RESUMEN
Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses.
Asunto(s)
Bacteriófago lambda , Cromatografía de Afinidad/métodos , Glutatión Transferasa/metabolismo , ARN/química , ARN/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genética , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Control de Calidad , ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.
Asunto(s)
ARN/aislamiento & purificación , Bacteriófago lambda/genética , Secuencia de Bases , Técnicas Genéticas , Indicadores y Reactivos , ARN/química , ARN Catalítico , ARN Viral/químicaRESUMEN
Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual (31)P and (15)N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface.
Asunto(s)
Endorribonucleasas/metabolismo , Neurospora/metabolismo , ARN Catalítico/metabolismo , ARN de Hongos/metabolismo , Secuencia de Bases , Endorribonucleasas/química , Endorribonucleasas/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Neurospora/genética , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/genética , ARN de Hongos/química , ARN de Hongos/genética , Especificidad por SustratoRESUMEN
On the basis of the structure of TTA-386, a specific antagonist of the endothelin-A receptor subtype (ET(A)), photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing, in position 6, the photoreactive amino acid D- or L-p-benzoyl-phenylalanine showed pharmacological properties very similar to those of TTA-386. Affinity of the probes were also evaluated on transfected CHO cells overexpressing the human ET(A) receptor. Data showed that binding of the radiolabeled peptides were inhibited by ET-1 and BQ-610. Therefore, these photolabile probes were used to label the ET(A) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 66 kDa. An excess of ET-1 or BQ-610 completely abolished the formation of the complex showing the selectivity of the photoprobes. Digestions of the [125I-Tyr5, D- or L-Bpa6]TTA-386-ET(A) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacted. Results showed that Endo Lys-C digestion gave a 4.8 kDa fragment corresponding to the Asp256-Lys299 segment, whereas migration after V8 digestion revealed a fragment of 2.9 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp257-Glu281 stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.9 kDa corresponding to Glu249-Met278. Thus, the combined cleavage data strongly suggested that the binding domain of ET(A) includes a portion of the fifth transmembrane domain, between residues Trp257 and Met278.
Asunto(s)
Antagonistas de los Receptores de la Endotelina A , Endotelina-1/metabolismo , Proteínas de la Membrana/metabolismo , Oligopéptidos/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Receptor de Endotelina A/metabolismo , Secuencia de Aminoácidos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Células CHO , Cricetinae , Cobayas , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Etiquetas de Fotoafinidad/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A/biosíntesis , Receptor de Endotelina A/genética , TransfecciónRESUMEN
On the basis of the structure of IRL-1620, a specific agonist of the endothelin-B receptor subtype (ET(B)), a few photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing the photoreactive amino acid, p-benzoyl-l-phenylalanine in position 5 showed, as assessed with endothelin-A (ET(A)) and ET(B) receptor paradigms, pharmacological properties very similar to those of IRL-1620. The binding capacity of the probe was also evaluated on transfected Chinese hamster ovary (CHO) cells overexpressing the human ET(B) receptor. Data showed that binding of the radiolabeled peptide was inhibited by ET-1 and IRL-1620. Therefore, this photolabile probe was used to label the ET(B) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 49 kDa. An excess of ET-1 or IRL-1620 completely abolished the formation of the complex, showing the selectivity of the photoprobe. Digestions of the [Bpa(5),Tyr((125)I)(6)]IRL-1620-ET(B) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacts. Results showed that Endo Lys-C digestion gave a 3.8-kDa fragment corresponding to the Asp(274)-Lys(303) segment, whereas migration after V8 digestion revealed a fragment of 4.6 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp(275)-Asp(313) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.6 kDa, corresponding to Gln(267)-Met(296). Thus, the combined cleavage data strongly suggested that the agonist binding domain of ET(B) includes a portion of the fifth transmembrane domain, between residues Trp(275) and Met(296).
Asunto(s)
Endotelinas/metabolismo , Fragmentos de Péptidos/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Receptor de Endotelina B/metabolismo , Secuencia de Aminoácidos , Animales , Aorta Torácica/metabolismo , Células CHO , Cricetinae , Endotelinas/síntesis química , Cobayas , Humanos , Hidrólisis , Radioisótopos de Yodo/metabolismo , Pulmón/metabolismo , Masculino , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Etiquetas de Fotoafinidad/síntesis química , Unión Proteica , Estructura Terciaria de Proteína , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina B/agonistas , TransfecciónRESUMEN
(1) Endothelin-1 (ET-1) is a bicyclic 21-amino-acid peptide causing a potent and sustained vasoconstriction, mainly through the ET(A) receptor subtype. So far, no selective ET(A) agonists are described in the literature. (2) A series of truncated and chemically modified ET-1 analogues were obtained through solid-phase peptide synthesis and their biological activity was assessed on rat thoracic aorta rings (ET(A) receptors) and guinea-pig lung parenchyma strips (ET(B) receptors). (3) Structure-activity studies led to the identification of ET-1 fragments exhibiting an ET(A) selective agonistic activity. (4) In particular, [D-Lys(9)]cyclo(11-15) ET-1(9-21) was the most potent peptide. It appeared as a full agonist of ET(A) receptors, being under two orders of magnitude less potent than ET-1 (EC(50): 2.3 x 10(-7) vs 6.8 x 10(-9) M). Interestingly, even a linear formylated analogue, [Ala(11,15), Trp(For)(21)]ET-1(9-21), showed a selective ET(A) activity (EC(50): 3.0 x 10(-6) M). None of the numerous analogues of the series exhibited substantial effects in the guinea-pig lung parenchyma bioassay. (5) Thus, this study describes the first compounds showing a significant bioactivity in an ET(A) pharmacological preparation while being inactive in an ET(B) paradigm. They show that the ET-1 pharmacophores, responsible for the ET(A)-mediated actions, are located within the 9-21 segment of the molecule. Moreover, the bicyclic structure of ET-1 does not appear as essential for the ET(A)-related vasoconstriction. Results also suggest that the positive charge of the Lys(9) side chain participates in an intramolecular ionic bond with the carboxylate function of Asp(18).