Antisense & nucleic acid drug development最新文献_第4页

Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. 细胞依赖的PNA、多肽和PNA-肽偶联物的细胞摄取差异。

Antisense & nucleic acid drug development Pub Date : 2002-04-01 DOI: 10.1089/108729002760070795

U. Koppelhus, S. Awasthi, V. Zachar, H. U. Holst, P. Ebbesen, P. Nielsen

{"title":"Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates.","authors":"U. Koppelhus, S. Awasthi, V. Zachar, H. U. Holst, P. Ebbesen, P. Nielsen","doi":"10.1089/108729002760070795","DOIUrl":"https://doi.org/10.1089/108729002760070795","url":null,"abstract":"Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"149 1","pages":"51-63"},"PeriodicalIF":0.0,"publicationDate":"2002-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83812026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 198

Antisense-mediated inhibition of ICAM-1 expression: a therapeutic strategy against inflammation of human periodontal tissue. 反义介导的ICAM-1表达抑制:对抗人类牙周组织炎症的治疗策略。

Antisense & nucleic acid drug development Pub Date : 2002-04-01 DOI: 10.1089/108729002760070812

W. Nedbal, P. Tomakidi, M. J. Lehmann, C. Dörfer, A. Kohl, G. Sczakiel

{"title":"Antisense-mediated inhibition of ICAM-1 expression: a therapeutic strategy against inflammation of human periodontal tissue.","authors":"W. Nedbal, P. Tomakidi, M. J. Lehmann, C. Dörfer, A. Kohl, G. Sczakiel","doi":"10.1089/108729002760070812","DOIUrl":"https://doi.org/10.1089/108729002760070812","url":null,"abstract":"Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa. Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective. In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues. The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses. Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis. Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin. Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases. This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"80 1","pages":"71-8"},"PeriodicalIF":0.0,"publicationDate":"2002-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88272425","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 14

Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. 寡核苷酸缀合物作为潜在的反义药物，具有改善的摄取、生物分布、靶向递送和作用机制。

Antisense & nucleic acid drug development Pub Date : 2002-04-01 DOI: 10.1089/108729002760070849

M. Manoharan

引用次数: 175

Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions. 通过环环RNA相互作用将发夹核酶锚定在长靶RNA上。

Antisense & nucleic acid drug development Pub Date : 2002-02-01 DOI: 10.1089/108729002753670210

Elena Puerta-Fernández, A. Barroso-deljesús, A. Berzal-Herranz

{"title":"Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions.","authors":"Elena Puerta-Fernández, A. Barroso-deljesús, A. Berzal-Herranz","doi":"10.1089/108729002753670210","DOIUrl":"https://doi.org/10.1089/108729002753670210","url":null,"abstract":"Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"8 1","pages":"1-9"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88878182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells. HeLa细胞中亲脂性前寡核苷酸的摄取和细胞内浓度的定量。

Antisense & nucleic acid drug development Pub Date : 2002-02-01 DOI: 10.1089/108729002753670247

J. Bologna, E. Vivés, J. Imbach, F. Morvan

引用次数: 24

Phosphodiester oligonucleotides inhibit mitosis and trigger apoptosis by a non-antisense, p53-mediated mechanism. 磷酸二酯寡核苷酸通过非反义p53介导的机制抑制有丝分裂并引发细胞凋亡。

Antisense & nucleic acid drug development Pub Date : 2002-02-01 DOI: 10.1089/108729002753670238

L. Papucci, N. Schiavone, M. Donnini, A. Lapucci, E. Luzi, A. Tempestini, E. Witort, S. Capaccioli

{"title":"Phosphodiester oligonucleotides inhibit mitosis and trigger apoptosis by a non-antisense, p53-mediated mechanism.","authors":"L. Papucci, N. Schiavone, M. Donnini, A. Lapucci, E. Luzi, A. Tempestini, E. Witort, S. Capaccioli","doi":"10.1089/108729002753670238","DOIUrl":"https://doi.org/10.1089/108729002753670238","url":null,"abstract":"Oligodeoxyribonucleotides (ODNs) are currently employed to switch-off genes selectively routinely in the laboratory practice. The drawback of ODN application is that they have been often reported to elicit non-antisense effects by different mechanisms. Recently, it has been shown that double-stranded DNA oligonucleotides (30-mers) with protruding ends activate p53 in a cell-free system. In a previous work, we described that simple addition to the culture medium of heterogeneous DNA combined with cationic lipids culminated in inhibition of mitosis and induction of apoptosis. Here, we report that the same effects are achieved by lipotransfecting cultured cells with phosphorodiester ODNs (30-mers). Such effects of ODN were mediated by a non-antisense mechanism that required the wild-type form of the p53 oncosuppressor protein and was dependent on ODN concentration. Mitosis inhibition and apoptosis induction appeared to be determined by the 3' and 5' free ends of ODNs, which activated p53 independently from their sequence. Most probably, this mechanism is analogous to that evoked by genotoxic agent-induced DNA damage or by lipotransfecting cells with heterogeneous DNA.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"73 1","pages":"21-31"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78098390","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size. c-Raf反义硫代寡核苷酸的体内促凋亡和抗肿瘤作用:与肿瘤大小的关系。

Antisense & nucleic acid drug development Pub Date : 2002-02-01 DOI: 10.1089/108729002753670229

Q. Lau, T. Achenbach, Oliver Borchers, R. Müller, E. Slater

{"title":"In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size.","authors":"Q. Lau, T. Achenbach, Oliver Borchers, R. Müller, E. Slater","doi":"10.1089/108729002753670229","DOIUrl":"https://doi.org/10.1089/108729002753670229","url":null,"abstract":"Previously, we have shown that a phosphorothioate antisense oligonucleotide (ODN) targeted against c-raf RNA (ISIS5132; cRaf-AS) induces apoptosis in human tumor cells. We now show that the same ODN also efficiently triggers apoptosis in human tumor xenografts in nu/nu mice. Although cRaf-AS showed a clearly inhibitory effect on the growth of established tumors (approximately 150 mm3) compared to a mismatched control ODN (MM), tumor progression was not prevented. This correlated with a partial refractoriness of the tumor to cRaf-AS-induced cell killing, which seemed to be due to an inhomogeneous and inefficient penetration of the ODN into the tumor tissue rather than cellular resistance. In agreement with this conclusion, we found that growth of small tumors (<50 mm3) was completely inhibited concomitantly with an accumulation of the ODN throughout the tumor. These data show that the cRaf-AS is a highly efficacious antitumor agent, provided accessibility into the tumor tissue is warranted, and suggest that PS-AS-ODN treatment may be particularly useful in an adjuvant setting.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"1 1","pages":"11-20"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88206360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Sequence-specific gene cleavage in intact mammalian cells by 125I-labeled triplex-forming oligonucleotides conjugated with nuclear localization signal peptide. 用125i标记的与核定位信号肽结合的三聚体寡核苷酸在完整哺乳动物细胞中进行序列特异性基因切割。

Antisense & nucleic acid drug development Pub Date : 2002-02-01 DOI: 10.1089/108729002753670256

O. Sedelnikova, Valeri N. Karamychev, I. Panyutin, Ronald D. Neumann

{"title":"Sequence-specific gene cleavage in intact mammalian cells by 125I-labeled triplex-forming oligonucleotides conjugated with nuclear localization signal peptide.","authors":"O. Sedelnikova, Valeri N. Karamychev, I. Panyutin, Ronald D. Neumann","doi":"10.1089/108729002753670256","DOIUrl":"https://doi.org/10.1089/108729002753670256","url":null,"abstract":"Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a \"ballast\" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the \"ballast\" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the \"ballast.\" We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"4 1","pages":"43-9"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90162508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 34

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites. 在特定位点含有5-氮胞嘧啶残基的DNA(胞嘧啶- c5)甲基转移酶寡核苷酸抑制剂的合成。

Antisense & nucleic acid drug development Pub Date : 2001-12-01 DOI: 10.1089/108729001753411335

Ramon Güimil García, A. Brank, J. Christman, V. Marquez, R. Eritja

引用次数: 17

A method to select chemically modified aptamers directly. 一种直接选择化学修饰适配体的方法。

Antisense & nucleic acid drug development Pub Date : 2001-12-01 DOI: 10.1089/108729001753411344

C. Boiziau, J. Toulmé

{"title":"A method to select chemically modified aptamers directly.","authors":"C. Boiziau, J. Toulmé","doi":"10.1089/108729001753411344","DOIUrl":"https://doi.org/10.1089/108729001753411344","url":null,"abstract":"In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"205 1","pages":"379-85"},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77093273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 12