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Competing Interests: CK ist Berater von Innotope und hält eine Beteiligung an Innotope, einem Unternehmen, das Produkte zur Markierung stabiler RNA‐Isotope anbietet. Die übrigen Autoren erklären, dass sie keine konkurrierenden Interessen haben.

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. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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- 15 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., Supplementary Information: The online version contains supplementary material available at 10.1007/s00706-022-02892-1., (© The Author(s) 2022.)

N 6 -methyladenosine (m 6 A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m 6 A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m 6 A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m 6 A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes., (© 2021. The Author(s).)

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. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2═O carbonyl. We use solvatochromatic studies to show that the TC2═O signal's position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2═O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH···O H-bond between adenine and thymine in both Watson-Crick and Hoogsteen base pairs in DNA.

We present the access to [5- 19 F, 5- 13 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- 19 F, 5- 13 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. The main stimulus to introduce the aromatic 19 F- 13 C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13 C- 19 F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5- 19 F, 5- 13 C]/[5- 19 F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19 F- 13 C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [ 19 F, 13 C]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

N 6 -Methyladenosine (m 6 A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m 6 A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m 6 A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R 1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m 6 A alters the hybridization kinetics in two RNA duplexes. The results show that m 6 A minimally impacts the rate constant for duplex dissociation, changing k off by ∼1-fold but significantly slows the rate of duplex annealing, decreasing k on by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg 2+ . We propose that rotation of the N 6 -methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m 6 A slows down tRNA selection and more generally suggest that m 6 A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

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. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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. mRNA modifications within coding sequences can either directly or indirectly interfere with protein synthesis. In order to investigate the roles of various natural occurring modified nucleotides, we site-specifically introduced them into the coding sequence of reporter mRNAs and subsequently translated them in HEK293T cells. The analysis of the respective protein products revealed a strong position-dependent impact of RNA modifications on translation efficiency and accuracy. Whereas a single 5-methylcytosine (m⁵C) or pseudouridine () did not reduce product yields, N ¹-methyladenosine (m¹A) generally impeded the translation of the respective modified mRNA. An inhibitory effect of 2' O -methlyated nucleotides (Nm) and N ⁶-methyladenosine (m⁶A) was strongly dependent on their position within the codon. Finally, we could not attribute any miscoding potential to the set of mRNA modifications tested in HEK293T cells., Competing Interests: The authors declare no conflicts of interest.

The structure of [Ir{(4-Cl-C 6 H 4 N 3 )C(dppm)-κ 3 P , C , N }(dppm-κ 2 P , P ')]Cl·1.5CH 2 Cl 2 ·0.5C 7 H 8 (C 57 H 48 Cl 2 IrN 3 P 4 ·1.5CH 2 Cl 2 ·0.5C 7 H 8 ) ( 2 ), dppm = bis-(di-phenyl-phosphino)methane {systematic name: [7-(4-chloro-phen-yl)-1,1,3,3-tetra-phenyl-5,6,7-tri-aza-κ N 7 -1,3λ 4 -diphospha-κ P 1 -hepta-4,6-dien-4-yl][methyl-ene-bis(di-phenyl-phosphine)-κ 2 P , P ']iridium(I) chloride-di-chloro-methane-toluene (2/3/1)}, resulting from the reaction of [IrClH{C(dppm) 2 -κ 3 P , C , P )(MeCN)]Cl ( 1a ) with 1-azido-4-chloro-benzene, shows a monocationic five-coordinate Ir I complex with a distorted trigonal-bipyramidal geometry. In 2 , the iridium centre is coordinated by the neutral triazeneyl-idene-phospho-rane (4-Cl-C 6 H 4 N 3 )C(dppm) acting as a PCN pincer ligand, and a chelating dppm unit. The structure of the coordination compound [IrCl(CN)H(C(dppm) 2 -κ 3 P , C , P )]·CH 3 CN, (C 52 H 45 ClIrNP 4 ·CH 3 CN) ( 1b ) [systematic name: chlorido-cyanidohydrido(1,1,3,3,5,5,7,7-octa-phenyl-1,3λ 5 ,5λ 4 ,7-tetra-phospha-κ 2 P 1 , P 7 -hept-3-en-4-yl)iridium(III) aceto-nitrile monosolvate], prepared from 1a and KCN, reveals an octa-hedral Ir III central atom with a meridional PCP pincer carbodi-phospho-rane (CDP) ligand; the chloride ligand is located trans to the central carbon of the CDP functionality while the hydrido and cyanido ligands are situated trans to each other. The chiral coordination compound [Ir(CN)((4-Cl-C 6 H 4 N 3 )CH(CH(P(Ph) 2 ) 2 )-κ 3 P , C , N )(dppm-κ 2 P , P ')]·2CH 3 OH, (C 58 H 48 ClIrN 4 P 4 ·2CH 3 OH) ( 3 ) (systematic name: {4-[3-(4-chloro-phen-yl)triazenido-κ N 3 ]-1,1,3,3-tetra-phenyl-1,3λ 5 -diphospha-κ P 1 -but-2-en-4-yl}cyanido[methyl-enebis(di-phenyl-phosphine)-κ 2 P , P ']iridium(III) methanol disolvate), formed via prolonged reaction of 1-azido-4-chloro-benzene with 1b , features a six-coordinate Ir III central atom. The iridium centre is coordinated by the dianionic facial PCN pincer ligand [(4-Cl-C 6 H 4 N 3 )CH(CH(P(Ph 2 ) 2 ) 2 )], a cyanido ligand trans to the central carbon of the PCN pincer ligand and a chelating dppm unit. Complex 2 exhibits a 2:1 positional disorder of the Cl - anion. The CH 2 Cl 2 and C 7 H 8 solvent mol-ecules show occupational disorder, with the toluene mol-ecule exhibiting additional 1:1 positional disorder with some nearly overlying carbon atoms.

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 13 C labeled RNA building blocks - a 5- 13 C-uridine and a 2,8- 13 C 2 -adenosine building block - are used to introduce isolated 13 C- 1 H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5- 13 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. Then, the 2,8- 13 C 2 -adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8- 13 C 2 -adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via 13 C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. The 13 C-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by 15 N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Compound [Ir(C 8 H 12 )(C 51 H 45 P 4 )]Cl 2 or [Ir(cod)(CH(dppm) 2 -κ 3 P , C , P )]Cl 2 ( 1a ), was obtained from [IrCl(cod)] 2 and the carbodi-phospho-rane (CDP) salt [CH(dppm) 2 ]Cl [where cod = cyclo-octa-1,5-diene and dppm = bis-(di-phenyl-phosphino)methane]. Treatment of 1a with thallium(I) tri-fluoro-methane-sulfonate [Tl(OTf)] and subsequent crystallization gave complex [Ir(C 8 H 12 )(C 51 H 45 P 4 )](OTf) 2 ·CH 3 CO 2 C 2 H 5 ·CH 2 Cl 2 or [Ir(cod)(CH(dppm) 2 -κ 3 P , C , P )](OTf) 2 ·CH 3 CO 2 C 2 H 5 ·CH 2 Cl 2 ( 1b ) [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. Compound 1b represents the first example of a non- meridional coordination of the PCP pincer ligand [CH(dppm) 2 ] + with a P-Ir-P angle of 98.08 (2)°. Compound 2 , [IrCl 2 H(C 51 H 44 P 4 )]·(CH 3 ) 2 CO or [IrCl 2 H(C(dppm) 2 -κ 3 P , C , P )]·(CH 3 ) 2 CO [systematic name: di-chlorido-hydrido(1,1,3,3,5,5,7,7-octa-phenyl-1,5λ 5 ,7-triphospha-3-phospho-niahept-4-en-4-yl)iridium(III) acetone monosolvate], crystallizes as an acetone monosolvate. It is a six-coordinate Ir III coordination compound. Here, the PCP pincer ligand is coordinated in a meridional manner; one chlorido ligand is positioned trans to the carbon donor, the remaining two coordination sites being occupied by the second chlorido and a hydrido ligand trans to each other. Complex 3 , [IrCl 2 H(C 51 H 45 P 4 )]Cl·5H 2 O or [IrCl 2 H(CH(dppm) 2 -κ 3 P , C , P )]Cl·5H 2 O [systematic name: di-chlorido-hydrido(1,1,3,3,5,5,7,7-octa-phenyl-1,7-diphospha-3,5-di-phospho-niaheptan-4-yl)iridium(III) chloride penta-hydrate], represents the conjugate CH acid of 2 . The ligand [CH(dppm) 2 ] + is coordinated in a meridional manner. In the cationic six-coordinate Ir III complex 4 , [IrClH(CO)(C 51 H 44 P 4 )]Cl·2CH 3 OH·H 2 O or [IrClH(CO)(C(dppm) 2 -κ 3 P , C , P )]Cl·2CH 3 OH·H 2 O [systematic name: carbonyl-chlorido-hydrido(1,1,3,3,5,5,7,7-octa-phenyl-1,5λ 5 ,7-triphospha-3-phos-pho-niahept-4-en-4-yl)iridium(III) chloride-methanol-water (1/2/1)], the chlorido ligand is found in the plane defined by the Ir center and the meridional PCP ligand; the H and CO ligands are positioned axially to this plane and trans to each other.

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 13 C scalar couplings, and line broadening. Herein, a strategic combination of solid-phase synthesis, site-specific isotopic labeled phosphoramidites, and enzymatic ligation is introduced. This approach allowed the position-specific insertion of isotopic probes into a 96 nt CCR5 RNA fragment. Accurate measurements of functional dynamics using the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments enabled extraction of the exchange rates and populations of this RNA. NMR chemical shift perturbation analysis of the RNA/microRNA-1224 complex indicated that A90-C1' of the pseudoknot exhibits similar changes in chemical shift observed in the excited state. This work demonstrates the general applicability of a NMR-labeling strategy to probe functional RNA structural dynamics., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2'deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min-1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s-1 and with a backward rate constant of 10.6 s-1., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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. As the main characteristic laboratory abnormalities of this syndrome are highly elevated serum IgE levels and eosinophilia; both features have a broad spectrum of differential diagnosis. The purpose of this essay was presenting the best way for diagnosis management of HIES. Based on the genetic reports of patients of the Center for Chronic Immunodeficiency (CCI) as a single center experience, and applying project management (PM) in health care research projects, we sought the best way for a rapid diagnosis of HIES. The combination of project management principles with immunologic and genetic knowledge to better define the laboratory and clinical diagnosis lead to an improvement of the management of patients with HIES. These results are shown in one "Decision Tree" which is based on 342 genetic reports of the CCI during the past ten years. It is necessary to facilitate the diagnostic analysis of suspected HIES patients; applying project management in health care research projects provides a better and more accurate diagnosis eventually leading to a better patients' care. This Abstract was presented at 16th Biennial Meeting of the European Society for Immunodeficiencies (ESID 2014), Prague, Czech Republic.

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. For an accurate diagnosis, clinical signs and symptoms, laboratory markers of infection, microbiology, histology and imaging examinations are needed. The treatment goal is eradication of infection and an optimal functional result. Successful treatment requires adequate surgical procedure combined with long-term antimicrobial therapy, ideally with an agent acting on biofilms.

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. The system was then tested on 10 patients with unilateral shoulder pathology (rotator cuff disease, osteoarthritis) before and after surgery (3, 6 months). In order to evaluate the system, nine tests based on the Simple Shoulder Test (SST) were performed on each shoulder for each patient. Three scores were defined: the P score was based on the angular velocities and accelerations of the humerus; the RAV score was based only on the angular velocities of the humerus; the M score was based on the sum of all moments of the humerus. Our kinematic scores indicated significant differences between baseline and follow-up (p<0.05) and differentiated between patients with varying severity of the same condition. We demonstrated a reliable technique of evaluating shoulder pathology and the results of surgery.