden RNA-Aptamers gebunden an GTP mit einer sehr komplexen Tertiarstruktur. Es enthalt ein neuartiges, durch ein protoniertes A stabilisiertes Basenquartett. Aufgrund der besonderen strukturellen Umgebung innerhalb des Basenquartetts im GTP-Komplex ist der pKa-Wert fur die Protonierung dieses A-Nukleotides im Vergleich zum pKa-Wert fur A-Nukleotide in einzelstrangiger RNA um mehr als funf pH-Einheiten verschoben. Dies ist die groste bisher beschriebene Verschiebung des pKa-Wertes fur ein A in strukturierten Nukleinsauren. Sowohl die Vorfaltung der RNA als auch die Ligandenbindung tragen zu dieser Verschiebung des pKa-Wertes bei.

In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear (13) C and (1) H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site-specific (2) H and (13) C labeling, spin topologies are introduced into DNA and RNA that make (1) H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A-site RNA was previously shown to undergo a two site exchange process in the micro- to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV-1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for (1) H RD experiments of nucleic acids.

The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

Renal transplantation is well established as the best and often the only treatment for many patients with end-stage kidney failure. Because of an increasing shortfall between the diminishing number of deceased donor organs available and the increasing waiting list of patients in need of transplantation the shortage of suitable donors remains one of the most pressing challenges. The success of organ transplantation can be attributed to many factors but ultimately depends upon retrieval and preservation techniques to maintain the quality of an organ. Although the literature on organ retrieval is extensive, the level of evidence provided is mainly low. But as techniques and treatments improve, it may be possible to use organs from donors who were previously thought to be unsuitable. This article provides the reader an overview on the topic of organ donation.

The structures of RNA-aptamer-ligand complexes solved in the last two decades were instrumental in realizing the amazing potential of RNA for forming complex tertiary structures and for molecular recognition of small molecules. For GTP as ligand the sequences and secondary structures for multiple families of aptamers were reported which differ widely in their structural complexity, ligand affinity and ligand functional groups involved in RNA-binding. However, for only one of these families the structure of the GTP-RNA complex was solved. In order to gain further insights into the variability of ligand recognition modes we are currently determining the structure of another GTP-aptamer--the so-called class II aptamer--bound to GTP using NMR-spectroscopy in solution. As a prerequisite for a full structure determination, we report here (1)H, (13)C, (15)N and partial (31)P-NMR resonance assignments for the class II GTP-aptamer bound to GTP.

Protein–protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein–protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins.

A straightforward and efficient alumination of functionalized arenes by using the frustrated Lewis pair Et3 Al and TMPMgCl⋅LiCl (TMP=2,2,6,6-tetramethylpiperidyl) has been developed. In particular, halogenated electron-rich aromatics can be smoothly functionalized by using the frustrated Lewis pair Et3 Al and TMPMgCl⋅LiCl. Compared with previously described alumination methods, this procedure avoids extensive cooling and the need for an excess of base. This in situ procedure has proven to be most practical and allows for regio- and chemoselective metalation of a wide range of aromatics with sensitive functional groups (CONEt2 , CO2 Me, CN, OCONMe2 ) or halogens (F, Cl, Br, I). The resulting aromatic aluminates, which were characterized by using NMR spectroscopy, were subjected to allylations, acylations, and palladium-catalyzed cross-coupling reactions after transmetalation to zinc. It was shown that the nature of the Zn salt used for transmetalation is crucial. Thus, compared with ZnCl2 (2 equiv), the use of Zn(OPiv)2 (2 equiv; OPiv=pivalate) allows the subsequent quenching reactions to be performed with only a slight excess of electrophile (1.2 equiv) and provides interesting functionalized aromatics in good yields.

An improved process for the preparation of aromatic and heteroaromatic diorganozinc reagents and their subsequent reaction with electrophiles is presented. The new method, featuring the use of a 2,2,6,6-tetramethylpiperidyl (TMP) magnesium base in the presence of zinc chloride (ZnCl2), is superior to the previous methods, which require the preparation of zinc bases. Specifically, the shorter reaction times under mild conditions provide an easier and more practical process, while the use of only a slight excess of the amide allows the isolation of products in high yields. These improvements are particularly significant for the large-scale preparation of organozincs and their subsequent reactions. Remarkably, beside the high kinetic activity, a wide range of functional groups is tolerated and sensitive heteroaromatics can easily be converted into the corresponding organometallic reagents and reacted with various electrophiles.

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pK

We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed.

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 Å X-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

The development of semiconductor quantum dot (QD) devices particularly for the infrared requires information on their intraband energy levels. For most of the samples FTIR spectroscopy does not provide reliable results on intraband transitions in the mid-infrared due to very low absorption signals. In recent years photocurrent spectroscopy was demonstrated as an alternative method. In this paper we present a modified photocurrent method, which does not require complicated contact structures or contact layers. The samples are laterally contacted by simply soldering wires and are biased by several 10 V. The intraband resonances are monitored via current changes induced by optical excitation with intense picosecond mid-infrared laser pulses. Both bound to bound and bound to continuum transitions enhance the current. We present data taken on two highly n-doped GaAs/InAs QD samples with different doping concentration at 77 K and at room temperature. In this way, a nearly complete picture of intraband transitions between excited levels in the conduction band is obtained. The intraband spectra obtained with this technique nicely agree with calculated QD intraband levels.

Highly functionalized organometallics are efficiently prepared in larger quantities (up to 4 g) by directed ortho-metalation using the previously reported amide bases TMP 2 Mn·2MgCl 2 ·4LiCl (TMP = 2,2,6,6-tetramethylpiperidyl), TMP 2 Fe·2MgCl 2 ·4LiCl and TMP 3 La·3MgCl 2 ·5LiCl. The resulting organometallics undergo various reactions with electrophiles like acid chlorides, alkyl iodides, or aldehydes and provide the corresponding products in good to excellent yields.

A range of aryl and heteroaryl organometallics are efficiently prepared in THF via directed ortho metalation by using the previously reported amide bases tmpMgCl·LiCl (tmp = 2,2,6,6-tetramethylpiperidyl), tmp2Mg·2LiCl and tmp2Zn·2MgCl2·2LiCl. These metalation reactions are carried out at 80−100 mmol scale. The unsaturated organometallic compounds undergo smooth reactions with various electrophiles, e.g. additions to carbonyl groups, Pd-catalyzed cross-coupling reactions or Cu-catalyzed acylations. In all cases, the metalation rates have been compared with corresponding small-scale reactions (1−2 mmol). Moreover, a procedure for the recovery of the valuable tmp-H from the aqueous layer is reported.

New mixed Li/Mg and Li/Mg/Zn amides have been synthesized starting from readily prepared secondary amines. They allow a highly chemoselective directed magnesiation or zincation of various polyfunctional aromatics and heteroaromatics. The kinetic basicity, solubility and stability of these new bases have been compared with those of the corresponding 2,2,6,6-tetramethylpiperamide-derived bases.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Selective C-H activation with a series of neutral aluminum trisamide bases led to a wide range of polyfunctional aryl and heteroaryl aluminum reagents. Ester and cyano groups are stable under the reaction conditions for this direct alumination, and donor oxygen substituents are efficient directing groups. High metalation regioselectivities were observed with O,S and N,S heterocycles (see example).

O am Steuer: Vielfaltige funktionalisierte (hetero)aromatische Aluminiumreagentien wurden in THF effizient uber eine dirigierte Aluminierung mithilfe neuartiger, neutraler Aluminiumtrisamidbasen hergestellt. Ester- und Cyanfunktionen werden gut toleriert. Bemerkenswerterweise sind Donor-Sauerstoffsubstituenten effektive dirigierende Gruppen fur diese Aluminierungen. An O,S- und N,S-Heterocyclen werden bei der Metallierung hohe Regioselektivitaten beobachtet.

A wide range of polyfunctional aryl and heteroaryl zinc reagents were efficiently prepared in THF by using (TMP)(2)Mg2 LiCl (TMP=2,2,6,6-tetramethylpiperamidyl) in the presence of ZnCl(2). The possible pathways of this metalation procedure as well as possible reactive intermediates are discussed. This experimental protocol expands the tolerance of functional groups and allows an efficient zincation of sensitive heterocycles such as quinoxaline or pyrazine. The zincated arenes and heteroarenes react with various electrophiles providing the expected products in 60-95 % yield.

PURPOSE To assess the diagnostic value of abdominal computed tomography in the preoperative staging of renal cell carcinoma. METHODS Computed tomograms of 87 renal cell carcinomas were classified according to the TNM-System. The results were correlated with the histopathological categories. The usual parameters for diagnostic tests were calculated and chance correction of the observed agreement was performed using Cohen's Kappa (kappa) test. RESULTS T-category staging showed an overall accuracy of 60% (kappa = 0.44). The pT1 category was correctly predicted in all cases. For perirenal invasion, an accuracy of 60% (kappa = 0.27), a sensitivity of 90.5%, and a specificity of 51% were found. For venous involvement, accuracy was 92% (kappa = 0.59), sensitivity 86%, and specificity 92%. All inconspicuous adrenals on CT were histologically normal as well. An accuracy of 80% for lymphadenopathy staging was attributable to chance (kappa = 0.04). 7 distant metastases were detected in the scanned volume. CONCLUSIONS Good CT staging results are obtained with discrimination between T1 tumors and higher categories, the assessment of venous invasion, the exclusion of adrenal involvement, and the detection of metastatic spread to abdominal organs. Insufficient results are seen with lymphadenopathy staging and the distinction of organ-confined and invasive tumors.

The synthesis of neutral mono- and cationic bis-aziridine complexes of ruthenium(II), rhodium(III) and iridium(III) are described. The dimeric complexes [MCl2L]2 (M = RuII, L = C6Me6; M = RhIII/IrIII, L = C5Me5) (1-3) react with a series of aziridines (Az = C2H4NH, C2H3MeNH, C2H2Me2NH, C2H3EtNH, C2H3PhNH) (a-e) in a 1:2 or 1:5 molar ratio to give the neutral mono-aziridine complexes [MCl2L(Az)] (4e-6e) or cationic bis-aziridine complexes [MClL(Az)2]Cl (7a-9d), respectively. After purification, all of the complexes were fully characterized and the IR, MS, 1H and 13C NMR spectra are reported and discussed. The single crystal structure analysis revealed a distorted octahedral structure for all complexes.

Renal transplantation is well established as the best and often the only treatment for many patients with end-stage kidney failure. Because of an increasing shortfall between the diminishing number of deceased donor organs available and the increasing waiting list of patients in need of transplantation the shortage of suitable donors remains one of the most pressing challenges. The success of organ transplantation can be attributed to many factors but ultimately depends upon retrieval and preservation techniques to maintain the quality of an organ. Although the literature on organ retrieval is extensive, the level of evidence provided is mainly low. But as techniques and treatments improve, it may be possible to use organs from donors who were previously thought to be unsuitable. This article provides the reader an overview on the topic of organ donation.

Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-(13)C-pyrimidine phosphoramidites to introduce isolated (13)C-(1)H spin pairs into RNAs up to 40 nucleotides long. The method allows the incorporation of 6-(13)C-uridine and -cytidine residues at any desired position within a target RNA. By site-specific positioning of the (13)C-label using RNA solid phase synthesis, these stable isotope-labeling patterns are especially well suited to resolve resonance assignment ambiguities. Of even greater importance, the labeling pattern affords accurate quantification of important functional transitions of biologically relevant RNAs (e.g., riboswitch aptamer domains, viral RNAs, or ribozymes) in the μs- to ms time regime and beyond without complications of one bond carbon scalar couplings. We outline the chemical synthesis of the 6-(13)C-pyrimidine building blocks and their use in RNA solid phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dispersion, ZZ exchange, and chemical exchange saturation transfer NMR experiments.

Purpose: We evaluated the pathological features of tumor size, lymph node and distant metastases, cell type, growth pattern, infiltration pattern, histological grade, local invasion and venous involvement of organ-confined renal carcinomas. The aim of this study was the re-evaluation of the TNM classification and the tumor cut-off point between T1 and T2 for renal cell carcinomas from the 1987 to the 1997 versions. Materials and Methods: (1) Patients with renal cell carcinoma who had been operated between October 1992 and August 2001 were evaluated. 437 of 691 patients showed T1 and T2 tumors. These organ-confined tumors have been divided into five groups: group 1: tumor-size of 20 mm or less (n = 16), group 2: 21–30 mm (n = 79); group 3: 31–40 mm (n = 83; group 4: 41–70 mm (n = 184), and group 5: more than 70 mm in diameter (only T2, n = 75). Follow-up ranged from 0 to 100 months (average 28.63 months). (2) Of 15,347 autopsies performed in Jena between 1985 and 1996, 272 renal cell carcinomas were revealed. In 145 of these 272 cases renal cell carcinomas were limited to the kidney. These 145 tumors were divided accordingly into 5 groups: group 1: 20 mm or less (n = 33), group 2: 21– 30 mm (n = 31); group 3: 31–40 mm (n = 29); group 4: 41–70 mm (n = 42), and group 5: T2 (n = 10). Clinicopathological criteria examined were lymph node and distant metastases, cell type, growth pattern, infiltration pattern, histological grade, local invasion and venous involvement. To identify the optimal cut-off point between T1 and T2 disease the χ2 test was used. Results: (1) In the clinical series only 1.8% (n = 8) of all cases showed lymph node metastases. Distant metastases were shown in 57 cases (13.04%); within group 1: 0%, group 2: 7.59%, group 3: 1.20%, group 4: 15.76%, group 5: 28%. The tumor grading was statistically correlated with tumor size. (2) In the pathological series 94 of the evaluated 145 patients were downstaged from T21987 to T11997. Lymph node and distant metastases were well correlated with tumor size. Lymph node metastases were seen in 0, 12.9, 31, 29.3 and 40% (group 1 to group 5) and distant metastases in 12.1, 25.8, 41.4, 47.7 and 60%. There were no statistically significant differences between T21997 and T13–7 cm. The tumor grading was statistically correlated with tumor size (grade 1: in 66.7, 25.8, 17.2, 9.5 and 0%). Conclusion: Our data suggest that the current cut-off diameter between T1 and T2 renal cell carcinomas (7 cm) is too high. Lowering the cut-off level will result in better discriminatory power of the TNM classification. From our data, we conclude that the cut-off diameter should be lowered to 3.5 cm (p < 0.001).

In this chapter, we briefly describe the history of biomolecular solution NMR spectroscopy focusing on the milestones making it possible to study large molecules with high molecular weight. We then discuss the fundamentals and perspectives of NMR spectroscopy as a tool in the drug discovery, for example, for the characterization of the binding of small molecules isolated from plants to biomolecules. First, the currently most popular NMR methods for ligand screening and hit validation are discussed followed by a short description of NMR spectroscopic tools for hit and lead optimization. In the last two sections, magnetic resonance methods for monitoring ligand binding to the two major classes of biomolecules, proteins and nucleic acids, are described. Keywords: biomolecular NMR; drug targeting; fragment-based drug design; proteins; nucleic acids

A straightforward and efficient alumination of functionalized arenes by using the frustrated Lewis pair Et3 Al and TMPMgCl⋅LiCl (TMP=2,2,6,6-tetramethylpiperidyl) has been developed. In particular, halogenated electron-rich aromatics can be smoothly functionalized by using the frustrated Lewis pair Et3 Al and TMPMgCl⋅LiCl. Compared with previously described alumination methods, this procedure avoids extensive cooling and the need for an excess of base. This in situ procedure has proven to be most practical and allows for regio- and chemoselective metalation of a wide range of aromatics with sensitive functional groups (CONEt2 , CO2 Me, CN, OCONMe2 ) or halogens (F, Cl, Br, I). The resulting aromatic aluminates, which were characterized by using NMR spectroscopy, were subjected to allylations, acylations, and palladium-catalyzed cross-coupling reactions after transmetalation to zinc. It was shown that the nature of the Zn salt used for transmetalation is crucial. Thus, compared with ZnCl2 (2 equiv), the use of Zn(OPiv)2 (2 equiv; OPiv=pivalate) allows the subsequent quenching reactions to be performed with only a slight excess of electrophile (1.2 equiv) and provides interesting functionalized aromatics in good yields.

Summary Background: Cystoscopy is still considered to be the ‘Gold Standard’ in the diagnosis or urinary bladder cancer. However, this invasive technique is not free fro

3,4-Dilithio-2,5-dimethyl-2,4-hexadiene (4a) rearranges to the cross-conjugated 2,5-dimethylhexadienediyl dianion 11a. A mechanistic investigation proves the intermolecularity of this rearrangement, which is also observed when starting from 4b. The 3-lithio-2,5-dimethylhexadienyl anion 10a with one vinyllithium and one allyllithium group, is a true intermediate in this rearrangement, its synthetic potential is employed in the reaction with dichlorosilanes to form 2-isopropylidene-2,5-dihydrosilols 8.

An improved protocol for the zincation and the subsequent reaction with different substituted electrophiles is presented.