Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by F NMR.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes.

Synthesis of [7-N]-GTPs for RNA structure and dynamics by NMR spectroscopy.

Unlabelled: Several isotope-labeling strategies have been developed for the study of RNA by nuclear magnetic resonance (NMR) spectroscopy. Here, we report a combined chemical and enzymatic synthesis of [7-N]-guanosine-5'-triphosphates for incorporation into RNA via T7 RNA polymerase-based in vitro transcription. We showcase the utility of these labels to probe both structure and dynamics in two biologically important RNAs.

Probing the Hydrogen-Bonding Environment of Individual Bases in DNA Duplexes with Isotope-Edited Infrared Spectroscopy.

Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy.

Aromatic F- C TROSY-[ F, C]-Pyrimidine Labeling for NMR Spectroscopy of RNA.

We present the access to [5- F, 5- C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5- F, 5- C]-pyrimidine labels into five RNAs-the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21.

Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing.

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step.

Crystal structures of two PCN pincer iridium complexes and one PCP pincer carbodi-phospho-rane iridium inter-mediate: substitution of one phosphine moiety of a carbodi-phospho-rane by an organic azide.

The structure of [Ir{(4-Cl-CHN)C(dppm)-κ ,,}(dppm-κ ,')]Cl·1.5CHCl·0.5CH (CHClIrNP·1.

Eukaryotic Translation Elongation is Modulated by Single Natural Nucleotide Derivatives in the Coding Sequences of mRNAs.

RNA modifications are crucial factors for efficient protein synthesis. All classes of RNAs that are involved in translation are modified to different extents. Recently, mRNA modifications and their impact on gene regulation became a focus of interest because they can exert a variety of effects on the fate of mRNAs.

Crystal structures of four new iridium complexes, each containing a highly flexible carbodi-phos-phorane PCP pincer ligand.

Compound [Ir(CH)(CHP)]Cl or [Ir(cod)(CH(dppm)-κ,,)]Cl (), was obtained from [IrCl(cod)] and the carbodi-phospho-rane (CDP) salt [CH(dppm)]Cl [where cod = cyclo-octa-1,5-diene and dppm = bis-(di-phenyl-phosphino)methane]. Treatment of with thallium(I) tri-fluoro-methane-sulfonate [Tl(OTf)] and subsequent crystallization gave complex [Ir(CH)(CHP)](OTf)·CHCOCH·CHCl or [Ir(cod)(CH(dppm)-κ,,)](OTf)·CHCOCH·CHCl () [systematic name: (cyclo-octa-1,5-diene)(1,1,3,3,5,5,7,7-octa-phenyl-1,7-diphospha-3,5-di-phospho-niaheptan-4-yl)iridium(I) bis-(tri-fluoro-methane-sulfonate)-ethyl acetate-di-chloro-methane (1/1/1)]. This five-coordinate iridium(I) complex cation adopts a trigonal-bipyramidal geometry with the CDP carbon and one cod double bond in axial sites.

Studying sparsely populated conformational states in RNA combining chemical synthesis and solution NMR spectroscopy.

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific C labeled RNA building blocks - a 5-C-uridine and a 2,8-C-adenosine building block - are used to introduce isolated C-H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme.

CCR5 RNA Pseudoknots: Residue and Site-Specific Labeling correlate Internal Motions with microRNA Binding.

Conformational dynamics of RNA molecules play a critical role in governing their biological functions. Measurements of RNA dynamic behavior sheds important light on sites that interact with their binding partners or cellular stimulators. However, such measurements using solution-state NMR are difficult for large RNA molecules (>70 nt; nt=nucleotides) owing to severe spectral overlap, homonuclear C scalar couplings, and line broadening.

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA.

We showcase the high potential of the 2'-cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectroscopic applications. The method was applied to synthesize RNAs with sizes ranging between 60 to 80 nucleotides. The presented approach gives the possibility to selectively modify larger RNAs (>60 nucleotides) with atom-specifically C/N-labelled building blocks.

Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR.

We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA.

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy.

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.

DOCK8 deficiency in six Iranian patients.

DOCK8 deficiency is a rare autosomal recessive combined immunodeficiency with high IgE level, eosinophilia, severe eczema, extensive cutaneous viral, and respiratory bacterial infections, mostly in populations with higher prevalence of consanguinity. Molecular diagnosis of this gene is a useful approach for early diagnosis and timely HSCT due to deleterious consequences.

The diagnosis of hyper immunoglobulin e syndrome based on project management.

Hyperimmunoglobulin E Syndrome (HIES) is a complex primary immunodeficiency characterized by both immunologic and non-immunologic manifestations. High serum IgE level, eosinophilia, eczema, recurrent skin and lung infections constitute the immunologic profile of HIES, whereas characteristic facial appearance, scoliosis, retained primary teeth, joint hyperextensibility, bone fractures following minimal trauma and craniosynostosis are the main non-immunologic manifestations. The diagnosis of HIES cannot be made by routine immunologic tests.

[Update on implant related infections in orthopaedic surgery. Diagnosis and treatment].

Infections associated with implants are increasingly important in modem medicine. Biofilms are the cause that these infections are more difficult to diagnose and to cure. Particularly low-grade infections are difficult to distinguish from aseptic failure, because they often present with early loosening and persisting pain.

Outcome evaluation in shoulder surgery using 3D kinematics sensors.

A new method of scoring systems for the functional assessment of the shoulder is presented. 3D accelerometers and gyroscopes attached on the humerus were used to differentiate a healthy from a painful shoulder. The method was first tested on 10 healthy volunteer subjects with no shoulder pathology.