Oligonucleotides最新文献

{"title":"Noncovalent peptide-mediated delivery of chemically modified steric block oligonucleotides promotes splice correction: quantitative analysis of uptake and biological effect.","authors":"Sandra D Laufer,&nbsp;Anna Lena Recke,&nbsp;Sandra Veldhoen,&nbsp;Alexander Trampe,&nbsp;Tobias Restle","doi":"10.1089/oli.2008.0160","DOIUrl":"https://doi.org/10.1089/oli.2008.0160","url":null,"abstract":"<p><p>Despite numerous encouraging reports in the literature, the efficiency of cell penetrating peptides (CPPs) in promoting cellular delivery of bioactive cargos is still limited. To extend our current understanding of the underlying limitations of such approaches, we performed quantitative uptake studies of different chemically modified (2'-O-methyl, LNA and PNA) steric block oligonucleotides, targeted against a mutated splice site inserted in a firefly luciferase reporter gene construct, applying the peptide carrier MPGalpha as a model system. The peptide formed stable noncovalent complexes with phosphorothioate oligonucleotide (PTO) and locked nucleic acid (LNA) modified oligonucleotides, whereas the neutral peptide nucleic acid (PNA) had to be hybridized to an unmodified DNA to allow for complex formation. Detailed quantitative uptake studies revealed comparable numbers of intracellular PTO and LNA oligonucleotides after peptide-mediated delivery. Surprisingly, the PTO derivative showed the strongest upregulation of reporter gene activity of about 100-fold followed by the PNA (40-fold) and LNA (10-fold). Electroporation and microinjection studies proved that delivery itself was not the limiting factor for the low activity of the LNA derivative. Maximal achievable reporter gene activity could be observed only after addition of chloroquine (CQ), indicative of an endosomal pathway involved. This is in line with nuclear microinjection experiments, which show that the minimal number of steric block molecules needed to trigger the observed reporter upregulation is about two orders of magnitude lower than determined after peptide or cationic lipid delivery.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1","pages":"63-80"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0160","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27968551","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}

{"title":"Knockdown of caveolin-1 by siRNA inhibits the transformation of mouse hepatoma H22 cells in vitro and in vivo.","authors":"Shujing Wang,&nbsp;Li Jia,&nbsp;Huimin Zhou,&nbsp;Wei Shi,&nbsp;Jianing Zhang","doi":"10.1089/oli.2008.0166","DOIUrl":"https://doi.org/10.1089/oli.2008.0166","url":null,"abstract":"<p><p>Caveolin-1 (Cav-1) is a main structural protein of caveolae and plays important roles in signal transduction and tumorigenesis. We previously showed that Cav-1 was highly expressed in mouse hepatoma cell lines and positively correlated with cell invasion capability. Thus, interfering with the expression and activity of Cav-1 might be a potential way to intervene with hepatoma progression. We used RNA interference to study the biological effects of silencing Cav-1 expression in hepatoma H22 cells, to validate its potential as a therapeutic target. Using small-interfering RNAs (siRNAs) targeting the mRNA region of Cav-1, we effectively suppressed Cav-1 mRNA and protein levels. This resulted in the decreased transformation ability of H22 cells in vitro and in vivo. In addition, downregulation of Cav-1 expression promoted the apoptosis of H22 cells in vitro and in vivo. These results suggest that the use of siRNA could be an efficient molecular therapeutic method for hepatoma with high expression of Cav-1.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1","pages":"81-8"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0166","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27968549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct CNS delivery of siRNA mediates robust silencing in oligodendrocytes.","authors":"William Querbes,&nbsp;Pei Ge,&nbsp;Wenjun Zhang,&nbsp;Yupeng Fan,&nbsp;Jason Costigan,&nbsp;Klaus Charisse,&nbsp;Martin Maier,&nbsp;Lubomir Nechev,&nbsp;Muthiah Manoharan,&nbsp;Victor Kotelianski,&nbsp;Dinah W Y Sah","doi":"10.1089/oli.2008.0165","DOIUrl":"https://doi.org/10.1089/oli.2008.0165","url":null,"abstract":"<p><p>The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery in vivo. The majority of reported studies describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1","pages":"23-29"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0165","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27909605","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}

{"title":"Therapeutic potential of splice-switching oligonucleotides.","authors":"John A. Bauman, Natee Jearawiriyapaisarn, R. Kole","doi":"10.1089/oli.2008.0161","DOIUrl":"https://doi.org/10.1089/oli.2008.0161","url":null,"abstract":"Alternative splicing enables a single pre-messenger RNA transcript to yield multiple protein isoforms, making it a major contributor to the diversity of the proteome. While this process is essential for normal development, aberrations in alternative splicing are the cause of a multitude of human diseases. Methods for manipulating alternative splicing would thus be of therapeutic value. Chemically modified antisense oligonucleotides that alter alternative splicing by directing splice site selection have been developed to achieve this end. These splice-switching oligonucleotides (SSOs) have been applied to correct aberrant splicing, induce expression of a therapeutic splice variant, or induce expression of a novel therapeutic splice variant in a number of disease-relevant genes. Recently, in vivo efficacy of SSOs has been reported using animal disease models, as well as in results from the first clinical trial.","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1 1","pages":"1-13"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0161","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60513899","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}

{"title":"Graft protection in bypass surgery: siRNA-mediated silencing of adhesion molecules.","authors":"T. Walker, H. Wendel, Claudia Raabe, Patrycja Wiechnik, L. Spranger, O. Heidenreich, A. Scheule, A. Nordheim, G. Ziemer","doi":"10.1089/oli.2008.0148","DOIUrl":"https://doi.org/10.1089/oli.2008.0148","url":null,"abstract":"The outcome of patients after coronary bypass grafting is greatly influenced by the type of graft material employed, especially regarding the rate of graft restenosis. Besides direct thrombotic events, the leukocyte-endothelial interaction modulated by adhesion molecules is identified to be the central cause leading to graft alterations. This study deals with a new therapeutic concept in order to achieve superior protection of a new bypass graft by blocking the adhesion molecule expression pathway with RNA interference to inhibit the initial leukocyte adhesion and transmigration. Leukocyte binding to adhesion molecules on activated human venous endothelial cells (HVECs) was determined by video-assisted microscopy in a flow chamber mimicking physiological conditions. The cells under study were sequentially transfected in a nonviral manner with specific short interfering RNA-sequences (siRNA) targeting E-selectin, intercellular adhesion molecule, and vascular adhesion molecule. After stimulation of adhesion molecule expression by tumor necrosis factor, a leukocyte-rich suspension was run through the chamber and the attaching leukocytes were counted. Transfection with specific siRNA targeting three different adhesion molecules resulted in a highly significant reduction of leukocyte attachment to activated HVECs in each case compared to the controls (p < 0.05). Transfection with a mixture out of all three siRNA-sequences showed the lowest leukocyte adhesion (p < 0.05) compared to the controls. siRNA-sequences inhibit the adhesion molecule expression on HVECs in an extremely effective way; not only in a single transfection of specific molecules but also in a parallel transfection of multiple sequences in one transfection. Accordingly, siRNA treatment significantly reduced adhesion of leukocyte cells to HVECs compared to controls. This study showed for the first time an effective knockdown of the leukocyte-endothelium interactions by transfection of HVECs with a cocktail consisting of three highly specific siRNAs against three different endothelial adhesion molecules.","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1 1","pages":"15-21"},"PeriodicalIF":0.0,"publicationDate":"2009-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0148","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60513806","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}

{"title":"Inhibition of human cancer-cell proliferation by long double-stranded RNAs.","authors":"I. A. Akimov, T. Kabilova, V. Vlassov, E. Chernolovskaya","doi":"10.1089/oli.2008.0151","DOIUrl":"https://doi.org/10.1089/oli.2008.0151","url":null,"abstract":"Three different enzymatically synthesized long double-stranded RNAs (dsRNAs) [448 bp homologous to the third exon of c-myc messenger RNA (mRNA) (dsMyc); 473 bp homologous to enhanced green fluorescent protein (EGFP) mRNA (dsEGFP) and control interferon inducer poly(I:C)] were studied for antiproliferative and gene-silencing activities in KB-3-1, SK-N-MC, and IMR-32 human cancer cell lines. Simple incubation with these dsRNAs did not affect the expression of c-myc gene and the proliferation of KB-3-1 and IMR-32 cells, but inhibited the proliferation of SK-N-MC cells. Transfection of KB-3-1 and SK-N-MC cells using Oligofectamine-dsRNAs complexes resulted in dose-dependent inhibition of c-myc and beta-actin genes expression and proliferation. The data show that dsMyc, acting both as interferon inducer and as gene-specific interfering RNA, is more effective as c-myc inhibitor than other tested dsRNAs. The most efficient inhibition of proliferation was displayed by dsEGFP RNA, dsMyc and poly(I:C) were effective only when used in higher concentrations. Our data indicate that transfection of studied dsRNAs causes an increase in apoptotic and dead cells number in the cell population. This proapoptotic activity correlates with dsRNAs-induced antiproliferative activity. However the difference in cell growth between dsRNA-treated and Oligofectamine-only treated cells can not be attributed only to the loss of cells due to the apoptosis; it also indicates some retardation of cell cycle progression caused by dsRNA.","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1 1","pages":"31-40"},"PeriodicalIF":0.0,"publicationDate":"2009-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0151","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60513886","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}

{"title":"Chemical modification of siRNAs for in vivo use.","authors":"Mark A Behlke","doi":"10.1089/oli.2008.0164","DOIUrl":"https://doi.org/10.1089/oli.2008.0164","url":null,"abstract":"<p><p>Well over a hundred reports have been published describing use of synthetic small-interfering RNAs (siRNAs) in animals. The majority of these reports employed unmodified RNA duplexes. While unmodified RNA is the natural effector molecule of RNA interference, certain problems arise with experimental or therapeutic use of RNA duplexes in vivo, some of which can be improved or solved through use of chemical modifications. Judicious use of chemical modifications can improve the nuclease stability of an RNA duplex, decrease the likelihood of triggering an innate immune response, lower the incidence of off-target effects (OTEs), and improve pharmacodynamics. This review will examine studies that document the utility of various chemical modifications for use in siRNAs, both in vitro and in vivo, with close attention given to reports demonstrating actual performance in animal model systems.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"18 4","pages":"305-19"},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0164","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27852573","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}

{"title":"Enhanced cytokine secretion owing to multiple CpG side chains of DNA duplex.","authors":"Jusaku Minari,&nbsp;Shinichi Mochizuki,&nbsp;Kazuo Sakurai","doi":"10.1089/oli.2008.0145","DOIUrl":"https://doi.org/10.1089/oli.2008.0145","url":null,"abstract":"<p><p>Unmethylated CpG sequences (CpG ODN) stimulate Toll-like receptor 9 (TLR9) to activate innate immunity. We made DNA duplexes from poly(dT)320 and CpG ODN with (dA)40 attached at the 3' end. Circular dichroism and gel electrophoresis indicated that the CpG parts turned outward from the duplex. When we changed the CpG ODN/poly(dT) molar ratio, the amount of IL-12 secreted from J774A.1 cells (murine macrophage-like) reached the maximum at the compositions with two to four CpG portions in one duplex, while the maximum loading was eight CpG ODNs per one poly(dT)320. When the residual free dT parts were hybridized with its control GpC ODN with (dA)40 tail or just (dA)40, the maximum disappeared and the secretion increased with increasing the CpG molar ratio. These results indicated that there is a particular DNA higher-order structure to activate TLR9 more efficiently than single CpG ODN.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"18 4","pages":"337-44"},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0145","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27719918","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}

{"title":"Silencing DNA methyltransferase (DNMT) enhances glioma chemosensitivity.","authors":"Amal Shervington,&nbsp;Rahima Patel","doi":"10.1089/oli.2008.0128","DOIUrl":"https://doi.org/10.1089/oli.2008.0128","url":null,"abstract":"<p><p>Previously, we demonstrated that demethylation with 5-Aza-2'-deoxycytidine (5azadC) resulted in reduced levels of telomerase that led to telomere shortening, enhanced MGMT expression and enhanced chemosensitivity. Although the results were encouraging, the fact that 5azadC is highly toxic and nonspecific, thus is not favored as a therapeutic molecule. The aim of this research is to downregulate the DNA methyltransferase (DNMT1) gene using three sets of double-stranded RNA oligos designed to align different regions of DNMT1 sequence. Results showed the small-interfering RNA (siRNA) 1 and 3 demonstrated significant levels of silencing DNMT1 and hTERT transcription after 24-hour treatment (p = 0.01) and approximately 90% and 70% transcriptional downregulation of DNMT1 and hTERT, respectively after 48 hours. However, siRNA 2 downregulated DNMT1, hTERT, and MGMT in GOS-3 and U87-MG cells that was attributed to sequence homology between oligo 2 and MGMT complementary DNA. The siRNA-treated glioma cell lines GOS-3 and U87-MG were subjected to two chemotherapeutic agents; taxol and Temozolomide (TMZ). Results suggest that either a combination of siRNA 1 or 3 followed by taxol (2-6 muM) after 48 hours or a combination of siRNA 1 or 3 followed by TMZ (600-1000 microM) after 24 hours would be novel and effective glioma therapies.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"18 4","pages":"365-74"},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0128","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27798548","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}

{"title":"Label-free electrochemical detection of short sequences related to the hepatitis B virus using 4,4'-diaminoazobenzene based on multiwalled carbon nanotube-modified GCE.","authors":"Xue-Mei Li,&nbsp;Zhi-Ming Zhan,&nbsp;Heng-Qiang Ju,&nbsp;Shu-Sheng Zhang","doi":"10.1089/oli.2008.0143","DOIUrl":"https://doi.org/10.1089/oli.2008.0143","url":null,"abstract":"<p><p>A novel label-free electrochemical DNA biosensor based on 4,4'-diaminoazobenzene (4,4'-DAAB) and multiwalled carbon nanotube (MWNT)-modified glassy carbon electrode (GCE) for short DNA sequences related to the hepatitis B virus (HBV) hybridization detection was presented. Differential pulse voltammetry (DPV) was used to investigate hybridization event. The decrease in the peak current of 4,4'-DAAB was observed on hybridization of probe with the target. This electrochemical approach was sequence specific as indicated by the control experiments, in which no peak current change was observed when a noncomplementary DNA sequence was used. Numerous factors affecting the target hybridization were optimized to maximize the sensitivity. Under optimal conditions, this sensor showed a good calibration range between 7.94 x 10(-8) M and 1.58 x 10(-6) M, with HBV DNA sequence detection limit of 1.1 x 10(-8) M.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"18 4","pages":"321-7"},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0143","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27798546","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}