RESUMO
Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of (18)O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging (18)O atom into the phosphate group during the oxidation step of the synthetic cycle by using (18)O water as the oxygen donor. The (18)O label in the RNA was stable at pH 3-8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The (18)O/(16)O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of (18)O-labeled RNA, and this technique was used to determine the blood concentration of (18)O-labeled RNA after administration to mice. (18)O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.
Assuntos
Isótopos de Oxigênio , RNA Interferente Pequeno/síntese química , Animais , Linhagem Celular , Humanos , Cinética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia/métodos , Interferência de RNA , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/químicaRESUMO
Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.
Assuntos
Neoplasias da Mama/terapia , Receptores ErbB/genética , Exossomos/metabolismo , MicroRNAs/administração & dosagem , Animais , Sequência de Bases , Western Blotting , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Humanos , Camundongos , Microscopia Imunoeletrônica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used as RNA interference (RNAi) reagents. Recently, truncated shRNAs that trigger RNAi in a Dicer-independent manner have been developed. We generated a novel class of RNAi reagent, designated enforced strand bias (ESB) RNA, in which an siRNA duplex was chemically bridged between the 3' terminal overhang region of the guide strand and the 5' terminal nucleotide of the passenger strand. ESB RNA, which is chemically bridged at the 2' positions of ribose (2'-2' ESB RNA), functions in a Dicer-independent manner and was highly effective at triggering RNAi without the passenger strand-derived off-target effect. In addition, the 2'-2' ESB RNA exhibited a unique target sequence preference that differs from siRNA and silenced target sequences that could not be effectively suppressed by siRNA. Our results indicate that ESB RNA has the potential to be an effective RNAi reagent even when the target sequence is not suitable for siRNA.
RESUMO
Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2'-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5'-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5'-cap and a 3'-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/genética , RNA Mensageiro/síntese química , Regiões 3' não Traduzidas , Animais , Células CHO , Cricetinae , Cricetulus , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Indicadores e Reagentes , Metiltransferases/metabolismo , Fosforilação , Poli A/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
Prostatic inflammation plays a role in the progression of benign prostatic hyperplasia (BPH). Eviprostat is an antioxidant, antiinflammatory phytotherapeutic agent widely used to treat lower urinary tract symptoms in BPH. Because Eviprostat is a mixture of compounds from multiple natural sources, however, its mechanism of action has been difficult to investigate. Here, we describe the use of oligonucleotide microarrays to investigate changes in gene expression in the prostate of rats with surgically induced partial bladder-outlet obstruction and the effect of Eviprostat on those changes. Several dozen proinflammatory genes were activated in obstructed rats, including cytokine, arachidonic acid cascade enzyme, Toll-like receptor (TLR), and transcription factor genes, and their expression was suppressed by Eviprostat. Pathway analysis revealed that several proinflammatory pathways were activated, including cytokine and TLR signaling pathways. The differential expression of selected genes was verified by real-time reverse-transcriptase polymerase chain reaction. Our findings suggest that prostate inflammation in our rat model of partial bladder-outlet obstruction is related to the increased expression of nuclear factor kappaB (NF-kappaB) and the induction of proinflammatory cytokines, and that Eviprostat suppresses their expression at the transcriptional level. The prostate inflammation seen in BPH and the clinical benefits of Eviprostat may be similarly explained.
Assuntos
Anti-Inflamatórios/farmacologia , Etamsilato/farmacologia , Mediadores da Inflamação/metabolismo , Extratos Vegetais/farmacologia , Próstata/efeitos dos fármacos , Prostatite/genética , Animais , Análise por Conglomerados , Combinação de Medicamentos , Perfilação da Expressão Gênica , Genoma , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Próstata/metabolismo , Prostatite/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Bexiga Urinária/cirurgiaRESUMO
A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-mumol scale corresponds to 1 mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100 nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.
Assuntos
Éteres/química , Inativação Gênica , MicroRNAs/síntese química , Nitrilas/química , Oligorribonucleotídeos/síntese química , Precursores de RNA/síntese química , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Humanos , MicroRNAs/química , MicroRNAs/isolamento & purificação , Oligorribonucleotídeos/química , Oligorribonucleotídeos/isolamento & purificação , Compostos Organofosforados/química , Precursores de RNA/química , Ribonucleosídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We have synthesized diastereomerically pure diadenosine 3',5'-boranophosphates (Ap(b)A) by using the boranophosphotriester method from ribonucleosides protected with the 2'-hydroxy protecting group 2-cyanoethoxymethyl (CEM). Melting curves of the triple-helical complex of the dimer Ap(b)A and 2poly(U) at high ionic strength revealed that presumptive (Sp)-Ap(b)A had a much higher affinity and presumptive (Rp)-Ap(b)A a much lower affinity for poly(U) than the natural dimer ApA did. In contrast, the affinities of these dimers for poly(dT) were similar. Both the (Rp)- and the (Sp)-boranophosphate diastereomers showed much higher resistance to digestion by snake venom phosphodiesterase and nuclease P1 than ApA did. They have potential for use as synthons to be incorporated into boranophosphate oligonucleotides. In particular, because oligonucleotides containing Sp boranophosphate nucleotides are expected to bind more strongly and specifically to RNA than natural oligoribonucleotides do, they may find application in the isolation and detection of functional RNA in basic research and diagnostics.
Assuntos
Adenosina/química , Boranos/síntese química , Cianetos/química , Etil-Éteres/química , Fosfatos/síntese química , Boranos/química , Boranos/metabolismo , Esterases/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , Estrutura Molecular , Desnaturação de Ácido Nucleico , Fosfatos/química , Fosfatos/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
A novel method for the synthesis of RNA oligomers with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group has been developed. The new method allows the synthesis of oligoribonucleotides with an efficiency and final purity comparable to that obtained in DNA synthesis. [structure: see text]
Assuntos
Nitrilas/química , RNA/síntese química , Sequência de Bases , Modelos Químicos , Dados de Sequência Molecular , Estrutura Molecular , RNA/químicaRESUMO
PURPOSE: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel cationic liposome, LIC-101. EXPERIMENTAL DESIGN: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. RESULTS: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. CONCLUSIONS: By combining siRNA with our cationic liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.
Assuntos
Antineoplásicos/farmacologia , Cátions/química , Lipossomos/química , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/química , Animais , Western Blotting , Linhagem Celular Tumoral , DNA/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias/genética , Oligonucleotídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , RNA Interferente Pequeno/metabolismo , Fatores de TempoRESUMO
Small interfering RNAs (siRNAs) are a potential treatment of atopic dermatitis (AD) because they can specifically silence the gene expression of AD-related factors. However, siRNA alone cannot exert a sufficiently strong therapeutic effect due to low delivery efficiency to the target tissues and cells; simply increasing the amount used is not possible due to the possibility of off-target effects. We previously reported a novel class of therapeutic RNA interference (RNAi) agents called nkRNA(®) and PnkRNA(®), which have been shown to be effective in several disease models, have greater resistance to nuclease degradation than canonical siRNAs, and do not induce any immunotoxicity. In the present study, we describe a non-invasive and effective transdermal RNAi therapeutic system for atopic dermatitis that uses the functional cell-penetrating stearoyl-oligopeptide OK-102 as a cytoplasm-responsive nanocarrier for nkRNA(®) and PnkRNA(®). The two RNAi agents were targeted against RelA, a subclass of NF-κB (nuclear factor kappa B), and, as part of OK-102 complexes, they strongly silenced RelA mRNA in macrophage cells and demonstrated a significant therapeutic effect in a mouse model of AD. It was shown that OK-102-complexed RNAi agents were an efficient therapeutic system for AD and caused no adverse reactions.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Dermatite Atópica/terapia , Oligopeptídeos/administração & dosagem , RNA/administração & dosagem , Fator de Transcrição RelA/genética , Administração Tópica , Animais , Linhagem Celular , Peptídeos Penetradores de Células/química , Dermatite Atópica/imunologia , Dinitrofluorbenzeno , Interleucina-6/imunologia , Camundongos , Oligopeptídeos/química , Peptídeos , RNA/química , Interferência de RNA , Fator de Necrose Tumoral alfa/imunologiaRESUMO
MicroRNAs (miRNAs) serve important roles in regulating various physiological activities through RNA interference (RNAi). miR-122 is an important mediator of RNAi that is known to control hepatitis C virus (HCV) replication and is being investigated in clinical trials as a target for anti-HCV therapy. In this study, we developed novel oligonucleotides containing non-nucleotide residues, termed iMIRs, and tested their abilities to inhibit miR-122 function. We compared the inhibitory effects of iMIRs and locked nucleic acids (LNAs) on HCV replication in OR6 cells, which contained full-length HCV (genotype 1b) and a luciferase reporter gene. We found that RNA-type iMIRs with bulge-type, imperfect complementarity with respect to miR-122 were 10-fold more effective than LNAs in inhibiting HCV replication and functioned in a dose-dependent manner. Moreover, iMIR treatment of OR6 cells reduced HCV replication without inducing interferon responses or cellular toxicity. Based on these results, we suggest that iMIRs can inhibit HCV replication more effectively than LNAs and are therefore promising as novel antiviral agents.
RESUMO
ISG20 is an interferon-induced antiviral exoribonuclease that acts on single-stranded RNA and also has minor activity towards single-stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5'-monophosphate (UMP) at 1.9 A resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh-group DNases, the epsilon subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2'-OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.
Assuntos
Antivirais/química , Exonucleases/química , Interferons/fisiologia , Sítios de Ligação , Cristalografia por Raios X , Exorribonucleases , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
We have investigated the mechanism of resistance of leukemia cells to Ara-C using an in-house cDNA microarray designed for the analysis of leukemia cells. We produced Ara-C-resistant cells from the CCRF-CEM (acute lymphoblastic leukemia) cell line and compared their gene-expression profile with that of wild-type cells. The adenosine deaminase (ADA) gene was highly up-regulated in Ara-C-resistant cells, while equilibrative nucleoside transporter 1 (ENT1) and several cell-cycle-related genes were down-regulated. Of all these genes, ENT1 seemed the most likely to be relevant to Ara-C resistance. To investigate the role of ENT1 in Ara-C-resistant cells, we transfected the cells with the gene. ENT1-transfected Ara-C-resistant cells resembled wild-type CCRF-CEM cells more closely than untransfected Ara-C-resistant cells in terms of growth rate, Ara-C-uptake characteristics, and ADA expression levels. The down-regulation of the ENT1 gene is expected to result in nucleotide deficiency in addition to blockage of Ara-C influx. Accordingly, Ara-C-resistant cells showed low growth rates, which were restored by transfection with ENT1. These low growth rates were also correlated with the phosphorylation level of cell-cycle checkpoint kinase 2. In this study we identified down-regulation of ENT1 as the factor responsible for Ara-C resistance, and this knowledge may be used to devise a clinical regimen that will overcome the resistance.
Assuntos
Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Perfilação da Expressão Gênica , Leucemia/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Separação Celular/métodos , Regulação para Baixo , Humanos , Leucemia/genética , Fosforilação , Fatores de TempoRESUMO
Small interfering RNA (siRNA)-based therapeutics have been used in humans and offer distinct advantages over traditional therapies. However, previous investigations have shown that there are several technical obstacles that need to be overcome before routine clinical applications are used. Currently, we are launching a novel class of RNAi therapeutic agents (PnkRNA™, nkRNA) that show high resistance to degradation and are less immunogenic, less cytotoxic, and capable of efficient intracellular delivery. Here, we develop a novel platform to promote naked RNAi approaches administered through inhalation without sophisticated delivery technology in mice. Furthermore, a naked and unmodified novel RNAi agent, such as ribophorin II (RPN2-PnkRNA), which has been selected as a therapeutic target for lung cancer, resulted in efficient inhibition of tumor growth without any significant toxicity. Thus, this new technology using aerosol delivery could represent a safe, potentially RNAi-based strategy for clinical applications in lung cancer treatment without delivery vehicles.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Hexosiltransferases/genética , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/patologia , Complexo de Endopeptidases do Proteassoma/genética , RNA Interferente Pequeno/uso terapêutico , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Administração por Inalação , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/análise , Portadores de Fármacos/uso terapêutico , Humanos , Luciferases/genética , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos SCID , Sprays Nasais , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno/genética , Transplante HeterólogoRESUMO
RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-ß1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-ß1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.
Assuntos
Lesão Pulmonar Aguda/terapia , Fibrose Pulmonar/terapia , Interferência de RNA , Animais , Sequência de Bases , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Camundongos , RNA Mensageiro/genética , RNA Interferente Pequeno , Fator de Crescimento Transformador beta1/genéticaAssuntos
DNA Complementar/genética , Proteínas de Ligação ao GTP/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores de Superfície Celular/genética , Animais , Sequência de Bases , DNA Complementar/isolamento & purificação , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Genoma , Técnicas In Vitro , RatosRESUMO
An RNA synthetic method with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group allows the synthesis of long oligoribonucleotides from CEM-amidites with an efficiency and final purity comparable to that obtained in DNA synthesis. The CEM-amidites give a high coupling efficiency, because the CEM group minimizes steric hindrance in the coupling reaction. The CEM group shows satisfactory stability under solid-phase synthetic conditions, avoids the generation of asymmetric centers, and is easily cleaved to give the final product. This unit describes the synthesis of the four CEM-amidites, the preparation of reagents, the solid-phase synthesis of oligoribonucleotides on an automated DNA synthesizer, and their deprotection.
Assuntos
Oligorribonucleotídeos/síntese química , Compostos Organofosforados/síntese química , Química Orgânica/métodos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oligorribonucleotídeos/química , Compostos Organofosforados/químicaRESUMO
The pharmacokinetics and antitumor activity of pegylated small interfering RNA (siRNA)/cationic liposome complexes were studied after systemic administration to mice. We designed pegylated-lipid carriers for achieving increased plasma concentrations of RNA and hence improved accumulation of RNA in tumors by the enhanced permeability and retention effect. We compared the pharmacokinetics of siRNA complexed with liposomes incorporating pegylated lipids with longer (C-17 or C-18), shorter (C-12 to C-16), or unsaturated (C-18:1) acyl chains. When longer acyl chains were used, the plasma concentrations of siRNA obtained were dramatically higher than when shorter or unsaturated chains were used. This may be explained by the higher gel-to-liquid-crystalline phase-transition temperature (Tc) of lipids with longer acyl chains, which may form more rigid liposomes with reduced uptake by the liver. We tested a siRNA that is sequence specific for the antiapoptotic bcl-2 mRNA complexed with a pegylated liposome incorporating a C-18 lipid (PEG-LIC) by i.v. administration in a mouse model of human prostate cancer. Three-fold higher accumulation of RNA in the tumors was achieved when PEG-LIC rather than nonpegylated liposomes was used, and sequence-specific antitumor activity was observed. Our siRNA/PEG-LIC complex showed no side effects on repeated administration and the strength of its antitumor activity may be attributed to its high uptake by the tumors. Pegylation of liposomes improved the plasma retention, uptake by s.c. tumors, and antitumor activity of the encapsulated siRNA. PEG-LIC is a promising candidate for siRNA cancer therapy.
Assuntos
Lipossomos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/administração & dosagem , Animais , Sequência de Bases , Cátions , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/farmacocinéticaRESUMO
A long RNA oligomer, a 110mer with the sequence of a precursor-miRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We confirmed the identity of the synthetic 110mer by MALDI-TOF mass spectrometry, as well as HPLC, electrophoretic methods, RNase-digestion experiments, and its in vitro gene-silencing activity. The chemical synthesis of RNA oligomers of more than 100 nucleotides, which has until now been extremely difficult, can be practically realized by the CEM method.