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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 [7-15N]-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. Graphical abstract

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 19F NMR methods to characterize complex exchange processes with multiple excited states.

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.

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-The online version contains supplementary material available at 10.1007/s00706-022-02892-1.

The rheology of mixed carrageenan gels was investigated at 15 mM calcium, 50 mM potassium and 20 mM sodium concentration. Used carrageenans were of ι-, κ- and Furcellaran-type and mixtures of two types were prepared. We found phase separation in all three possible mixtures showing different degrees of phase separation. ι-/κ-mixture's follow the biphasic model approach with phase parameter k = (0.24 ± 0.01) being close to the theoretical value of k = 0.2 for bicontinuity. For furcellaran/ι-carrageenan phase separation is less distinct, resulting in a lower phase parameter compared to ι-/κ-mixtures. We assume less sulphate ester groups on the furcellaran molecules enable a higher degree of intermixing and more counter phase dilution during gel preparation procedure. Furcellaran/κ-carrageenan gels show synergistic behaviour with respect to storage modulus. Whether the synergy is derived from enforcing phase borders or intermolecular binding could not be clarified.

N6-methyladenosine (m6A) 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 m6A 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 m6A 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 m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes., m6A RNA post-transcriptional modification changes RNA hybridization kinetics. Here the authors show that the methylamino group can adopt syn-conformation pairing with uridine with a mismatch-like conformation in RNA duplex. They also develop a quantitative model that predicts how m6A affects the kinetics of hybridization.

N6-methyladenosine (m6A) 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 NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in theanticonformation 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 issyn.This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with differentsyn:antiisomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.

We present the access to [5‐19F, 5‐13C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐19F, 5‐13C]‐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 19F–13C‐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 13C–19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐19F, 5‐13C]/[5‐19F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19F–13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]‐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., 19 F and 13 C in RNA—a perfect match. An aromatic 19F–13C isotope labeling protocol is introduced for uridines and cytidines in RNA up to 60 nucleotides. The favorable TROSY properties of this spin pair will be useful to probe large RNAs by NMR spectroscopy.

The syntheses and crystal structures of two PCN pincer iridium complexes, prepared from the reaction of their respective PCP pincer carbodi­phospho­rane iridium precursors with an organic azide, are reported. Crystal data for one of the precursors is also discussed., The structure of [Ir{(4-Cl-C6H4N3)C(dppm)-κ3 P,C,N}(dppm-κ2 P,P′)]Cl·1.5CH2Cl2·0.5C7H8 (C57H48Cl2IrN3P4·1.5CH2Cl2·0.5C7H8) (2), dppm = bis­(di­phenyl­phosphino)methane {systematic name: [7-(4-chloro­phen­yl)-1,1,3,3-tetra­phenyl-5,6,7-tri­aza-κN 7-1,3λ4-diphospha-κP 1-hepta-4,6-dien-4-yl][methyl­ene­bis(di­phenyl­phosphine)-κ2 P,P′]iridium(I) chloride–di­chloro­methane–toluene (2/3/1)}, resulting from the reaction of [IrClH{C(dppm)2-κ3 P,C,P)(MeCN)]Cl (1a) with 1-azido-4-chloro­benzene, shows a monocationic five-coordinate IrI complex with a distorted trigonal–bipyramidal geometry. In 2, the iridium centre is coordinated by the neutral triazeneyl­idene­phospho­rane (4-Cl-C6H4N3)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-κ3 P,C,P)]·CH3CN, (C52H45ClIrNP4·CH3CN) (1b) [systematic name: chlorido­cyanidohydrido(1,1,3,3,5,5,7,7-octa­phenyl-1,3λ5,5λ4,7-tetra­phospha-κ2 P 1,P 7-hept-3-en-4-yl)iridium(III) aceto­nitrile monosolvate], prepared from 1a and KCN, reveals an octa­hedral IrIII central atom with a meridional PCP pincer carbodi­phospho­rane (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-C6H4N3)CH(CH(P(Ph)2)2)-κ3 P,C,N)(dppm-κ2 P,P′)]·2CH3OH, (C58H48ClIrN4P4·2CH3OH) (3) (systematic name: {4-[3-(4-chloro­phen­yl)triazenido-κN 3]-1,1,3,3-tetra­phenyl-1,3λ5-diphospha-κP 1-but-2-en-4-yl}cyanido[methyl­enebis(di­phenyl­phosphine)-κ2 P,P′]iridium(III) methanol disolvate), formed via prolonged reaction of 1-azido-4-chloro­benzene with 1b, features a six-coordinate IrIII central atom. The iridium centre is coordinated by the dianionic facial PCN pincer ligand [(4-Cl-C6H4N3)CH(CH(P(Ph2)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 CH2Cl2 and C7H8 solvent mol­ecules show occupational disorder, with the toluene mol­ecule 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 13C labeled RNA building blocks - a 5-13C-uridine and a 2,8-13C2-adenosine building block - are used to introduce isolated 13C-1H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-13C-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 2,8-13C2-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (e) RNA element of the duck Hepatitis B virus. The 2,8-13C2-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 (

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 non-invasively 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.

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 13C/15N-labelled building blocks. The method harbors the unique potential to address structural as well as dynamic features of these RNAs with NMR spectroscopy but also using other biophysical methods, such as mass spectrometry (MS), or small angle neutron/X-ray scattering (SANS, SAXS).

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 synthesis and crystal structures of four iridium–PCP pincer complexes, each containing a highly flexible carbodi­phospho­rane PCP pincer ligand, are discussed., Compound [Ir(C8H12)(C51H45P4)]Cl2 or [Ir(cod)(CH(dppm)2-κ3 P,C,P)]Cl2 (1a), was obtained from [IrCl(cod)]2 and the carbodi­phospho­rane (CDP) salt [CH(dppm)2]Cl [where cod = cyclo­octa-1,5-diene and dppm = bis­(di­phenyl­phosphino)methane]. Treatment of 1a with thallium(I) tri­fluoro­methane­sulfonate [Tl(OTf)] and subsequent crystallization gave complex [Ir(C8H12)(C51H45P4)](OTf)2·CH3CO2C2H5·CH2Cl2 or [Ir(cod)(CH(dppm)2-κ3 P,C,P)](OTf)2·CH3CO2C2H5·CH2Cl2 (1b) [systematic name: (cyclo­octa-1,5-diene)(1,1,3,3,5,5,7,7-octa­phenyl-1,7-diphospha-3,5-di­phospho­niaheptan-4-yl)iridium(I) bis­(tri­fluoro­methane­sulfonate)–ethyl acetate–di­chloro­methane (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, [IrCl2H(C51H44P4)]·(CH3)2CO or [IrCl2H(C(dppm)2-κ3 P,C,P)]·(CH3)2CO [systematic name: di­chlorido­hydrido(1,1,3,3,5,5,7,7-octa­phenyl-1,5λ5,7-triphospha-3-phospho­niahept-4-en-4-yl)iridium(III) acetone monosolvate], crystallizes as an acetone monosolvate. It is a six-coordinate IrIII 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, [IrCl2H(C51H45P4)]Cl·5H2O or [IrCl2H(CH(dppm)2-κ3 P,C,P)]Cl·5H2O [systematic name: di­chlorido­hydrido(1,1,3,3,5,5,7,7-octa­phenyl-1,7-diphospha-3,5-di­phospho­niaheptan-4-yl)iridium(III) chloride penta­hydrate], represents the conjugate CH acid of 2. The ligand [CH(dppm)2]+ is coordinated in a meridional manner. In the cationic six-coordinate IrIII complex 4, [IrClH(CO)(C51H44P4)]Cl·2CH3OH·H2O or [IrClH(CO)(C(dppm)2-κ3 P,C,P)]Cl·2CH3OH·H2O [systematic name: carbonyl­chlorido­hydrido(1,1,3,3,5,5,7,7-octa­phenyl-1,5λ5,7-triphospha-3-phos­pho­niahept-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.

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.