反义技术在抗HBV感染中的应用

反义技术是近些年来随着现代分子生物学技术的发展而产生的新的生物医学治疗技术。它采用反义核酸分子抑制、封闭或破坏靶基因组的技术手段,包括反义寡核苷酸、核酶及RNA干扰等。反义分子通过与靶基因异性互补配对结合,阻断靶基因的复制、转录或翻译过程,从而发挥抗病毒作用。针对乙型肝炎病毒的反义技术也有了广泛而深入的研究。根据反义技术在分子、细胞以及动物水平上的研究表明：反义技术能够高效、特异地抑制HBV的复制与表达。     

反义核酸技术应用难点及研究进展

反义核酸技术已被广泛用于治疗药物、药物靶点确认、探知病理基因的表达。目前对其作用原理的研究集中于其被吸收入细胞的机制、在细胞内的分布、反义核酸序列的最佳长度和性质,并针对体内可能抑制反义核酸活性的影响因素,采取了各种相应的反义核酸优化技术,如对反义核酸的化学修饰、联结高效的转运载体、确定最佳的反义结合位点等,通过这些技术来提高其体内稳定性、跨细胞转运的效率,识别靶序列的特异性,以获得更多更好的反义药物投入实用。     

反义RNA技术

反义RNA(antisence RNA)是一种与特异mRNA互补的RNA分子,它通过配对碱基间氢键作用与对应的RNA形成双链复合物,抑制RNA的翻译过程。利用人工合成或生物体合成特定互补RNA片段(或其化学修辞产物)抑制或封闭基因表达技术,称为反义RNA技术。本文综述了反义RNA作用机理,合成途径,并展示了该技术在研究基因功能,防治肿瘤、遗传病以及人工免疫等方面广阔的应用前景。一、反义RNA抑制基因表达机理许多实验证明,原核类生物如细菌、粘菌,     

反义RNA研究新进展

近年来,人们不断发现原核生物和真核生物中自然存在的反义RNA,这可能揭示另一个新的基因调控方式。 反义RNA通过碱基配对,特异性地与mRNA结合,阻止mRNA的翻译,从而抑制细胞中内源性或外源性基因的表达。因此,反义RNA技术为基因表达的功能研究以及基因定位和表达量检测提供了一种比常规遗传分析更为有效的方法,也为肿瘤病、病毒病等预防和治疗提供了可能途径。     

真核基因反义RNA研究进展

反义RNA是指能与特定mRNA互补的RNA片段。本文介绍了近年来真核基因反义RNA研究的一些进展,包括不同基因反义RNA的作用,反义RNA抑制作用的特点,以及反义RNA的抑制机理。反义RNA对基因表达具有高度专一性的调控作用,因此可利用它研究特定基因在细胞生长、分化中的作用,同时,反义RNA系统也可用于抑制有害基因的表达,从而为治疗提供新的途径。     

反义RNA及其在植物基因工程领域的应用

随着反义RNA的发现及对其研究的深入,反义RNA技术已被广泛应用于基因调控的研究中。本介绍了反义RNA的概念,并就反义RNA的作用机理和在植物基因工程领域的应用进行了综述。其作用机理包括：在原核生物中反义RNA与引物RNA前体及mRNA分子5′的不同区域进行互补,从而抑制其复制、转录和翻译;在其核生物中反义RNA影响mRNA前体拼接、转移及mRNA分子5′和3′正常修饰。在植物基因工程领域,反义RNA主要应用于抑制果实成熟、抗病、作为反向筛选标记基因、控制花色、控制淀粉合成、控制油料种子中脂肪酸的合成、控制雄性不育等方面。     

反义RNA 技术在花色育种中的应用

反义RNA技术是用反义RNA链去抑制靶基因的活性, 从而达到对目的基因调控的一项分子生物学技术。该项技术应用于观赏植物的花色育种已有16年的历史并且取得了一定的成就。到目前为止, 已经利用该技术对14种花卉花色形成过程中的3大类基因进行了正义和反义导入, 获得了花色改变的转基因植株。本文简要回顾了反义RNA技术的产生与发展, 并在介绍花色形成的分子生物学的基础上, 综述 了国际园艺育种中利用反义RNA技术调控花色基因表达的研究进展, 以期为花色改良的分子育种提供参考资料。     

反义RNA技术在花色育种中的应用

反义RNA技术是用反义RNA链去抑制靶基因的活性,从而达到对目的基因调控的一项分子生物学技术.该项技术应用于观赏植物的花色育种已有16年的历史并且取得了一定的成就.到目前为止,已经利用该技术对14种花卉花色形成过程中的3大类基因进行了正义和反义导入,获得了花色改变的转基因植株.本文简要回顾了反义RNA技术的产生与发展,并在介绍花色形成的分子生物学的基础上,综述了国际园艺育种中利用反义RNA技术调控花色基因表达的研究进展,以期为花色改良的分子育种提供参考资料.     

反义技术应用在细菌代谢调控中的研究进展

随着基因工程技术的蓬勃发展和代谢调控研究的深入,反义技术作为一种温和调控的基因工程技术,开始向世人展示其无穷的魅力.与基因敲除等功能缺失性研究方法相比,反义技术具有投入少、周期短、操作简单等优点,受到广泛的关注,成为细菌代谢调控的有力工具.以下对反义RNA、反义寡核苷酸,核酶这几种反义技术在细菌代谢工程操作中的研究进展及存在的问题进行了概述.     

功能基因组学技术在慢性疼痛研究中的应用

慢性疼痛是一个世界性难题,其治疗效果不佳,与其机制不明有很大关系。解决机制问题,探索有效的治疗方法已经成为研究的焦点。伴随着后基因时代的到来,以及分子生物学,生物信息学等多门生物相关学科的发展,RNA干扰技术,反义寡核苷酸技术,基因芯片等这些功能基因组学中常用的实验手段,通过在基因组或系统水平上全面分析基因的功能,为研究慢性疼痛发生机制,发现新的疼痛调节相关基因以及探索疼痛治疗的新途径开辟了更加广阔的空间。     

A Role for Oligonucleotide-Based RNA-Knock Down Technologies in Functional Genomics

Abstract

Functional genomics is inundating the pharmaceutical industry with large numbers of potential gene targets from several sources such as gene expression profiling experiments (DNA microchips, proteomics) or database mining. Oligonucleotide-based RNA-knock down technologies such as antisense or RNA interference can aid in the filtering and prioritization of target candidates in the drug discovery process.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

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.     

Selective binding of trisamine-modified phosphorothioate antisense DNA to target mRNA improves antisense activity and reduces toxicity

Antisense activity in living cells has been thought to occur via a mechanism involving both DNA-mediated hybridization arrest of target mRNA and RNase H-mediated mRNA digestion. Therefore an ideal antisense agent should be permeable to the cell and possess capacities (1) to form a thermally stable duplex in vivo with its target, (2) to discriminate between mRNAs with different degrees of complementarity, and (3) to form antisense/RNA complexes that are susceptible to RNase H hydrolysis. A trisamine-modified deoxyuridine derivative of a novel phosphorothioate DNA 15-mer that meets all these criteria is described here. Compared with the unmodified phosphorothioate oligomer, the phosphorothioate derivative exhibits a higher antisense activity as well as reduced cytotoxicity in cells infected with HIV-1. Our data suggest that the melting temperature (T(m)) between antisense DNA and the target mRNA is not only one of the factors contributing to this derivative's improved antisense activity. Also important are an enhanced ability to discriminate between sequences and an increased susceptibility of the DNA/mRNA complex to RNase H hydrolysis. These results will be useful in designing more active, clinically useful antisense drugs.     

人U6基因POI Ⅲ启动子表达反义VEGF cDNA抑制SMMC-7721肝癌细胞VEGF表达的观察

 由 RNA聚合酶启动子在细胞内转录高浓度反义 RNA是抑制靶蛋白的一种有效手段 ,有报道 POI 启动子转录小分子 RNA存在效率不高 ,带有较多的非特异性序列等缺点 ,为了克服这些问题 ,表达反义 VEGF RNA的人 U6基因表达盒对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制作用进行了研究 .首先 PCR扩增 2 0 0 bp VEGF c DNA以正、反向插入人 U6 sn RNA.通过测序证实反向插入的正确性 .采用细胞原位杂交 ,RNA酶保护分析 ,Northern印迹来证实反义 RNA表达的情况 ,利用 RT- PCR方法研究了其对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制效果 .细胞原位杂交结果显示 U6启动子转录产物主要分布于细胞核内 ,细胞浆内亦有表达 ,RNA酶保护分析显示 U6基因 POI 启动子能高表达所需大小反义 RNA,Northern印迹结果显示脂质体 lipo-fectamine介导含 U6基因 POI 启动子的质粒转染人肝癌细胞株 SMMC- 772 1后 1 2 h即有表达且可持续表达 6 d. RT- PCR证实 U6基因 POI 启动子转录的反义 VEGF RNA能有效抑制SMMC- 772 1细胞 VEGF的 m RNA表达 .已有的研究结果揭示 POI 启动子是反义基因治疗中一种好的选择     

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。     

Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor

Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.     

Bcl-2 Antisense Therapy for Cancer: The Art of Persuading Tumour Cells to Commit Suicide

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis induced by numerous physiological and pathological stimuli, and uncontrolled cell survival due to Bcl-2 overexpression has been shown to contribute to tumour formation and the development of autoimmune diseases. The multifunctional action of Bcl-2 is thought to prevent activation of the ced3/caspase-3 subfamily of ICE proteases, resulting in suppression of the death effector machinery. Since most conventional anti-cancer agents act by triggering this suicide pathway, overexpression of Bcl-2 in cancer cells has also been associated with drug resistance. The antisense approach to inhibition of gene expression relies on the binding of small synthetic oligodeoxynucleotides to a complementary base sequence on a target mRNA. As a consequence, expression of the corresponding gene is downregulated due to endonuclease-mediated hydrolysis of the mRNA strand, or to translational arrest arising from sterie hindrance by the RNA:DNA heterodimer. Since these mechanisms of action differ from those exerted by conventional anticancer agents, antisense oligodeoxynucleotides designed to specifically inhibit bcl-2 gene expression hold great promise as agents that could overcome clinical drug resistance, and improve the treatment outcome of many hitherto incurable cancer diseases.     

Pseudo-cyclic oligonucleotides: in vitro and in vivo properties

We have designed and studied antisense oligodeoxynucleotides (oligonucleotides; oligos) which we call ‘pseudo-cyclic oligonucleotides’ (PCOs). PCOs contain two oligonucleotide segments attached through their 3′-3′- or 5′-5′-ends. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5–8 nucleotides long and complementary to the 3′- or 5′-end of the antisense oligo. As a result of complementarity between the antisense and protective oligo segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies described here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RI (PKA-RI). Thermal melting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the target RNA dissociate, bind to the target RNA, and form heteroduplexes. The results of RNase H cleavage assays suggest that PCOs bind to complementary RNA and activate RNase H in a manner similar to that of an 18-mer conventional antisense PS-oligo. In snake venom (a 3′-exonuclease) or spleen (a 5′-exonuclease) phosphodiesterase digestion studies, PCOs are more stable than conventional antisense oligos because of the presence of 3′-3′- or 5′-5′-linkages and the formation of intramolecular pseudo-cyclic structures. PCOs with a phosphorothioate antisense oligo segment inhibited cell growth of MDA-MB-468 and GEO cancer cell lines similar to that of the conventional antisense PS-oligo, suggesting efficient cellular uptake and target binding. The nuclease stability studies in mice suggest that PCOs have higher in vivo stability than antisense PS-oligos. The studies in mice showed similar pharmacokinetic and tissue distribution profiles for PCOs to those of antisense PS-oligos in general, but rapid elimination from selected tissues.     

RNA complementary to the 5' UTR of mRNA triggers effective silencing in Saccharomyces cerevisiae

Conditional silencing of target genes in Saccharomyces cerevisiae by antisense RNAs expressed in vivo has been challenged. The MFalpha1::lacZ fusion present in S. cerevisiae SF51-3 was chosen as a model target, and fragments of this gene were cloned in reverse orientation into the expression vector pYES2, bearing the GAL1 promoter. Among the different antisense constructs tested, only the one complementary to the 5' UTR of target mRNA featured effective silencing. Nevertheless, the expression in vivo of this antisense RNA could not be properly tuned by the absence or presence of galactose in the culture medium. Accordingly, conditional silencing could not be attained by this antisense hosted into pYES2. On the contrary, cloning the same antisense construct into the expression vector pSAL4 yielded a fully conditional silencing linked to the control of antisense expression by the absence or presence of Cu(2+) into the culture medium.