RÉSUMÉ
Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.
Sujet(s)
Lumière , ARN de transfert/synthèse chimique , Techniques de synthèse en phase solide , ARN de transfert/composition chimiqueRÉSUMÉ
acp3U is a hypermodified base that is found in the tRNAs of prokaryotes and eukaryotes and also in the ribosomal RNA of mammals. Its function has so far been unknown but it is speculated that acp3U complexes Mg ions, which may contribute to the stabilization of the RNA structure. As a hypermodified base in which a nucleoside is covalently connected to an amino acid, acp3U is a natural nucleoside between genotype and phenotype and hence is also of particular importance for theories about the origin of life. Herein, we report the development of a phosphoramidite building block and of a solid phase protocol that allows synthesis of RNA containing acp3U.
Sujet(s)
Nucléosides/synthèse chimique , Acides aminés/composition chimique , Séquence nucléotidique , Cyclisation , Isomérie , Structure moléculaire , Oligonucléotides/composition chimique , ARN de transfert/synthèse chimique , Techniques de synthèse en phase solide/méthodesRÉSUMÉ
We report here the synthetic route of two constrained dinucleotides and the determination of the sugar puckering by NMR analyses of the starting nucleosides. Enzymatic ligation to microhelix-RNAs provide access to tRNA analogues containing a 3' terminal A76 locked in South conformation. Biological evaluation of our tRNA analogues has been performed using amino-acyl tRNA-dependent transferase FemXWv, which mediates non-ribosomal incorporation of amino acids into the bacterial cell wall. We have shown that our tRNA analogues inhibited the aminoacyl transfer reaction catalyzed by FemXWv with IC50s of 10 and 8 µM. These results indicate that FemXWv displays a moderate preference for tRNAs containing a terminal A76 locked in the South conformation and that a South to North switch in the conformation of the terminal ribose might contribute to the release of the uncharged tRNAAla product of the aminoacyl transfer reaction catalyzed by FemXwv.
Sujet(s)
Techniques de chimie synthétique/méthodes , ARN de transfert/composition chimique , Ribonucléotides/composition chimique , Ribose/analogues et dérivés , Aminoacyltransferases/antagonistes et inhibiteurs , Aminoacyltransferases/métabolisme , Protéines bactériennes/métabolisme , Modèles moléculaires , Conformation d'acide nucléique , ARN de transfert/synthèse chimique , ARN de transfert/métabolisme , Ribonucléotides/synthèse chimique , Ribonucléotides/métabolisme , Ribose/synthèse chimique , Ribose/métabolisme , Weissella/enzymologie , Weissella/métabolismeRÉSUMÉ
Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.
Sujet(s)
ARN bactérien/génétique , ARN de transfert/génétique , 4-Thiouridine/analogues et dérivés , 4-Thiouridine/composition chimique , Amino acyl-tRNA synthetases/composition chimique , Appariement de bases , Séquence nucléotidique , Sites de fixation , Glucosides/synthèse chimique , Magnésium/composition chimique , ARN bactérien/synthèse chimique , ARN de transfert/synthèse chimique , Thermodynamique , Température de transitionRÉSUMÉ
Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.
Sujet(s)
Alanine/analogues et dérivés , Alanine/synthèse chimique , Purines/synthèse chimique , Pyrimidinones/synthèse chimique , Dihydrofolate reductase/composition chimique , Alanine/génétique , Codon , DNA polymerase I/génétique , Escherichia coli , ARN messager/composition chimique , ARN messager/génétique , ARN de transfert/synthèse chimique , ARN de transfert/génétique , Dihydrofolate reductase/génétique , Aminoacylation des ARN de transfertRÉSUMÉ
In times, when drug seeking assays focus on the natural molecular triggers and their analogs, a deeper insight into molecular mechanisms governing the initial step of intrinsic apoptosis (cytochrome c release) is essential to suppress the immortality of pathologically changed cells. In this study, we examined RNA molecules mimicking mitochondrial tRNAs interacting with cytochrome c and possibly affecting its cellular function. tRNA analogs were designed and synthesized prior to the conformational analysis and gel assays clearly stating the nucleic acid-protein complex formation. The circular dichroism spectroscopic (CD) and microscale thermophoresis examination revealed the structural and conformational differences between four tRNA analogs in their interactions with cytochrome c. Obtained CD spectra and gel studies resulted in the complex ratio estimation and conclusion that not only the complex formation may be preferential towards specific tRNAs present in the cell, but nucleobase modifications are not essential for such interaction.
Sujet(s)
Cytochromes c/métabolisme , Mitochondries/métabolisme , Conformation d'acide nucléique , ARN de transfert/métabolisme , Dichroïsme circulaire , Humains , ARN de transfert/synthèse chimiqueRÉSUMÉ
An efficient approach for the desulfuration of C5-substituted 2-thiouridines (R5S2U) bound in the RNA chain exclusively to 4-pyrimidinone nucleoside (R5H2U)-containing RNA products is proposed. This post-synthetic transformation avoids the preparation of a suitably protected H2U phosphoramidite, which otherwise would be necessary for solid-phase synthesis of the modified RNA. Optimization of the desulfuration, which included reaction stoichiometry, time and temperature, allowed to transform a set of ten R5S2U-RNAs into their R5H2U-RNA congeners in ca. 90% yield.
Sujet(s)
Pyrimidinones/composition chimique , ARN/composition chimique , 4-Thiouridine/analogues et dérivés , Nucléosides/composition chimique , ARN/analyse , ARN/synthèse chimique , ARN de transfert/synthèse chimique , ARN de transfert/composition chimique , Techniques de synthèse en phase solide , Spectrométrie de masse MALDI , Acides sulfuriques/composition chimique , Température , 4-Thiouridine/composition chimiqueRÉSUMÉ
Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.
Sujet(s)
Biosynthèse des protéines , ARN de transfert/génétique , Séquence d'acides aminés , Séquence nucléotidique , Codon/génétique , Escherichia coli/cytologie , Escherichia coli/génétique , Escherichia coli/métabolisme , Protéines à fluorescence verte/biosynthèse , Protéines à fluorescence verte/composition chimique , Modèles moléculaires , Conformation d'acide nucléique , Oligopeptides/composition chimique , Oligopeptides/génétique , Conformation des protéines , ARN de transfert/synthèse chimique , ARN de transfert/composition chimiqueRÉSUMÉ
The ability to specifically engineer metal binding sites into target proteins has far-reaching consequences ranging from the development of new biocatalysts and imaging reagents to the production of proteins with increased stability. We report the efficient tRNA-mediated incorporation of the hydroxamate containing amino acid, N(ε)-acetyl-N(ε)-hydroxy-L-lysine, into a transcription factor (TFIIIA). Because this amino acid is compact, hydrophilic, and uncharged at physiological pH, it should have little or no effect on protein folding or solubility. The N(ε)-hydroxy group of the hydroxamate is refractory to photodeprotection and required the identification of reagents for O-protection that are compatible with the synthesis of acylated tRNA. Tetrahydrofuranyl and tetrahydropyranyl O-protecting groups can be removed using mild acid conditions and allowed for an orthogonal protection strategy in which deprotection of the amino acid side chain precedes ligation of an acylated dinucleotide to a truncated suppressor tRNA. These protecting groups will provide a valuable alternative for O-protection, especially in cases where photodeprotection cannot be used.
Sujet(s)
Acides aminés/composition chimique , Furanes/composition chimique , Acides hydroxamiques/composition chimique , Pyrannes/composition chimique , ARN de transfert/synthèse chimique , Aminoacylation , Conformation moléculaire , ARN de transfert/composition chimiqueRÉSUMÉ
Tandemly activated tRNAs participate effectively in protein synthesis and exhibit superior chemical and biochemical stability compared to the more commonly used singly aminoacylated tRNAs. While several bisaminoacylated tRNAs have been prepared via the T4 RNA ligase-mediated condensation of bisaminoacylated pdCpAs and abbreviated tRNA transcripts (tRNA-C(OH)), the bisaminoacylated pdCpAs are difficult to prepare when using bulky amino acids. Described herein is a new strategy for preparing bisaminoacylated tRNAs, applicable even for bulky amino acids.
Sujet(s)
ARN de transfert/synthèse chimique , Acylation , Structure moléculaire , ARN de transfert/composition chimique , StéréoisomérieRÉSUMÉ
Major attention has been focused on dendrimer-DNA complexes because of their applications in gene delivery systems. Dendrimers are also used to transport miRNA and siRNA in vitro. We examine the interaction of tRNA with several dendrimers of different compositions, mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) under physiological conditions using constant tRNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on RNA stability, aggregation, particle formation, and conformation. Structural analysis showed that dendrimer-tRNA complexation occurred via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(mPEG-G3) = 7.6 (± 0.9) × 10(3) M(-1), K(mPEG-G4) = 1.5 (± 0.40) × 10(4) M(-1), and K(PAMAM-G4) = 5.3 (± 0.60) × 10(4) M(-1) show stronger polymer-RNA complexation by PAMAM-G4 than pegylated dendrimers. RNA remains in the A-family structure, whereas biopolymer aggregation and particle formation occurred at high polymer concentrations.
Sujet(s)
Dendrimères/composition chimique , ARN de transfert/synthèse chimique , Interactions hydrophobes et hydrophiles , Structure moléculaire , Taille de particule , ARN de transfert/composition chimique , Propriétés de surfaceRÉSUMÉ
The 2-thiouridine (S2U) unit in the RNA strand is predominantly desulfured with H(2)O(2) to 4-pyrimidinone nucleoside (H2U). The resulting H2U-RNA exhibits significantly lower binding affinity to its complementary strand and in certain conditions undergoes strand scission. These results may explain the tRNA loss of biological function in oxidative stress conditions.
Sujet(s)
ARN de transfert/métabolisme , Soufre/métabolisme , 4-Thiouridine/analogues et dérivés , Appariement de bases , Peroxyde d'hydrogène/métabolisme , Oxydoréduction , Stress oxydatif , Pyrimidinones/métabolisme , ARN de transfert/synthèse chimique , Spectrométrie de masse MALDI , 4-Thiouridine/métabolisme , Température de transitionRÉSUMÉ
The naturally occurring tRNA nucleoside preQ(0), 7-cyano-7-deazaguanosine, which is a central intermediate for other natural occurring 7-deazapurine nucleosides was synthesized via a copper(I)-ion-mediated iodoâcarbonitrile exchange. The reaction was performed on the easily accessible 7-iodo-7-deazaguanosine under microwave conditions. The overall reaction yield was 30% starting with the glycosylation reaction of the nucleobase. Corresponding 2'-deoxyribonucleosides were prepared following the same route.
Sujet(s)
Guanosine/analogues et dérivés , Micro-ondes , Nitriles/composition chimique , Nucléoside Q/synthèse chimique , ARN de transfert/synthèse chimique , Cuivre/composition chimique , Glycosylation , Guanosine/synthèse chimique , Guanosine/composition chimique , Spectroscopie par résonance magnétique , Structure moléculaire , Nucléoside Q/composition chimique , ARN de transfert/composition chimiqueRÉSUMÉ
Supramolecular assembly is a powerful strategy used by nature to build nanoscale architectures with predefined sizes and shapes. With synthetic systems, however, numerous challenges remain to be solved before precise control over the synthesis, folding and assembly of rationally designed three-dimensional nano-objects made of RNA can be achieved. Here, using the transfer RNA molecule as a structural building block, we report the design, efficient synthesis and structural characterization of stable, modular three-dimensional particles adopting the polyhedral geometry of a non-uniform square antiprism. The spatial control within the final architecture allows the precise positioning and encapsulation of proteins. This work demonstrates that a remarkable degree of structural control can be achieved with RNA structural motifs for the construction of thermostable three-dimensional nano-architectures that do not rely on helix bundles or tensegrity. RNA three-dimensional particles could potentially be used as carriers or scaffolds in nanomedicine and synthetic biology.
Sujet(s)
Structures macromoléculaires/composition chimique , ARN de transfert/composition chimique , Structures macromoléculaires/synthèse chimique , Structures macromoléculaires/métabolisme , Microscopie à force atomique , Microscopie électronique à transmission , Modèles moléculaires , Nanostructures , Conformation d'acide nucléique , ARN de transfert/synthèse chimique , Ribonuclease T1/composition chimique , Ribonuclease T1/métabolismeRÉSUMÉ
Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes.
Sujet(s)
Lanthane/composition chimique , Ovocytes/métabolisme , ARN de transfert aminoacylés/synthèse chimique , ARN de transfert/synthèse chimique , Xenopus/métabolisme , Acylation , Animaux , Codon non-sens/génétique , Souris , Structure moléculaire , ARN de transfert/composition chimique , ARN de transfert aminoacylés/composition chimique , Xenopus/génétiqueRÉSUMÉ
A fragment-based approach for the synthesis of ligands of tRNA(Lys) (3), the HIV reverse-transcription primer, is described. The use of NMR spectroscopy has proved to be very useful in this approach, not only to detect low-affinity complexes between small compounds and RNA, but also to provide information on their binding mode and on the way they can be connected. This NMR-spectroscopy-guided analysis enabled us to design micromolar ligands after the optimisation and connection of millimolar fragments with an appropriate linker. The influence of the linker region on the binding affinity and selectivity outlines the importance of having a flexible assemblage strategy with a variety of linkers in such an approach.
Sujet(s)
Spectroscopie par résonance magnétique/méthodes , Fragments peptidiques/composition chimique , ARN de transfert aminoacylés/synthèse chimique , ARN de transfert/synthèse chimique , Séquence nucléotidique , Sites de fixation , Conception de médicament , Ligands , Données de séquences moléculaires , Structure moléculaire , ARN de transfert/composition chimique , ARN de transfert aminoacylés/composition chimiqueRÉSUMÉ
We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.
Sujet(s)
Carbocyanines/composition chimique , Colorants fluorescents/composition chimique , ARN de transfert/synthèse chimique , Uridine/composition chimique , Transfert d'énergie par résonance de fluorescence , Méthodes , Conformation d'acide nucléique , Facteur Tu d'élongation de la chaîne peptidique/métabolisme , Peptides/métabolisme , Poly U/métabolisme , Biosynthèse des protéines , ARN fongique/synthèse chimique , ARN fongique/composition chimique , ARN de transfert/composition chimique , ARN de transfert/métabolisme , ARN de transfert aminoacylés/synthèse chimique , ARN de transfert aminoacylés/composition chimique , ARN de transfert aminoacylés/métabolisme , ARN de transfert de la méthionine/synthèse chimique , ARN de transfert de la méthionine/composition chimique , ARN de transfert de la méthionine/métabolisme , Petite sous-unité du ribosome/métabolismeRÉSUMÉ
Described herein is the preparation of new aminoacylated derivatives of the dinucleotide pdCpA, and of transfer RNAs. The focus of the present work is the synthesis of amino acid analogs related to aspartic acid and cysteine species that have important functional roles in many proteins. The activated aminoacyl-tRNAs prepared can be utilized for the elaboration of proteins containing modified aspartic acid and cysteine derivatives at predetermined sites. Of particular interest is definition of functional group protection strategies that can be used for the preparation of the aminoacylated pdCpAs and tRNAs.
Sujet(s)
Acide aspartique/composition chimique , Cystéine/composition chimique , Dinucléoside phosphates/synthèse chimique , ARN de transfert/synthèse chimique , Aminoacylation des ARN de transfert , Acide aspartique/métabolisme , Cystéine/métabolisme , Dinucléoside phosphates/métabolisme , ARN de transfert/métabolisme , Relation structure-activitéRÉSUMÉ
Modified nucleosides in tRNAs play an important role in the translational process. They fine tune the codon-anticodon interactions and they influence the folding and stabilisation of the tRNA structure. Herein, we present a novel synthetic route to the highly modified nucleosides PreQ(0) and archaeosine. The synthesis involves coupling of a protected 7-cyano-7-deazaguanosine nucleobase with a TBDMS and isopropylidene protected chloro-ribose unit yielding the PreQ(0) nucleoside after deprotection. This PreQ(0) nucleoside is then used as the starting material for the synthesis of archaeosine providing the first total synthetic access to this hypermodified RNA nucleoside.
Sujet(s)
Guanosine/analogues et dérivés , ARN de transfert/synthèse chimique , Guanosine/synthèse chimique , Spectroscopie par résonance magnétique , Spectrométrie de masse ESIRÉSUMÉ
The synthesis and NMR analysis of a 2'-O-alanyl, 3'-O-[1-(13)C]valyl-pdCpA derivative has permitted the definitive assignment of the positions of acylation of tandemly activated pdCpAs, and the bisaminoacylated transfer RNAs derived therefrom.