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1.
Mol Ther Nucleic Acids ; 13: 483-492, 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30388622

RESUMO

We report the evaluation of 18-mer 2'-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5' end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.

2.
Mol Pharm ; 13(8): 2718-28, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27398779

RESUMO

Oligospermine-siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine-siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA-tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3'-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5'-end of the antisense strand abolished the activity. 2'-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3'-end of both strands and at the 5'-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine-siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5'-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine-siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine-siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5'-end of the sense strand.


Assuntos
Inativação Gênica/fisiologia , Oligonucleotídeos/química , RNA Interferente Pequeno/química , Espermina/química , Células A549 , Cátions/química , Imunofluorescência , Células HeLa , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Luciferases/genética , Luciferases/metabolismo , RNA Interferente Pequeno/fisiologia
3.
BMC Cancer ; 13: 338, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23835136

RESUMO

BACKGROUND: Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics. METHODS: We used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development. RESULTS: We confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition. CONCLUSION: PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment.


Assuntos
Ciclina B1/metabolismo , Terapia Genética/métodos , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/secundário , Melanoma Experimental/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Animais , Western Blotting , Ciclina B1/genética , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Técnicas de Transferência de Genes , Proteínas Inibidoras de Apoptose/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/secundário , Camundongos , Camundongos Nus , Polietilenoimina , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Control Release ; 170(2): 183-90, 2013 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-23727288

RESUMO

RNA interference allows the design of new inhibitors that target deregulated pathways in cancer. However systemic delivery of siRNA for the treatment of solid tumors still remains an issue. In our study, in order to suppress the progression of lung cancer metastasis in mice, we developed sticky siRNA (ssiRNA) to inhibit survivin and cyclin B1, two candidates involved in cell survival and proliferation. We exploited the linear polyethylenimine (PEI) as potent non-viral carrier to efficiently deliver our inhibitors. As a proof of concept, we have chosen a very aggressive mammary adenocarcinoma model (TSA-Luc cells), which forms lung metastases upon systemic cell injection. We confirmed in vitro, that the ssiRNAs delivered with PEI are not only able to inhibit our target genes at the mRNA and protein levels, but are also able to block the cell cycle and cell proliferation through a mechanism of RNA interference. More importantly, we showed in vivo by luciferase dosage, bioimaging and tissue section, an inhibition of lung tumor metastases after systemic delivery of cyclin B1 and survivin ssiRNA complexed with PEI. Alternating treatment with cisplatin and ssiRNA/PEI showed an additive effect between the two anticancer drugs on lung tumor inhibition leading to a significant increase in animal survival. Moreover a promising window between activity (IC50) and toxicity (LD50), essential for therapeutic application, was observed. Our data show that systemic delivery of ssiRNA/PEI complexes targeting the cell cycle is a valuable strategy for the treatment of lung tumor metastasis and that it can be combined with chemotherapy.


Assuntos
Adenocarcinoma/terapia , Ciclina B1/genética , Proteínas Inibidoras de Apoptose/genética , Neoplasias Pulmonares/terapia , Neoplasias Mamárias Experimentais/terapia , RNA Interferente Pequeno/administração & dosagem , Proteínas Repressoras/genética , Adenocarcinoma/patologia , Animais , Antineoplásicos/uso terapêutico , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/uso terapêutico , Feminino , Dose Letal Mediana , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Polietilenoimina/química , RNA Interferente Pequeno/genética , Survivina
5.
J Control Release ; 170(1): 92-8, 2013 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-23639452

RESUMO

Despite its considerable interest in human therapy, in vivo siRNA delivery is still suffering from hurdles of vectorization. We have shown recently efficient gene silencing by non-vectorized cationic siRNA. Here, we describe the synthesis and in vitro evaluation of new amphiphilic cationic siRNA. C12-, (C12)2- and cholesteryl-spermine(x)-siRNA were capable of luciferase knockdown at nanomolar concentrations without vectorization (i.e. one to two orders of magnitude more potent than commercially available cholesteryl siRNA). Moreover, incubation in the presence of serum did not impair their efficiency. Finally, amphiphilic cationic siRNA was pre-loaded on albumin. In A549Luc cells in the presence of serum, these siRNA conjugates were highly effective and had low toxicity.


Assuntos
Albuminas/farmacologia , Inativação Gênica , RNA Interferente Pequeno/administração & dosagem , Soro , Linhagem Celular Tumoral , Humanos , Lipídeos/química , Luciferases de Vaga-Lume/genética , Oligonucleotídeos/química , RNA Interferente Pequeno/química , Espermina/química
6.
Mol Pharm ; 9(12): 3464-75, 2012 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-23148419

RESUMO

siRNAs are usually formulated with cationic polymers or lipids to form supramolecular particles capable of binding and crossing the negatively charged cell membrane. However, particles hardly diffuse through tissues when administered in vivo. We therefore are developing cationic siRNAs, composed of an antisense sequence annealed to an oligophosphospermine-conjugated sense strand. Cationic siRNAs have been previously shown to display gene silencing activity in human cell line (Nothisen et al. J. Am. Chem. Soc.2009). We have improved the synthesis, purification and characterization of oligospermine-oligoribonucleotide conjugates which provide cationic siRNAs with enhanced biological activity. We show data supporting their carrier-free intracellular delivery in a molecular, soluble state. Additional results on the relationship between global charge, uptake and silencing activity confirm the requirement for an overall positive charge of the conjugated siRNA in order to enter cells. Importantly, conjugated siRNAs made of natural phosphodiester nucleotides are protected from nuclease degradation by the oligophosphospermine moiety, operate through the RNAi mechanism and mediate specific gene silencing at submicromolar concentration in the presence of serum.


Assuntos
Sistemas de Liberação de Medicamentos , Inativação Gênica , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Espermina/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Luciferases/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espermina/química , Survivina , Células Tumorais Cultivadas
7.
Angew Chem Int Ed Engl ; 51(34): 8478-84, 2012 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-22829421

RESUMO

An amphiphilic dendrimer bearing a hydrophobic alkyl chain and hydrophilic poly(amidoamine) dendrons is able to combine the advantageous features of lipid and dendrimer vectors to deliver a heat shock protein 27 siRNA and produce potent gene silencing and anticancer activity in vitro and in vivo in a prostate cancer model. This dendrimer can be used alternatively for treating various diseases.


Assuntos
Dendrímeros/administração & dosagem , Dendrímeros/química , Inativação Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Dendrímeros/síntese química , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Choque Térmico HSP27/genética , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , RNA Interferente Pequeno/genética
8.
Acc Chem Res ; 45(7): 980-4, 2012 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-22311735

RESUMO

The discovery of RNA interference has given a new lease on life to both the chemistry of oligonucleotides and chemical approaches for the intracellular delivery of nucleic acids. In particular, delivery of siRNA, whether in vitro for screening and target validation purposes or in humans as a new class of drugs, may revolutionize our approach to therapy. Their impact could equal that of the bioproduction and various uses of monoclonal antibodies today. Unfortunately, global pharmaceutical companies again seem to be waiting to buy the next Genentech or Genzyme of gene silencing rather than investing research and development into this promising area of research. Gene silencing encounters barriers similar to gene addition and hence may benefit from the extra decade of experience brought by gene therapy. "Chemical" transfection of cells in culture has become routine, and this Account discusses some of the reasons this success has not extended to nonviral gene therapy trials, most of which do not progress beyond the phase 2 stage. The author also discusses a (much debated) mechanism of nucleic acid cell entry and subsequent release of the polycationic particles into the cytoplasm. Both topics should be useful to those interested in delivery of siRNA. The move from gene therapy toward siRNA as an oligonucleotide-based therapy strategy provides a much wider range of druggable targets. Even though these molecules are a hundredfold smaller than a gene, they are delivered via similar cellular mechanisms. Their complexes with cationic polymers are less stable than those with a higher number of phosphate groups, which may be compensated by siRNA concatemerization or by chemical conjugation with the cationic carrier. Thus chemistry is again desperately needed.


Assuntos
Vetores Genéticos/metabolismo , Transfecção , Células 3T3 , Animais , Vetores Genéticos/genética , Camundongos , Polietilenoimina/química , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
9.
Bioconjug Chem ; 22(12): 2461-73, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22053932

RESUMO

With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.


Assuntos
DNA/administração & dosagem , Dendrímeros/química , Etanolaminas/química , Timo/metabolismo , Transfecção , Animais , DNA/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL
10.
Hum Gene Ther ; 21(7): 795-805, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19947826

RESUMO

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.


Assuntos
Adenoviridae/genética , Terapia Genética , Vetores Genéticos/genética , Neoplasias da Próstata/terapia , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Masculino
11.
Hum Gene Ther ; 21(7): 807-13, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20001452

RESUMO

Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.


Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Neoplasias da Próstata/terapia , Terapia Genética/tendências , Humanos , Masculino , Estadiamento de Neoplasias , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/cirurgia , Resultado do Tratamento
12.
J Am Chem Soc ; 131(49): 17730-1, 2009 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-19928854

RESUMO

siRNA-mediated gene silencing requires intracellular delivery of the nucleic acid. We have developed a carrierless molecular approach that follows the same cell entry route as cationic supramolecular complexes, yet should avoid the extracellular barriers encountered by nanoparticles. Cationic oligospermine-oligonucleotide conjugates (ZNAs, for Zip Nucleic Acids) were synthesized stepwise on an oligonucleotide synthesizer using a DMT-spermine phosphoramidite derivative. They were shown to enter cells and have access to the cytoplasm, provided their formal charge ratio N/P was >1.5. Cationic siRNAs that fulfilled this condition were shown to achieve selective inhibition of luciferase gene expression in the submicromolar concentration range in constitutively luciferase-expressing cells.


Assuntos
Inativação Gênica/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Cátions/química , Linhagem Celular , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/química , Espermina/química
13.
Nucleic Acids Res ; 37(19): e130, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19696078

RESUMO

Most nucleic acid-based technologies rely upon sequence recognition between an oligonucleotide and its nucleic acid target. With the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, novel modified oligonucleotides named Zip nucleic acids (ZNAs) were recently developed. ZNAs are oligonucleotide-oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotide. It was demonstrated that the melting temperature of a hybridized ZNA is easily predictable and increases linearly with the length of the oligocation. Furthermore, ZNAs retain the ability to discriminate between a perfect match and a single base-pair-mismatched complementary sequence. Using quantitative PCR, we show here that ZNAs are specific and efficient primers displaying an outstanding affinity toward their genomic target. ZNAs are particularly efficient at low magnesium concentration, low primer concentrations and high annealing temperatures, allowing to improve the amplification in AT-rich sequences and potentially multiplex PCR applications. In reverse transcription experiments, ZNA gene-specific primers improve the yield of cDNA synthesis, thus increasing the accuracy of detection, especially for genes expressed at low levels. Our data suggest that ZNAs exhibit faster binding kinetics than standard and locked nucleic acid-containing primers, which could explain why their target recognition is better for rare targets.


Assuntos
Primers do DNA , Reação em Cadeia da Polimerase , Transcrição Reversa , Sequência Rica em At , Reação em Cadeia da Polimerase/normas
14.
Mol Pharm ; 6(5): 1544-52, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19685893

RESUMO

Progress in the design of gene delivery systems is of utmost importance for cancer gene therapy since several physiological and intracellular barriers remain. We previously developed a technology for condensing a single gene into a single and stable globular nanometric system. In this manuscript, we have decorated the nanometric particles with cyclic RGD ligands in order to target endothelial cells. The potential of these artificial viruses as targeted gene delivery vehicles is demonstrated in vitro with alpha(v)beta(3) integrin-expressing primary endothelial cells.


Assuntos
Apresentação de Antígeno , Terapia Genética/métodos , Vetores Genéticos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Nanopartículas , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Células Endoteliais/imunologia , Humanos , Células KB , Ligantes , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Nanopartículas/ultraestrutura , Nanotecnologia , Oligopeptídeos , Vírus/genética
15.
J Am Chem Soc ; 130(40): 13500-5, 2008 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-18781743

RESUMO

Synthesis of oligonucleotide probes and control of their hybridization temperature are key aspects of polymerase chain reaction (PCR)-based detection of genetic sequences. A straightforward means to approach the last goal is to decrease the repulsion between the polyanionic probe and target strands. To this end, we have developed a versatile automated synthesis of oligonucleotide-oligospermine derivatives that gave fast access to a large variety of compounds. Plots of their hybridization temperatures T(m) vs overall charge provided a measure of the impact of interstrand phosphate repulsion (and of spermine-mediated attraction) on the main driving force of duplex formation, i.e., base pairing. It showed that stabilization brought about by excess cationic charges can be of larger absolute magnitude than interstrand repulsion, even in high salt media. Base sequence and conjugation site (3' or 5') hardly influenced the effect of spermine on T(m). In typical PCR probe conditions, the T(m) increased linearly with the number of grafted spermines (e.g., 6.2 degrees C per spermine for a decanucleotide probe). The large data set of T(m) vs number of spermines and oligonucleotide length allowed us to empirically derive a simple mathematical relation that is accurately predicting the T(m) of any oligonucleotide-oligospermine derivative. Zip nucleic acids (ZNA) are thus providing an interesting alternative to locked nucleic acids (LNA) or minor groove binders (MGB) for raising the stability of 8-12-mer oligonucleotides up to ca. 70 degrees C, the level required for quantitative PCR experiments.


Assuntos
Sondas Moleculares/química , Ácidos Nucleicos/química , Oligonucleotídeos/química , Espermina/química , Temperatura , Íons/química , Estrutura Molecular , Oligonucleotídeos/síntese química
16.
Proc Natl Acad Sci U S A ; 104(41): 16050-5, 2007 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-17913877

RESUMO

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery. We show here that making siRNAs "gene-like," via short complementary A(5-8)/T(5-8) 3' overhangs, increases complex stability, and hence RNase protection and gene silencing in vitro up to 10-fold. After decomplexation in the cytoplasm, sticky siRNA (ssiRNA) concatemers fall apart. ssiRNAs are therefore not inducing antiviral responses, as shown by the absence of IFN-beta production. Finally, transfection experiments in the mouse lung show that ssiRNA should be particularly suited to silencing with linear polyethylenimine in vivo.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Linhagem Celular , Feminino , Corantes Fluorescentes , Genes Reporter , Técnicas Genéticas , Vetores Genéticos , Glicosaminoglicanos , Células HeLa , Humanos , Lipídeos , Luciferases/genética , Pulmão/metabolismo , Camundongos , Células NIH 3T3 , Polietilenoimina , RNA Interferente Pequeno/administração & dosagem , Ribonucleases , Transfecção
17.
Nat Protoc ; 2(6): 1360-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17545974

RESUMO

A protocol for the rapid, automated synthesis of oligospermine-oligonucleotide sequences is described. To this end, a protected spermine phosphoramidite derivative was synthesized in six steps from spermine and used as the fifth synthon in an oligonucleotide synthesizer. Parameters were optimized to reach greater than 95% coupling yields. Cationic oligonucleotides show enhanced hybridization and strand invasion properties, and hence are an alternative to conventional oligonucleotides for molecular biology, diagnostic and potential therapeutic applications. A multi-gram-scale synthesis of the spermine phosphoramidite allowing several hundred coupling steps takes 2-3 weeks. Oligonucleotide synthesis and purification takes approximately 3 d.


Assuntos
Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/síntese química , Espermina/química , Estrutura Molecular , Hibridização de Ácido Nucleico , Compostos Organofosforados/química
18.
J Am Chem Soc ; 128(33): 10763-71, 2006 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-16910671

RESUMO

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems. Moreover, stability of the duplex formed with the complementary sequence increased without inhibiting oligonucleotide enzyme recognition as shown by the properties of the conjugate to prime chain elongation by Taq DNA polymerase in a linear amplification/sequencing process.


Assuntos
Oligonucleotídeos/química , Peptídeos/química , Sequência de Aminoácidos , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica
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