逆转录法筛选mRNA靶点设计核酶对GPA的表达干预实验研究

采用自行设计5’固定3’随机的文库对基因mRNA进行杂交用于逆转录反应以筛选mRNA的寡核苷酸结合靶点。对人I型跨膜糖蛋白-血型糖蛋白A （glycophorin A,GPA）的mRNA筛选了4个反义寡核苷酸可结合靶点,分别设计反义核酸（Antisense）,分别加入mRNA中用RNase H验证各靶点的核酸结合和切割效率,最终确定2个高效结合和切割靶点。再设计Ribozyme,构建表达核酶（Ribozyme）质粒,利用慢病毒（Lentivirus）包装技术,感染人源红系白血病细胞株K562细胞,在细胞水平验证其下调GPA基因表达的效果,对转染细胞mRNA进行反转录和Real Time PCR分析mRNA表达水平,并在蛋白水平进行了Western Blot分析。结果表明文库结合逆转录方法筛选靶点设计的Ribozyme具有高效率下调膜受体表达的作用,GPA为I型跨膜糖蛋白,该实验为筛选mRNA靶点提供参考方法,并对膜受体表达干预有参考价值。     

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。     

一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达

摘要：【目的】本研究旨在从重金属汞抗性细菌中分离鉴定汞抗性基因【方法】从北京凉水河河床底泥中分离抗汞细菌,采用16S rRNA基因序列分析结合生理生化特征对菌株进行鉴定。根据GenBank中已发表的多种抗汞细菌的merA基因序列设计引物,以抗性细菌基因组DNA为模板,扩增merA基因,并在大肠杆菌（Escherichia coli）BL21(DE3)表达。同时对表达菌株的重金属汞抗性进行测定。【结果】分离得到一株能在含HgCl2为70 mg/L的平板上生长良好的高抗汞细菌,编号为KHg2。16S rRNA     

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。     

miRNA-21反义核酸对人淋巴瘤Raji细胞的生长抑制作用研究

为研究以miRNA-21为靶标的反义核酸（AMO-miR-21）对淋巴瘤Raji细胞的生长抑制作用,使用LipofectamineTM 2000将化学合成的反义核酸转染Raji细胞,采用四甲基偶氮唑蓝（MTT）法和台盼蓝拒染法检测细胞生长抑制率;实时定量PCR技术检测细胞miRNA-21水平改变;PI和Annexin V双染,流式细胞仪检测细胞凋亡.结果显示转染Raji细胞48～72h,反义核酸组细胞生长受到明显抑制,反义核酸抑制细胞活性的最佳浓度是0.4μmol/L;细胞内miRNA-21的表达水平明显下调;细胞凋亡明显增加;此外,反义组细胞中抑癌基因Pdcd4的mRNA和蛋白质呈高水平表达.提示以miRNA-21为靶标的反义核酸是人淋巴瘤Raji细胞生长的有效抑制剂和凋亡诱导剂.miRNA-21可能是淋巴瘤治疗的潜在靶标,其通过下调抑癌基因Pdcd4的表达水平发挥抗肿瘤作用.     

癌症治疗的目的

端粒酶与癌症密切相关,抑制端粒酶的活性主抑制癌细胞的生长,反义核酸、核酶、细胞分化剂、逆录酶抑制剂和鸟嘌呤四联体等都可以在不同程度上抑制端粒酶活性,在癌症治疗中具有很大应用潜力。     

癌症治疗的目标——抑制端粒酶的策略

端粒酶与癌症密切相关,抑制端粒酶的活性可以抑制癌细胞的生长.反义核酸、核酶、细胞分化剂、逆转录酶抑制剂和鸟嘌呤四联体等都可以在不同程度上抑制端粒酶活性.在癌症治疗中具有很大应用潜力.     

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。     

功能核酸用于致病菌检测的研究进展

由食源性致病菌引发的疾病对人类健康构成巨大威胁。虽然一些致病菌如金黄色葡萄球菌、大肠杆菌和沙门氏菌等在诊断和预防方面已经取得了重大进展,但开发快速、高效、低成本的检测方法仍然是一项挑战。功能核酸（functional nucleic acids,FNAs）是一类功能超出核酸常规遗传作用的核酸,主要包括天然的核酶（RNAzymes）、核糖开关（riboswitches）以及体外通过指数富集配体系统进化技术（systematic evolution of ligands by exponential enrichment,SELEX）筛选的适配体（aptamers）、核酶（RNAzymes）和脱氧核酶（DNAzymes）。适配体和脱氧核酶因具有较高的稳定性、特异性和可设计性,使其成为病原微生物识别的理想工具,近年来在生物传感和医学诊断领域备受关注。综述了功能核酸的筛选原理和流程、适配体及具有RNA裂解活性的脱氧核酶（RNA cleavage deoxyribozymes,RCDs）在致病菌检测中的应用进展和面临的挑战,并对其未来的发展前景进行了展望。     

可产生铁载体的春兰根内生细菌多样性

摘要：【目的】了解可产生铁载体的春兰根内生细菌的多样性,以便筛选到高效的植物促生细菌。【方法】采用CAS检测法测定了189株春兰根内生细菌产生铁载体的能力,并结合16S rRNA基因系统发育分析对可产铁载体的春兰根内生细菌多样性进行了研究。【结果】从189株春兰内生细菌中筛选到47株可产生铁载体的细菌,占菌株总数的24.9%。16S rRNA基因系统发育分析结果表明,47株细菌分属于4个系统发育类群（Alphaproteobacteria,Betaproteobacteria,Firmicutes,Actinobacteria）,17个属的31个种。其中放线菌门为最优势类群（42.6%）,芽孢杆菌属（Bacillus）和贪噬菌属（Variovorax）为优势菌属,且贪噬菌属为高产铁载体的主体菌属。另外有2个菌株可能代表两个不同属的新物种。【结论】春兰根中可产生铁载体的内生细菌具有丰富的多样性。     

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.     

Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides

Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.     

计算机辅助设计联合斑点杂交筛选铜绿假单胞菌PAO1 motA基因反义寡核苷酸序列

为了筛选出能与铜绿假单胞菌PAO1 motA基因的mRNA结合紧密的反义寡核苷酸序列,采用全基因寻靶技术（full length gene targeting,FLGT）,运用计算机软件（Mfold和RNA Structure4.6）模拟铜绿假单胞菌PAO1 motA基因mRNA的二级结构,根据最小自由能原理设计出8条寡核苷酸探针序列;PCR扩增出全长motA基因,克隆motA基因并进行体外转录,同时用地高辛标记mRNA,以斑点杂交方法筛选出与motA基因mRNA结合紧密、杂交信号较强的寡核苷酸序列。斑点杂交结果显示8条寡核苷酸中的4条有较强的杂交信号,从而成功筛选到了能与motA mRNA牢固结合的反义序列,为进一步研究以motA基因为靶的反义技术抑制生物膜形成打下基础。     

Evaluation of dynamic features of Escherichia coli 16S ribosomal RNA in homogeneous physiological solution

There is no methodology for the estimation of the dynamic features of large-molecular-weight RNAs in homogeneous physiological media. In this report, a luminescence anisotropy-based method using a long-lifetime luminescent oligonucleotide probe for the estimation of the dynamic features of large-molecular-weight RNA is described. As a luminescent probe, Ru(II) complex-labeled oligonucleotides, which have a complementary sequence to the single-stranded regions of Escherichia coli 16S rRNA, were synthesized. After the hybridization of the probe to single-stranded regions of 16S rRNA, the segmental motions of the regions were evaluated by time-resolved luminescence anisotropy analysis. In 16S rRNA, the L2 site (323-332 nt) was found to be the most flexible among the seven sites chosen. From a comparison between the hybridization kinetics of oligonucleotides to these single-stranded regions and the rotational correlation times, it was suggested that the flexibility of the single-stranded region was closely correlated with the hybridization kinetics. Furthermore, results of the luminescence lifetime measurement and luminescence quenching experiments suggested that the highly flexible region was located on the surface of the 16S rRNA and that the less flexible region was located in the depths of 16S rRNA.     

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.     

An additional ribosome-binding site on mRNA of highly expressed genes and a bifunctional site on the colicin fragment of 16S rRNA from Escherichia coli: important determinants of the efficiency of translation-initiation.

For various genes of E. coli, three regions (-55 to -1; -35 to -1; -21 to -1) 5' to AUG codon on mRNA were searched for sites of interaction with colicin fragment of 16S rRNA. The detailed sequence comparison points out that apart from Shine-Dalgarno base pairing, an additional ribosome-binding site, a subsequence of 5'-UGAUCC-3' invariably exists in mRNA for highly expressed genes. Poorly expressed genes appear to be controlled by only Shine-Dalgarno base pairing. The analysis indicates that in the initiator region, the -55 to -1 region contains the signal which decides the efficiency of the translation-initiation. The site on 16S rRNA, 5'-GGAUCA-3' at position 1529, that can base pair to the above site, has a recognition site on 23S rRNA at position 2390. In the light of the conserved nature and accessibility of these sites, it is proposed that the site on 16S rRNA plays a bifunctional role--initially it binds to mRNA from highly expressed genes to form a stable 30S initiation complex, and upon association with 50S subunit it exchanges base pairing with 23S rRNA, thus leaving the site on mRNA free.