c-Myc功能及其下游靶点

c-Myc是一个在进化上较为保守的,具有b/HLH/LZ结构的转录调节因子,它可以与Max形成异源二聚体通过结合于启动子区的E盒结构对基因进行转录激活调控,也可以通过其他方式对基因进行正负调节,参与调控了细胞的增殖、分化、生长、凋亡、细胞周期进程、细胞内生物大分子的代谢以及细胞的恶性转化。近期,研究者通过采用微阵列芯片、生物信息学技术、染色质免疫沉淀(ChIP)、基因表达系列分析(SAGE)等高通量研究的新技术对c-Myc下游靶点进行研究,这对于揭示c-Myc结构与功能之间的关系具有重要的生物学意义。     

软件预测和MAST技术筛选mRNA反义核酸靶点的比较

基因mRNA的结构靶点筛选是反义核酸药物研发的一个难题 .兔 (Oryctolaguscuniculus) β珠蛋白基因mRNA的结构靶位点通过运用MAST技术筛选获得 ,和计算机软件RNAstructure3 71模拟分析的位点进行了比较 ,也和寡核苷酸微阵列杂交技术筛选获得的靶点结果 (M .Natalie ,1 997)进行了比较 ,显示 :据MAST技术获得的兔 β珠蛋白基因 2个反义核酸结合靶位点 ,和用RNAstructure3 71软件给出的模拟分析的 2个靶位点相同 ,且它们与寡核苷酸微阵列杂交技术的结果完全一致 .运用MAST技术筛选获得绿色荧光蛋白 (GFP)mRNA有 4个结构靶位点 ,体外分析表明这 4个靶位点均有效 ,其中有 3个与RNAstructure3 71软件分析的靶点相同 ,但计算机模拟推荐的结构靶位点较多 ,而且随着基因长度增加确认靶位点的难度增大 ,获得的靶位点还需要实验验证 ,计算机软件模拟分析对实验筛选靶点、设计反义核酸有辅助价值 .MAST方法能筛选各种长度基因mRNA的全部可及位点和准确给定核苷酸的起止位置以供设计反义核酸 ,具有简单快捷的优点 ,将能为反义核酸设计起重要作用 .     

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.     

小干扰RNA对庚型肝炎病毒基因在人Huh-7细胞中表达的抑制作用

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默现象, 是近年来兴起的一项研究生物基因调控与功能的崭新技术. 庚型肝炎病毒(HGV)是一单正链RNA病毒, 复制时不与宿主细胞基因组整合, 尤其适合用于RNAi的研究. 构建了含HGV完整结构基因并携带筛选标志潮霉素基因的真核表达载体pVAX.EH, 转染Huh-7细胞后, 筛选获得稳定表达HGV 结构蛋白的Huh-7细胞株(Huh-7-EH). RT-PCR和Western blot检测证实, HGV结构基因能在Huh-7-EH细胞中转录、表达, 并能进行剪切和翻译后修饰. 以体外转录法制备了2对靶向HGV E2基因的siRNA(1-E2 siRNA和2-E2 siRNA), 将其导入Huh-7-EH细胞中, 采用Western blot和克隆形成实验证实, HGV 1-E2 siRNA和2-E2 siRNA均能特异性抑制HGV结 构蛋白的表达, 抑制作用可维持1周以上. 其中2-E2 siRNA的抑制作用更强, 转染后对Huh-7-EH细胞潮霉素抗性克隆形成的抑制率达到了99%. Huh-7-EH细胞转染siRNA后对潮霉素敏感, 说 明HGV E2 siRNA不仅使HGV E2区的mRNA降解, 还可使融合在HGV E2区下游的潮霉素mRNA降解. 综上所述, 本实验建立的稳定表达HGV结构蛋白的Huh-7-EH细胞株, 能作为用于研究HGV复制和RNAi的细胞模型; HGV结构基因区的siRNA可同时抑制HGV结构蛋白及其下游的潮霉素基因的表达, 证明RNAi在真核细胞Huh-7-EH内可能存在放大作用.     

microRNA化学小分子抑制剂的研究进展

药物靶点的研究一直是新药研发中最重要的内容之一,microRNA(miRNA)作为调节基因与蛋白之间相互关系的纽带无疑是药物靶点研究的重要对象,以它们作为靶点的药物设计和药物研发工作也在不断深入,并获得了一系列新发现.其中,对细胞内miRNA的表达或者功能具有调控活性的化学小分子的研究很值得关注.这些化学小分子不但可以用于研究细胞内miRNA的调控网络,而且基于其对重大疾病,如肿瘤、心脏疾病、神经性疾病等相关的miRNA的调控,可能在将来的疾病治疗方面有着潜在的重要作用.本文围绕miRNA化学小分子抑制剂及其筛选方法进行综述,通过总结归纳现有的研究进展,希望对今后的miRNA特异性小分子抑制剂的发现和研究提供借鉴.     

结直肠癌组织特异性治疗靶点的生物信息学筛选

目的 预测与筛选结直肠癌组织特异性基因,作为靶向治疗的候选靶点.方法 利用自主开发的Python语言程序分析人类正常组织与结直肠癌组织mRNA表达的组织特异性,结合人类胚胎干细胞富集基因集以及文献挖掘结果,筛选可能的与结直肠癌发生或发展相关的基因作为候选靶点,并对其进行通路分析及基因富集分析.结果 获得了结直肠癌组织特异的且与肿瘤生物学通路密切相关的4个基因,作为进一步研究的候选靶点.结论 应用生物信息学方法从芯片数据进行挖掘,可以为结直肠癌的靶向治疗提供候选靶点,并为后续的药物设计奠定基础.     

细胞分子生物学技术在靶向药物筛选中的应用

随着细胞及分子生物学的发展,新技术方法越来越多地用于新靶点建立和药物筛选研究,为药物设计、靶点的选择和用药方案的确定提供理论依据,同时使药物筛选有了更高的特异性,对药物筛选和药理学研究起到了极大的促进作用. 论述了功能基因的筛选、高通量细胞筛选和高内涵筛选技术、反义核酸技术、转基因/基因敲除技术、基因芯片、蛋白质芯片、组织芯片、酶免疫分析、荧光免疫分析、流式细胞技术等方法在靶向药物筛选中的应用.     

靶向小分子创新药物

寻找药物新靶点是全球创新药物研究激烈竞争的焦点."组学"、生物信息学、系统生物学、药物筛选现代检测技术等新理论、新技术的发展使新的筛选模型和评价技术不断取得突破.靶向抗肿瘤药物的开发是靶向小分子创新药物的重点任务,多靶点的抗肿瘤药物开发及新靶点的发现是抗肿瘤药物研发的新趋势.     

强啡肽单抗融合蛋白的克隆、表达及功能分析

G蛋白偶联受体(GPCR)长期以来是最重要的药物靶点家族,小分子药物层出不穷。然而受研发难度的限制,针对GPCR的抗体或大分子类药物屈指可数。我们利用选择性靶向κ阿片受体(KOR)且无法激活下游信号的单克隆抗体连接强啡肽(Dynorphin)基因、HEK 293F系统进行表达,纯化获得KOR强啡肽单抗融合蛋白,我们将此融合蛋白命名为APF(antibody-peptide fusion)。结果显示,获得的单抗融合蛋白二级结构未显著改变,保持了Dynorphin活性,可激活KOR相关下游蛋白(Gi)活性,调动β-arrestin信号。结果证明,基因层面实现抗体药物改构的可行性,该法可指导新一代抗体偶联药物的改造,为以GPCR为靶点的大分子药物开发提供了新的空间。     

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.     

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。     

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.     

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.     

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.