RESUMO
Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas PrPC/química , Animais , Aptâmeros de Nucleotídeos/farmacologia , Bovinos , Linhagem Celular , Camundongos , Modelos Moleculares , Peptídeos/química , Proteínas PrPSc/análiseRESUMO
Microfold (M) cells are intestinal epithelial cells specialized for sampling and transport of luminal antigens to gut-associated lymphoid tissue for initiation of both mucosal and systemic immune responses. Therefore, M-cell targeted vaccination has the potential to be a better immunization strategy. Glycoprotein 2 (GP2), an antigen uptake receptor for FimH(+) bacteria on M cells, can be a good target for this purpose. Aptamers are oligonucleotides that bind to a variety of target molecules with high specificity and affinity. Together with its low toxic feature, aptamers serves as a tool of molecular-targeted delivery. In this study, we used Systematic Evolution of Ligands by EXponential enrichment (SELEX) to isolate aptamers specific to murine GP2 (mGP2). After ten rounds of SELEX, eleven different aptamer sequences were selected. Among them, the most frequently appeared sequence (~60%) were aptamer NO. 1 (Apt1), and the second most (~7%) were aptamer NO. 5 (Apt5). In vitro binding experiment confirmed that only Apt1 and Apt5 specifically bound to mGP2 among eleven aptamers initially selected. Apt1 showed the strongest affinity with mGP2, with the Kd value of 110±2.6 nM evaluated by BIACORE. Binding assays with mutants of Apt1 suggest that, in addition to the loop structure, the nucleotide sequence, AAAUA, in the loop is important for binding to mGP2. Furthermore, this aptamer was able to bind to mGP2 expressed on the cell surface. These results suggest that this mGP2-specific aptamer could serve as a valuable tool for testing M-cell-targeted vaccine delivery in the murine model system.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Proteínas Ligadas por GPI/metabolismo , Técnica de Seleção de Aptâmeros , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Especificidade por SubstratoRESUMO
RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its beta-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein. R12 forms an intramolecular parallel quadruplex. The quadruplex contains G:G:G:G tetrad and G(:A):G:G(:A):G hexad planes. Two quadruplexes form a dimer through intermolecular hexad-hexad stacking. Two lysine clusters of bPrP have been identified as binding sites for R12. The electrostatic interaction between the uniquely arranged phosphate groups of R12 and the lysine clusters is suggested to be responsible for the affinity of R12 to bPrP. The stacking interaction between the G:G:G:G tetrad planes and tryptophan residues may also contribute to the affinity. One R12 dimer molecule is supposed to simultaneously bind the two lysine clusters of one bPrP molecule, resulting in even higher affinity. The atomic coordinates of R12 would be useful for the development of R12 as a therapeutic agent against prion diseases and Alzheimer's disease.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Príons/química , Animais , Sítios de Ligação , Bovinos , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Príons/antagonistas & inibidores , Príons/metabolismoRESUMO
Prion diseases comprise a fatal neuropathy caused by the conversion of prion protein from a cellular (PrPC) to a pathological (PrPSc) isoform. Previously, we obtained an RNA aptamer, r(GGAGGAGGAGGA) (R12), that folds into a unique G-quadruplex. The R12 homodimer binds to a PrPC molecule, inhibiting PrPC-to-PrPSc conversion. Here, we developed a new RNA aptamer, r(GGAGGAGGAGGAGGAGGAGGAGGA) (R24), where two R12s are tandemly connected. The 50% inhibitory concentration for the formation of PrPSc (IC50) of R24 in scrapie-infected cell lines was ca. 100 nM, i.e., much lower than that of R12 by two orders. Except for some antibodies, R24 exhibited the lowest recorded IC50 and the highest anti-prion activity. We also developed a related aptamer, r(GGAGGAGGAGGA-A-GGAGGAGGAGGA) (R12-A-R12), IC50 being ca. 500 nM. The structure of a single R12-A-R12 molecule determined by NMR resembled that of the R12 homodimer. The quadruplex structure of either R24 or R12-A-R12 is unimolecular, and therefore the structure could be stably formed when they are administered to a prion-infected cell culture. This may be the reason they can exert high anti-prion activity.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas PrPSc/química , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/genética , Proteínas Priônicas , Relação Estrutura-AtividadeRESUMO
Hepatitis C virus (HCV) translation begins within the internal ribosome entry site (IRES). We have previously isolated two RNA aptamers, 2-02 and 3-07, which specifically bind to domain II and domain III-IV of the HCV IRES, respectively, and inhibit IRES-dependent translation. To improve the function of these aptamers, we constructed two conjugated molecules of 2-02 and 3-07. These bound to the target RNA more efficiently than the two parental aptamers. Furthermore, they inhibited IRES-dependent translation about 10 times as efficiently as the 3-07 aptamer. This result indicates that combining aptamers for different target recognition sites potentiates the inhibition activity by enhancing the domain-binding efficiency.
Assuntos
Aptâmeros de Nucleotídeos/genética , Hepacivirus/metabolismo , Biossíntese de Proteínas , RNA Viral/genética , Ribossomos/metabolismo , Eletroforese em Microchip , Ensaio de Desvio de Mobilidade Eletroforética , Hepacivirus/genética , Humanos , Conformação de Ácido Nucleico , RNA Viral/química , Ribossomos/genética , Ribossomos/virologiaRESUMO
In order to isolate RNA aptamers against the mouse prion protein (mPrP), we carried out in vitro selection from RNA pools containing a 30-nucleotide randomized region. Aptamer 60-3 was found to have a high affinity for mPrP (K(d) = 5.6 +/- 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (K(d) = 22 +/- 4 nM). Following 5' biotinylation, aptamer 60-3 specifically detected PrP in mouse brain homogenate in a Northwestern blotting assay. To determine the mPrP-aptamer binding region, we performed protein-deletion-mutant analysis and competition-binding analysis using heparin. The results showed that aptamer 60-3 appears to have binding sites located between amino acids 23-108.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Príons/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Animais , Aptâmeros de Nucleotídeos/química , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/fisiologia , RNA/químicaRESUMO
Non-structural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct activities, protease and helicase, which are essential for HCV proliferation. In previous work, we obtained RNA aptamers (G9-I, II and III) which specifically bound the NS3 protease domain (DeltaNS3), efficiently inhibiting protease activity in vitro. To utilize these aptamers in vivo, we constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken beta-actin globin (CAG) promoter. By conjugating the cis-acting genomic human hepatitis delta virus (HDV) ribozyme and G9-II aptamer, a chimeric HDV ribozyme-G9-II aptamer (HA) was constructed, which was used to produce stable RNA in vivo and to create tandem repeats of the functional unit. To target the transcribed RNA aptamers to the cytoplasm, the minimal mutant of constitutive transport element (CTE), derived from type D retroviruses, was conjugated at the 3' end of HA (HAC). Transcript RNAs from (HA)(n) and (HAC)(n) were processed into the G9-II aptamer unit by the cis-acting HDV ribozyme, both in vitro and in vivo. Efficient protease inhibition activity of HDV ribozyme-G9-II aptamer expression plasmid was demonstrated in HeLa cells. Protease inhibition activity level of tandem chimeric aptamers, (HA)(n) and (HAC)(n), rose with the increase of n from 1 to 4.
Assuntos
Oligorribonucleotídeos/metabolismo , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Ligação Competitiva , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Transporte Nucleocitoplasmático/genética , Oligorribonucleotídeos/genética , Plasmídeos/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , Sequências de Repetição em Tandem/genética , Transfecção , Células Tumorais Cultivadas , Proteínas não Estruturais Virais/genéticaRESUMO
DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (<3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Eletroforese em Microchip/métodos , Proteínas/metabolismo , RNA/metabolismo , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Eletroforese Capilar , Cinética , Ligantes , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas/química , RNA/química , Especificidade por SubstratoRESUMO
The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Delta, was conjugated via an oligo(U) tract to the 3'-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition.
Assuntos
Desenho de Fármacos , Hepacivirus/enzimologia , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , RNA Helicases/antagonistas & inibidores , RNA/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Aptâmeros de Nucleotídeos , Células HeLa , Hepacivirus/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Inibidores de Proteases/síntese química , RNA/síntese química , RNA/farmacologiaRESUMO
Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.
Assuntos
Interferon beta/metabolismo , Glicoproteínas de Membrana/metabolismo , Poli I-C/farmacologia , RNA de Cadeia Dupla/química , Receptores de Superfície Celular/metabolismo , Ribose/química , Células Cultivadas , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Interferon beta/genética , Interferon beta/farmacologia , Rim/metabolismo , Pulmão/metabolismo , RNA de Cadeia Dupla/metabolismo , Receptor 3 Toll-Like , Receptores Toll-LikeRESUMO
Ligand stimulation of the Toll-like receptors (TLRs) triggers innate immune response, cytokine production and cellular immune activation in dendritic cells. However, most TLR ligands are microbial constituents, which cause inflammation and toxicity. Toxic response could be reduced for secure immunotherapy through the use of chemically synthesized ligands with defined functions. Here we create an RNA ligand for TLR3 with no ability to activate the RIG-I/MDA5 pathway. This TLR3 ligand is a chimeric molecule consisting of phosphorothioate ODN-guided dsRNA (sODN-dsRNA), which elicits far less cytokine production than poly(I:C) in vitro and in vivo. The activation of TLR3/TICAM-1 pathway by sODN-dsRNA effectively induces natural killer and cytotoxic T cells in tumour-loaded mice, thereby establishing antitumour immunity. Systemic cytokinemia does not occur following subcutaneous or even intraperitoneal administration of sODN-dsRNA, indicating that TICAM-1 signalling with minute local cytokines sufficiently activate dendritic cells to prime tumoricidal effectors in vivo.
Assuntos
Adjuvantes Imunológicos/química , Células Matadoras Naturais/citologia , Ativação Linfocitária/efeitos dos fármacos , RNA/química , Linfócitos T Citotóxicos/citologia , Receptor 3 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Citocinas/metabolismo , Células Dendríticas/citologia , Feminino , Células HEK293 , Células HeLa , Humanos , Inflamação , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Oligonucleotídeos Fosforotioatos/química , Poli I-C/química , Transdução de SinaisRESUMO
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct domains, protease and helicase, that are essential for HCV proliferation. Therefore, NS3 is considered a target for anti-HCV treatment. To study RNA aptamers of the NS3 helicase domain, we carried out in vitro selection against the HCV NS3 helicase domain. RNA aptamers obtained after eight generations possessed 5' extended single-stranded regions and the conserved sequence (5'-GGA(U/C)GGAGCC-3') at stem-loop regions. Aptamer 5 showed strong inhibition of helicase activity in vitro. Deletion and mutagenesis analysis clarified that the conserved stem-loop is important and that the whole structure is needed for helicase inhibition. We compared the inhibition of helicase activity between aptamer 5 and 3'+-UTR of HCV.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , RNA Helicases/antagonistas & inibidores , RNA/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Sequência de Bases , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA/química , RNA/genética , RNA Helicases/química , RNA Helicases/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (DeltaNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of DeltaNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (DeltaNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8).C(+24)-G(-36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.
Assuntos
Hepacivirus/enzimologia , RNA Viral/química , RNA Viral/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/fisiologia , Sequência de Bases , Análise Mutacional de DNA , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Viral/genética , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genéticaRESUMO
Endosomal Toll-like receptor 3 (TLR3) serves as a sensor of viral infection and sterile tissue necrosis. Although TLR3 recognizes double-stranded RNA, little is known about structural features of virus- or host-derived RNAs that activate TLR3 in infection/inflammatory states. Here we demonstrate that poliovirus-derived single-stranded RNA segments harbouring stem structures with bulge/internal loops are potent TLR3 agonists. Functional poliovirus-RNAs are resistant to degradation and efficiently induce interferon-α/ß and proinflammatory cytokines in human and mouse cells in a TLR3-dependent manner. The N- and C-terminal double-stranded RNA-binding sites of TLR3 are required for poliovirus-RNA-mediated TLR3 activation. Like polyriboinosinic:polyribocytidylic acid, a synthetic double-stranded RNA, these RNAs are internalized into cells via raftlin-mediated endocytosis and colocalized with TLR3. Raftlin-associated RNA uptake machinery and the TLR3 RNA-sensing system appear to recognize an appropriate topology of multiple RNA duplexes in poliovirus-RNAs. Hence, TLR3 is a sensor of extracellular viral/host RNA with stable stem structures derived from infection or inflammation-damaged cells.
Assuntos
Conformação de Ácido Nucleico , Poliovirus/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Sítios de Ligação , Antígeno CD11c/metabolismo , Sistema Livre de Células , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Endocitose , Endorribonucleases/metabolismo , Endossomos/metabolismo , Proteínas de Escherichia coli/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Interferon Tipo I/biossíntese , Interferon beta/biossíntese , Camundongos , Estabilidade de RNA , RNA de Cadeia Dupla/metabolismo , Receptor 3 Toll-Like/deficiênciaRESUMO
Microchip electrophoresis (ME) coupled with fluorescence detection was used to estimate the binding activity of aptamer in each systematic evolution of ligands by exponential enrichment (SELEX) round for a target molecule. This approach is a non-radioisotopic, rapid and simple platform, and electrophoretic separation appears to be an effective technique for aptamers of oligonucleotide molecules. We tried to obtain gonadotropin-specific RNA aptamer by the above approach. As a result, the peaks of aptamers based on the conformational differences between them were separated and detected on the electropherograms. Moreover, the intensity of peak of unbound aptamer was decreased with progression through the SELEX rounds, suggesting that RNA aptamer with high affinity was obtained by the proposed method.
RESUMO
Nuclear magnetic resonance (NMR) studies have shown that RNA/DNA oligomers with GGA repeat sequences contain unique G-quadruplex structures in the presence of K(+) or Na(+) ions. In this study, we used microchip electrophoresis to study the structure of an RNA aptamer against bovine prion protein that possessed four GGA-triplet repeats (wt2). We analyzed the structural changes and characterized dimer formation of the aptamer. Mutational, circular dichroism, and one-dimensional NMR studies of wt2 revealed that K(+) ions induce wt2 to assume a thermostable dimer in an intramolecular G-quadruplex with parallel orientation.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Príons/antagonistas & inibidores , Repetições de Trinucleotídeos , Adenina/química , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Bovinos , Dicroísmo Circular , Eletroforese em Microchip , Guanina/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Príons/genética , Sódio/químicaRESUMO
In order to obtain RNA aptamers against bovine prion protein (bPrP), we carried out in vitro selection from RNA pools containing a 55-nucleotide randomized region to target recombinant bPrP. Most of obtained aptamers contained conserved GGA tandem repeats (GGA)(4) and aptamer #1 (apt #1) showed a high affinity for both bPrP and its beta isoform (bPrP-beta). The sequence of apt #1 suggested that it would have a G-quadruplex structure, which was confirmed using CD spectra in titration with KCl. A mutagenic study of this conserved region, and competitive assays, showed that the conserved (GGA)(4) sequence is important for specific binding to bPrP and bPrP-beta. Following 5'-biotinylation, aptamer #1 specifically detected PrP(c) in bovine brain homogenate in a Northwestern blotting assay. Protein deletion mutant analysis showed that the bPrP aptamer binds within 25-131 of the bPrP sequence. Interestingly, the minimized aptamer #1 (17 nt) showed greater binding to bPrP and bPrP-beta as compared to apt #1. This minimized form of aptamer #1 may therefore be useful in the detection of bPrP, diagnosis of prion disease, enrichment of bPr and ultimately in gaining a better understanding of prion diseases.
Assuntos
Aptâmeros de Nucleotídeos/química , Química Encefálica , Proteínas PrPC/química , Animais , Encéfalo/metabolismo , Bovinos , Proteínas PrPC/metabolismo , Doenças Priônicas/diagnóstico , Doenças Priônicas/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Repetições de TrinucleotídeosRESUMO
Antibody-based detection systems are widely used, but in the cases of immunoprecipitations and enzyme-linked immunoassays, they can be laborious. These techniques require the preparation of at least two kinds of non-cross-reactive immunoglobulin Gs (IgGs), usually made from different species against the single target molecule. Aptamers composed of nucleic acids possess strict recognition ability for the target molecule's three-dimensional structure and, thus, are considered to act like IgG. In this study, experimental trials were designed to combine the advantages of IgG and aptamers. For this purpose, aptamers against rabbit IgG were identified by in vitro selection. One of the obtained aptamers had a dissociation constant lower than 15 pM to the rabbit IgG. It bound specifically to the constant region of the rabbit IgG, and no binding was observed with mouse or goat IgG. Moreover, this aptamer recognized only the native form of rabbit IgG and could not bind the sodium dodecyl sulfate (SDS)-denatured form. These features show the advantage of using the aptamer as a secondary probing agent rather than the usual secondary antibodies.
Assuntos
Anticorpos/análise , Anticorpos/metabolismo , Aptâmeros de Nucleotídeos/análise , Aptâmeros de Nucleotídeos/metabolismo , Western Blotting/métodos , Animais , Anticorpos/imunologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Sítios de Ligação , Reações Cruzadas , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Desnaturação de Ácido Nucleico , Coelhos , Especificidade por SubstratoRESUMO
RNA aptamers for bovine prion protein (bPrP) were obtained by in vitro selection. It was found that the r(GGA) triplet repeat was frequently present in these aptamers. We already reported that both DNA and RNA containing the GGA repeat form unique quadruplex structures. The unique structures may be utilized for the recognition of bPrP by these aptamers. One of these aptamers contains four continuous repeat of r(GGA), r(GGA)(4). Here, we analyzed the structure of r(GGA)(4) under physiological ionic conditions by NMR. It was revealed on the basis of characteristic NOEs that an r(GGA)(4) strand forms the quadruplex composed of one G:G:G:G tetrad plane and one G(:A):G:G(:A):G hexad plane. Furthermore, two monomers form a dimeric structure in a tail-to-tail manner. The dimeric quadruplex structure of r(GGA)(4) is similar to that of d(GGA)(4), but the difference between two structures was also noticed.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Príons/metabolismo , Animais , Aptâmeros de Nucleotídeos/metabolismo , Bovinos , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
We performed in vitro selection against bovine prion protein (bPrP) using RNA pools with thirty-nucleotides randomized regions. Among the obtained RNA aptamers, RNA aptamer wt2 possessed four GGA triplets. It is known that RNA and DNA oligomers containing GGA repeat sequences show a G-quadruplex structure in the presence of K+ or Na+ ions using the NMR and crystallographic studies. Recently we have been studying RNA aptamer-protein, -peptide and -RNA interactions using microchip electrophoresis (ME). In this study, we used this system to detect structural changes of RNA aptamer wt2 in the presence of K+ and Na+ ions, and detected the dimer formation with simple operation.