Survivin反义核酸结合位点的体外筛选及鉴定

合成 2 0mer随机寡核苷酸文库 ,与体外转录出的全长survivincRNA杂交 ,RNaseH酶切割后 ,经引物延伸、放射自显影 ,共筛选出 13个针对survivin基因的反义结合位点 (antisenseaccessiblesites ,AAS) .运用RNADraw软件分析、选定具有显著茎环结构的 4个位点 ,合成互补性反义寡核苷酸AS ODN1、AS ODN2 、AS ODN3 、AS ODN4并转染高表达survivin基因的胃癌细胞株MKN 4 5 .逆转录聚合酶链反应和Western印迹检测发现MKN 4 5细胞的survivinmRNA和蛋白水平均有显著的下降 ;MTT比色法证实 6 0 0nmol LAS ODN1～AS ODN4转染 2 4h后细胞生长受到明显抑制 ,透射电镜、annexinⅤ FITC和PI双染色流式细胞术均检测到细胞凋亡 .说明运用随机寡核苷酸文库 RNaseH酶切割与计算机分析相结合的方法 ,在体外有效筛选出survivin的反义核酸结合位点 ,其相应的反义寡核苷酸能阻断survivin基因的生物学功能 .     

Rapid turnover of the "functional" Na(+)-Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach

Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na(+)-Ca2+ exchanger (NCX1) to correlate suppression of the Na(+)-Ca2+ exchange function with down-regulation of NCX1 protein expression. In a de-novo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in whole-cell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.     

Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.     

Post-transcriptional silencing of Notch2 mRNA in chronic lymhocytic leukemic cells of B-CLL patients

Environmental and genomic stresses induce different pathological conditions and one of them is blood cancer. This escalating load of disease with a constant threat to life requires an intensive comprehensive response. For our understanding about the cancer treatment capabilities, novel medicinal platforms should be strived to explore among the existing conventional and molecular approaches that have already been proven to be successful in fighting against genetic diseases. Several DNA therapeutics previously studied are currently in clinical settings. RNA interfering antisense oligonucleotide (AS-ODN) is the most experimentally advanced molecular therapeutic which has the potential to modify the gene activity resulting in the down regulation of particular protein. In this study, we focused on the inhibition of Notch2 function in B-cell chronic lymphocytic leukemia (B-CLL) by AS-ODN (phosphorothioate oligomers) targeted to the initiation codon region of the Notch2 mRNA. We investigated the in vitro ability of four such oligomers to reduce the expression of Notch2 gene in peripheral blood mononuclear cells from B-CLL patients. Our findings implicate that AS-ODNs specifically designed for the region of 314–333 neucleotides (AS1) of Notch2 inhibits its gene expression better than other AS-ODNs designed for other regions and respond in a dose dependent manner. The results of cell proliferation assay for the evaluation of AS1 in gene silencing, infer that the number of cells were reduced to 80% (P < 0.001). Our results implicate that using the AS-ODNs against specific Notch2 nucleotide sequence can be used as future therapeutic agent with the ability of Notch2 down regulation, which is the root problem in the pathogenicity of B-CLL.     

Controlled delivery of antisense oligodeoxynucleotide from cationically modified phosphorylcholine polymer films

Antisense strategy is a promising approach for the prevention of in-stent restenosis if therapeutic agents such as antisense oligodeoxynucleotides (AS-ODNs) can be successfully delivered to the implant site. Optimizing the routes and conditions for controlled loading and release of therapeutic agents from a biocompatible polymer coating is still required. In this study, phosphorylcholine (PC) polymer films bearing different cationic charge densities were deposited onto smooth silicon substrates. The thickness of these films was determined by spectroscopic ellipsometry (SE). Human c-myc AS-ODNs were incorporated into the PC polymer films by immersion in concentrated AS-ODN solution and eluted into PBS under physiological conditions. The elution profile was monitored by UV spectrometry and gel electrophoresis. Cellular uptake of the eluted AS-ODN into vascular smooth muscle cells (VSMCs) was evaluated by fluorescence microscopy. The results showed that ODN loading capacities increased with film thickness and were also strongly dependent on the cationic charge density. AS-ODN release was characterized by a slight initial burst in the first half hour followed by a period of sustained release up to 8 days. Gel electrophoresis demonstrated DNA integrity, and different transfection efficiencies were observed when the eluted ODNs were transfected into VSMCs. These results demonstrated that cationically modified PC polymers are capable of delivery of antisense ODNs in a controlled manner and that they are well suited for specific biomedical devices such as DNA-eluting stents.     

Effects of antisense oligodeoxynucleotides targeting CCR3 on the airway response to antigen in rats

Asthma is characterized by airway hyperresponsiveness (AHR) and inflammation, consisting predominantly of eosinophils within the airway lumen and walls. Eosinophil recruitment to the airways is mediated mainly by eotaxin and other chemokines that bind to the CC-chemokine receptor-3 (CCR3), which is highly expressed on eosinophils. This study assessed whether topical inhibition of CCR3 mRNA expression by phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) modifies pulmonary eosinophilia and AHR in an antigen-induced allergic asthma model in Brown Norway (BN) rats. Results show that specific inhibition of CCR3 expression in the lungs by an AS-ODN (AS4) reduced total eosinophil infiltration and the percentage of eosinophils into the airways of ovalbumin challenged rats. Moreover, reduction in CCR3 mRNA levels was correlated with a decrease in CCR3 protein in lung tissue. In addition, AS4 treatment had no effect on circulating eosinophils or on eosinophils in the bone marrow. Finally, AHR was significantly decreased in AS4-treated rats when compared with rats treated with a mismatch AS-ODN. In conclusion, inhibition of the expression of CCR3 decreased pulmonary eosinophilia and reduced AHR after antigen challenge in rats. Topical inhibition of CCR3 expression, using an AS-ODN, could represent a novel approach for the treatment of asthma.     

Volatility in mRNA secondary structure as a design principle for antisense

Designing effective antisense sequences is a formidable problem. A method for predicting efficacious antisense holds the potential to provide fundamental insight into this biophysical process. More practically, such an understanding increases the chance of successful antisense design as well as saving considerable time, money and labor. The secondary structure of an mRNA molecule is believed to be in a constant state of flux, sampling several different suboptimal states. We hypothesized that particularly volatile regions might provide better accessibility for antisense targeting. A computational framework, GenAVERT was developed to evaluate this hypothesis. GenAVERT used UNAFold and RNAforester to generate and compare the predicted suboptimal structures of mRNA sequences. Subsequent analysis revealed regions that were particularly volatile in terms of intramolecular hydrogen bonding, and thus potentially superior antisense targets due to their high accessibility. Several mRNA sequences with known natural antisense target sites as well as artificial antisense target sites were evaluated. Upon comparison, antisense sequences predicted based upon the volatility hypothesis closely matched those of the naturally occurring antisense, as well as those artificial target sites that provided efficient down-regulation. These results suggest that this strategy may provide a powerful new approach to antisense design.     

Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry

Alteration of gene expression by use of antisense oligonucleotides has considerable potential for therapeutic purposes and scientific studies. Although applied for almost 25 years, this technique is still associated with difficulties in finding antisense-effective regions along the target mRNA. This is mainly due to strong secondary structures preventing binding of antisense oligonucleotides and RNase H, playing a major role in antisense-mediated degradation of the mRNA. These difficulties make empirical testing of a large number of sequences complementary to various sites in the target mRNA a very lengthy and troublesome procedure. To overcome this problem, more recent strategies to find efficient antisense sites are based on secondary structure prediction and RNase H-dependent mechanisms. We were the first who directly combined these two strategies; antisense oligonucleotides complementary to predicted unpaired target mRNA regions were designed and hybridized to the corresponding RNAs. Incubation with RNase H led to cleavage of the RNA at the respective hybridization sites. Analysis of the RNA fragments by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which has not been used in this context before, allowed exact determination of the cleavage site. Thus the technique described here is very promising when searching for effective antisense sites.     

RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts

The use of antisense oligodeoxyribonucleotides (ODN) or ribozymes to specifically suppress gene expression is simple in concept and relies on efficient binding of the antisense strand to the target RNA. Although the identification of target sites accessible to base pairing is gradually being overcome by different techniques, it remains a major problem in the antisense and ribozyme approaches. In this study we have investigated the potential of a recent experimental and theoretical approach to predict the local accessibility of murine DNA-methyltransferase (MTase) mRNA in a comparative way. The accessibility of the native target RNA was probed with antisense ODN in cellular extracts. The results strongly correlated with the theoretically predicted target accessibility. This work suggests an effective two-step procedure for predicting RNA accessibility: first, computer-aided selection of ODN binding sites defined by an accessibility score followed by a more detailed experimental procedure to derive information about target accessibility at the single nucleotide level.     

TargetFinder: a software for antisense oligonucleotide target site selection based on MAST and secondary structures of target mRNA

SUMMARY: TargetFinder is a PC/Windows program for interactive effective antisense oligonucleotide (AO) selection based on mRNA accessible site tagging (MAST) and secondary structures of target mRNA. To make MAST result intuitive, both the alignment result and tag frequency profile is illustrated. As theoretical reference, secondary structure and single strand probability profile of target mRNA is also represented. All of these sequences and profiles are displayed in aligned mode, which facilitates identification of the accessible sites in target mRNA. Graphical, user-friendly interface makes TargetFinder a useful tool in AO target site selection. AVAILABILITY: The software is freely available at http://www.bioit.org.cn/ao/targetfinder.htm CONTACT: sqwang@nic.bmi.ac.cn.     

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond

Single-stranded regions in RNA secondary structure are important for RNA–RNA and RNA–protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals ‘well-determined’ single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit β-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA–RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.     

Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay. Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression.

Antisense oligodeoxynucleotides (AS-ODNs) are frequently used for the down-regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness, fast screening methods for the selection of effective AS-ODNs are needed. We describe a new cellular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of the gene of interest and the gene encoding the enhanced green fluorescent protein (EGFP) are cotransfected with AS-ODNs to COS-7 cells. Subsequently, cells are analysed for expression of the EGFP fusion protein by flow cytometry. With the assay, we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S-transferase Mu1 (GSTM1) and/or Mu2 (GSTM2). We found several AS-ODNs that demonstrated potent, sequence-specific, and concentration-dependent inhibition of fusion protein expression. At 0.5 microm, AS-6 and AS-8 inhibited EGFP-GSTM1 expression by 95 +/- 4% and 81 +/- 6%, respectively. AS-5 and AS-10 were selective for GSTM2 (82 +/- 4% and 85 +/- 0.4% decrease, respectively). AS-2 and AS-3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77-95% decrease). Other AS-ODNs were not effective or displayed non-target-specific inhibition of protein expression. The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity. As isoform-selective, chemical inhibitors of GSTM and GSTM knock-out mice are presently unavailable, the selected AS-ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protection against chemical-induced carcinogenesis.     

Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs.     

Oligonucleotide-based inhibition of embryonic gene expression

We describe a technique to define gene function using antisense oligonucleotide (AS-ODN) inhibition of gene expression in mice. A single intravenous injection of an AS-ODN targeting vascular endothelial growth factor (VEGF) into pregnant mice between E7.5-8.5 resulted in a lack of primary angiogenesis. This enabled us to define the critical window required to inhibit VEGF expression and recapitulate the primary loss of function phenotype observed in VEGF (-/-) embryos. This phenotype was sequence-specific and time- and dose-dependent. Injection of an AS-ODN targeting a second gene, E-cadherin, into pregnant mice at E10 confirmed a hypothesized secondary phenotype. This is the first report of AS-ODN inhibition of gene expression in utero and provides a new strategy for target validation in functional genomics.     

Trafficking of intracerebroventricularly injected antisense oligonucleotides in the mouse brain

Intracerebroventricular (icv) delivery of therapeutic molecules directly into the brain parenchyma has attracted considerable attention because of the advantage of bypassing the blood-brain barrier. Exogenous icv administration of antisense oligodeoxynucleotides (AS-ODNs) has been implicated in modifying gene expression within the targeted brain area. The biodistribution, tissue penetration, and stability of exogenously administered AS-ODNs are the major determinants with regard to their potential utility as agents for modifying gene expression. This report examined the distribution and clearance of labeled AS-ODNs with the aim of exploring the feasibility of icv administration of AS-ODNs as a targeted treatment approach to Alzheimer's disease. A single icv injection of fluorescein-labeled 2'-O-(methoxy) ethyl (2'MOE) ribosyl-modified AS-ODNs directed at the beta-secretase cleavage site of beta-amyloid precursor protein (APP) mRNA into the mouse brain showed rapid uptake by 15 minutes, overall gradual spread and retention by 30 minutes to 3 hours, and complete clearance by 8 hours postinjection. Labeled AS-ODNs were observed to penetrate across the cell membrane and accumulate in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cell populations. Current study provides a basic pattern of uptake, distribution, and stability of AS-ODNs in the mouse brain.     

mRNA靶点筛选方法研究进展

mRNA靶点筛选问题是反义核酸领域的一个难题。近年来出现了多种筛选mRNA上可接近位点以确定靶位点的方法,包括mRNA实测分析法和计算机模拟分析两大类。其中mRNA实测分析法又包括多种针对自然折叠mRNA的实验分析技术;即基因walk技术,RNaseH作图技术、寡核苷酸微阵列技术,酶作图法确定二级结构技术,核酶导向型随机RNA库位点筛选技术和随机寡核苷酸库结合逆转录位点筛选技术。这些方法在鉴定RNA可接近位点及反义核酸的设计方面均有重要作用。     

Fundamental differences in the equilibrium considerations for siRNA and antisense oligodeoxynucleotide design

Both siRNA and antisense oligodeoxynucleotides (ODNs) inhibit the expression of a complementary gene. In this study, fundamental differences in the considerations for RNA interference and antisense ODNs are reported. In siRNA and antisense ODN databases, positive correlations are observed between the cost to open the mRNA target self-structure and the stability of the duplex to be formed, meaning the sites along the mRNA target with highest potential to form strong duplexes with antisense strands also have the greatest tendency to be involved in pre-existing structure. Efficient siRNA have less stable siRNA–target duplex stability than inefficient siRNA, but the opposite is true for antisense ODNs. It is, therefore, more difficult to avoid target self-structure in antisense ODN design. Self-structure stabilities of oligonucleotide and target correlate to the silencing efficacy of siRNA. Oligonucleotide self-structure correlations to efficacy of antisense ODNs, conversely, are insignificant. Furthermore, self-structure in the target appears to correlate with antisense ODN efficacy, but such that more effective antisense ODNs appear to target mRNA regions with greater self-structure. Therefore, different criteria are suggested for the design of efficient siRNA and antisense ODNs and the design of antisense ODNs is more challenging.