Fas基因mRNA反义靶点筛选和体外验证

应用PARASS(poly-A anchored RNA accessible sites screening) 技术筛选Fas基因mRNA 获得3个潜在反义作用靶点,靶点1、2、3分别位于Fas基因297nt-317nt、619nt-639nt和662nt-682nt。设计了对应靶点的反义寡核苷酸A1、A2、A3,和10-23型DNAzyme D1、D2和D3。将反义寡核苷酸和Fas基因RNA结合再加入RNase H进行反应,10-23型DNAzyme则直接与Fas基因RNA作用,结果表明：3个靶点的反义寡核苷酸组及DNAzyme均能降解Fas基因RNA,为有效靶点,其靶点反应优势次序为靶点3>靶点1>靶点2;而非靶点对照组和有效靶点突变了2个碱基的对照组均没有反应。靶点2和靶点3与ISIS公司经过多次实验筛选到的Fas反义作用靶点位置基本相同,表明PARASS技术的有效性和可靠性。获得的有效反义寡核苷酸和DNAzyme为后续研究打下基础。     

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.     

绿色荧光蛋白基因mRNA反义寡核苷酸的筛选和应用

基因mRNA的靶点筛选是设计反义寡核苷酸的关键.建立了PARASS(polyAanchoredRNAaccessiblesitesscreening)方法,即通过在mRNA末端引入polyA,与生物素标记的polyT退火结合,将其同链亲和素磁珠混合,使mRNA通过3’末端得到固定,保持mRNA的自然伸展和折叠,与寡核苷酸文库杂交筛选mRNA的结合靶点.PARASS筛选获得了绿色荧光蛋白(GFP)mRNA的3个反义寡核苷酸结合靶点,据其设计了多条反义寡核苷酸,与对照组相比,体外RNaseH分析显示3个靶点均为有效,在HeLa细胞内针对靶点的反义寡核苷酸能抑制GFP的表达,得到了Northern印迹结果支持.PARASS对反义寡核苷酸药物设计具有应用价值.     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

A method for prediction of accessible sites on an mRNA sequence for target selection of hammerhead ribozymes.

Hammerhead ribozymes are short RNA molecules endowed with endoribonucleolytic activity. Their use as molecular tools for specific inhibition of gene translation is affected by many factors including the target accessibility. A method for the prediction of accessible target sites for hammerhead ribozymes within a given RNA sequence is described. This method maps all putative NUH cleavage sites (N = A, C, G, U and H = A, C, U) and picks out short flanking regions as the binding domain for the corresponding ribozyme. The probabilistic level of unfolding, accessibility score (AS), is then calculated for each target region on the basis of a comparison of all folding structures obtained for the target RNA and arranged according to the Boltzmann's distribution. At the end, a series of imposed limits gives the best target sequences endowed with highly probable accessibility and with a potentially active catalytic structure of the hammerhead sequence. A successive experimental approach to verify the effective accessibility of selected targets was used. For that, antisense oligonucleotides addressed to the coding region of bcl2 mRNA were synthesized and administered to the MCF7 human cell line. The inhibition of gene expression, as measured by western analysis of the BCL2 protein, demonstrated that all target sites selected on the basis of their putative accessibility were actually sensitive to antisense treatments while the inaccessible ones were not. The application of this target discovery method to ribozyme design is proposed in order to satisfy a crucial condition.     

大肠杆菌16S rRNA的核酶设计和初步应用

针对细菌rRNA研发抑制细菌增殖的新型抗菌素是抗生素研究领域的新课题。细菌rRNA与基因mRNA一样自然形成折叠卷曲高级结构,其结构上可以结合反义核酸的位点即靶点,靶点的阐明是设计有效反义核酸、核酶（Ribozyme）和脱氧核酶（DNAzyme）的关键。MAST方法固定16S rRNA,将其与寡核苷酸文库杂交筛选出靶点,获得了大肠杆菌16S rRNA的6个反义核酸结合靶点,并鉴定5个靶点有效,其中1个为高效。5个靶点的反义核酸能在通透性大肠杆菌菌株培养中不同程度地抑制其生长,针对高效靶点的核酶在转化大肠杆菌中表达而抑制其生长。     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides

The biological activity of siRNA seems to be influenced by local characteristics of the target RNA, including local RNA folding. Here, we investigated quantitatively the relationship between local target accessibility and the extent of inhibition of the target gene by siRNA. Target accessibility was assessed by a computational approach that had been shown earlier to be consistent with experimental probing of target RNA. Two sites of ICAM-1 mRNA predicted to serve as accessible motifs and one site predicted to adopt an inaccessible structure were chosen to test siRNA constructs for suppression of ICAM-1 gene expression in ECV304 cells. The local target-dependent effectiveness of siRNA was compared with antisense oligonucleotides (asON). The concentration dependency of siRNA-mediated suppression indicates a >1000-fold difference between active siRNAs (IC₅₀ ≈ 0.2–0.5 nM) versus an inactive siRNA (IC₅₀ ≥ 1 µM) which is consistent with the activity pattern of asON when relating target suppression to predicted local target accessibility. The extremely high activity of the siRNA si2B (IC₅₀ = 0.24 nM) indicates that not all siRNAs shown to be active at the usual concentrations of >10–100 nM belong to this highly active species. The observations described here suggest an option to assess target accessibility for siRNA and, thus, support the design of active siRNA constructs. This approach can be automated, work at high throughput and is open to include additional parameters relevant to the biological activity of siRNA.     

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。     

Potent growth inhibition of leukemic cells by novel ribbon-type antisense oligonucleotides to c-myb1

We studied the effects of antisense oligonucleotides (AS oligos) with a novel structure. The AS oligos were covalently closed to avoid exonuclease activities by enzymatic ligation of two identical molecules. The AS oligos of a ribbon type (RiAS oligos) consist of two loops containing multiple antisense sequences and a stem connecting the two loops. Three antisense sequences targeting different binding sites were placed in a loop that was designed to form a minimal secondary structure by itself. RiAS oligos were found to be stable because they largely preserved their structural integrity after 24 h incubation in the presence of either exonuclease III or serums. When a human promyelocytic cell line, HL-60, was treated with RiAS oligos to c-myb, c-myb expression was effectively ablated. Cell growth was inhibited by >90% determined by both the 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and [(3)H]thymidine incorporation. Further, when the leukemic cell line K562 was treated with c-myb RiAS oligos, colony formation on soft agarose was reduced by 92 +/- 2%. These results suggest that RiAS oligos may be employed for developing molecular antisense drugs as well as for the functional study of a gene.     

基因药物研究现状和对策

生物技术药物以人类体细胞的基因组、转录本组和蛋白质组三个层次生物大分子为目标 ,基因药物的研究主要针对致病基因的DNA和基因转录本mRNA两类生物大分子 .mRNA从结构上考虑是研发核酸药物的最理想靶标和策略之一 .反义寡核苷酸、特异水解基因mRNA的核酸酶(ribozyme和DNAzyme)以及具有干扰作用的双链RNA(siRNA)是药物设计的策略之二 .mRNA结构靶点研究是研发反mRNA基因药物的基础 ,mRNA分子具有高度折叠的二级及三级结构 ,阐明其可及性位点 ,筛选其结构靶位点序列是关键 .近年研究报道的靶点筛选有约 7种mRNA的实测新技术 ,以及计算机辅助软件预测分析 .但发展分子生物学实验新技术以分析、确认靶点是药物研发策略之三 .     

Predicting the efficacy of short oligonucleotides in antisense and RNAi experiments with boosted genetic programming

MOTIVATION: Both small interfering RNAs (siRNAs) and antisense oligonucleotides can selectively block gene expression. Although the two methods rely on different cellular mechanisms, these methods share the common property that not all oligonucleotides (oligos) are equally effective. That is, if mRNA target sites are picked at random, many of the antisense or siRNA oligos will not be effective. Algorithms that can reliably predict the efficacy of candidate oligos can greatly reduce the cost of knockdown experiments, but previous attempts to predict the efficacy of antisense oligos have had limited success. Machine learning has not previously been used to predict siRNA efficacy. RESULTS: We develop a genetic programming based prediction system that shows promising results on both antisense and siRNA efficacy prediction. We train and evaluate our system on a previously published database of antisense efficacies and our own database of siRNA efficacies collected from the literature. The best models gave an overall correlation between predicted and observed efficacy of 0.46 on both antisense and siRNA data. As a comparison, the best correlations of support vector machine classifiers trained on the same data were 0.40 and 0.30, respectively.     

Prediction of antisense oligonucleotides using structural and thermodynamic motifs

Specific gene expression regulation strategy using antisense oligonucleotides occupy significant space in recent clinical trials. The therapeutical potential of oligos lies in the identification and prediction of accurate oligonucleotides against specific target mRNA. In this work we present a computational method that is built on Artificial Neural Network (ANN) which could recognize and predict oligonucleotides effectively. In this study first we identified 11 major parameters associated with oligo:mRNA duplex linkage. A feed forward multilayer perceptron ANN classifier is trained with a set of experimentally proven feature vectors. The classifier gives an exact prediction of the input sequences under 2 classes – oligo or non-oligo. On validation, our tool showed comparatively significant accuracy of 92.48% with 91.7% sensitivity and 92.09% specificity. This study was also able to reveal the relative impact of individual parameters we considered on antisense oligonucleotide predictions.     

Target site selection for an RNA-cleaving catalytic DNA.

A small catalytic DNA, known as the 10-23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.     

Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells

RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.     

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.     

Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.     

Local RNA target structure influences siRNA efficacy: a systematic global analysis

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.