RESUMO
Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.
Assuntos
Trifosfato de Adenosina/síntese química , Guanosina Trifosfato/síntese química , Marcação por Isótopo/métodos , Nucleotídeos/síntese química , Bacillus anthracis/química , Bacillus anthracis/genética , Isótopos de Carbono , Coronavirus Humano 229E/química , Coronavirus Humano 229E/genética , Creatina Quinase/química , Creatina Quinase/genética , Espectroscopia de Ressonância Magnética , Pentosiltransferases/química , Pentosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Elementos de Resposta , Ribose/química , Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/genética , Riboswitch , Transcrição GênicaRESUMO
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 inâ 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 Aâ residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this Aâ residue in the complex is shifted by more than 5 pH units compared to the pKa for Aâ nucleotides in single-stranded RNA. This is the largest pKa shift for an Aâ 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.
Assuntos
Nucleotídeos de Adenina/química , Aptâmeros de Nucleotídeos/química , Guanosina Trifosfato/química , Prótons , Sítios de Ligação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Nucleotídeos de Pirimidina/síntese química , RNA/química , Marcadores de Spin/síntese química , Isótopos de Carbono/síntese química , Isótopos de Carbono/química , Citidina/química , HIV-1/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , Compostos Organofosforados/química , Nucleotídeos de Pirimidina/química , RNA Viral/química , Técnicas de Síntese em Fase Sólida/métodos , Uridina/químicaRESUMO
A ligand-observed 1H NMR relaxation experiment is introduced for measuring the binding kinetics of low-molecular-weight compounds to their biomolecular targets. We show that this approach, which does not require any isotope labeling, is applicable to ligand-target systems involving proteins and nucleic acids of variable molecular size. The experiment is particularly useful for the systematic investigation of low affinity molecules with residence times in the micro- to millisecond time regime.
Assuntos
Espectroscopia de Prótons por Ressonância Magnética , Relação Dose-Resposta a Droga , Cinética , Ligantes , Estrutura Molecular , Peso Molecular , Relação Estrutura-Atividade , Fatores de TempoRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Guanosina Trifosfato/metabolismo , Ressonância Magnética Nuclear Biomolecular , Sequência de Bases , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
RNA de Cadeia Dupla/química , RNA/química , Adenosina/química , Pareamento de Bases , Sequência de Bases , Guanosina/química , Ligação de Hidrogênio , Sequências Repetidas Invertidas , Modelos Moleculares , Estabilidade de RNARESUMO
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.
Assuntos
Marcação por Isótopo , Ressonância Magnética Nuclear Biomolecular/métodos , Compostos Organofosforados/química , RNA/químicaRESUMO
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.
RESUMO
In this work, we present a novel 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) radical phosphoramidite building block, which can be attached to the 5'-terminus of nucleic acids. To investigate the paramagnetic relaxation enhancement (PRE) emanating from this radical center, we incorporated the TEMPO label into various types of RNAs. We measured proton PREs for selectively (13)C-isotope labeled nucleotides to derive long-range distance restraints in a short 15 nucleotide stem-loop model system, underscoring the potential of the 5'-TEMPO tag to determine long-range distance restraints for solution structure determination. We subsequently applied the distance-dependent relaxation enhancement induced by the nitroxide radical to discern two folding states in a bistable RNA. Finally, we investigated the fast conformational sampling of the HIV-1 TAR RNA, a paradigm for structural flexibility in nucleic acids. With PRE NMR in combination with molecular dynamics simulations, the structural plasticity of this RNA was analyzed in the absence and presence of the ligand L-argininamide.