脱氧核酶研究进展

使用体外分子进化技术,从一个人工合成的随机多核苷酸单链DNA库中筛选出具有酶活性的DNA分子,称为脱氧核酶. 目前已经筛选出具有RNA切割作用、DNA切割作用、金属螯合作用和过氧化物酶活性、DNA激酶活性以及DNA连接酶活性等多种催化功能的脱氧核酶. 特别是脱氧核酶10～23,无论在体外应用于RNA限制性内切酶,还是在生物系统内作为RNA水平上的基因失活剂,都具有很好的应用前景.     

脱氧核酶的研究进展

具有RNA裂解活性的DNA分子称为脱氧核酶。它是经过体外选择技术经多次筛选获得的。脱氧核酶在切割与其互补的RNA底物分子时具有极高的特异性和切割效率,有望成为新的RNA灭活工具。     

脱氧核酶的研究进展

具有RNA裂解活性的DNA分子称为脱氧核酶,它是经过体外选择技术经多次筛选获得的。脱氧核酶在切割与其互补的RNA底物分子时具有极高的特异性和切割效率,有望成为新的RNA灭活工具。     

Deoxyribozymes的研究进展

具有RNA裂解活性的DNA分子称为脱氧核酶,它是经过体外选择技术经多次筛选获得的。脱氧核酶在切割与其互补的RNA底物分子时具有极高的特异性和切割效率,有望成为新的RNA灭活工具。     

10-23脱氧核酶介导的生物传感器研究进展

近20年间,DNA介导的生物传感器得到了快速的发展,DNA能够作为遗传信息重要载体的同时,其折叠成的空间构象也具有很多的功能。功能核酸的概念逐渐引入到了包括生物传感、生物成像、医疗在内的重要领域中。10-23脱氧核酶作为功能核酸的一种,是通过体外筛选技术得到的,Mg2+存在的条件下能够特异性识别并切割RNA,切割位点为RNA中的嘧啶与嘌呤间的磷酸二酯键。由于其独特的识别以及切割能力,10-23脱氧核酶介导的相关疾病治疗得到了广泛的应用,同时人们逐渐开始关注10-23脱氧核酶介导的生物传感器的搭建。本文对于10-23脱氧核酶的结构、性质、作用方式以及改进修饰进行了介绍,并对10-23脱氧核酶介导的生物传感器的搭建以及应用进行了综述,为人们在未来使用10-23脱氧核酶搭建新型快捷生物传感器奠定理论基础。     

10-23脱氧核酶介导的生物传感器研究进展

近20年间,DNA介导的生物传感器得到了快速的发展,DNA能够作为遗传信息重要载体的同时,其折叠成的空间构象也具有很多的功能。功能核酸的概念逐渐引入到了包括生物传感、生物成像、医疗在内的重要领域中。10-23脱氧核酶作为功能核酸的一种,是通过体外筛选技术得到的,Mg²⁺存在的条件下能够特异性识别并切割RNA,切割位点为RNA中的嘧啶与嘌呤间的磷酸二酯键。由于其独特的识别以及切割能力,10-23脱氧核酶介导的相关疾病治疗得到了广泛的应用,同时人们逐渐开始关注10-23脱氧核酶介导的生物传感器的搭建。对于10-23脱氧核酶的结构、性质、作用方式及改进修饰进行了介绍,并对10-23脱氧核酶介导的生物传感器的搭建及应用进行了综述,旨为人们在未来使用10-23脱氧核酶搭建新型快捷生物传感器奠定理论基础。     

脱氧核酶及其应用进展

脱氧核酶是利用体外分子进化技术合成的一种具有催化功能的单链DNA片段,具有高效的催化活性和结构识别能力,迄今为止,已发现大量具有催化功能的脱氧核酶。其中具有RNA切割活性的脱氧核酶,能催化RNA特定部位的切割反应,从mRNA水平对基因灭活．从而调控蛋白质的表达．可能成为治疗肿瘤、病毒感染性疾病以及其它相关疾病,基因功能研究,核酸突变分析等的新型工具。     

脱氧核酶的研究进展

脱氧核酶是的年来利用体外分子进化技术合成的一种具有催化功能的单链DNA片段,它能催化RNA特定部位的切割反应,从mRNA水平对基因灭活,从而调控蛋白的表达,可能成为对抗RNA病毒感染、肿瘤等疾病的新型工具。     

脱氧核酶抑制近日基因period 1表达的实验研究

研究脱氧核酶对近日钟基因period1(per1)表达的影响, 进而寻找治疗和近日节律有关疾病的基因疗法. 设计合成针对per 1的脱氧核酶DRz164, DRz256, 并构建pcDNA3-per1_164:256体外转录载体, 将转录产物和脱氧核酶混合, 在一定反应条件下进行体外切割反应, 地高辛酶联免疫及酶催化显色法检测脱氧核酶的体外切割效率. 将pcDNA3-per1和DRz164或DRz256在脂质体的介导下转染NIH3T3细胞, 通过逆转录-聚合酶链反应(RT-PCR)、流式细胞术(FCM)检测脱氧核酶对近日基因表达的影响. 于37℃孵育2 h后, DRz164对底物的剪切百分率为63%, DRz256为50.5%. RT-PCR半定量检测per1 mRNA表达水平明显下降, FCM结果显示细胞内Per1蛋白的合成受到抑制. 脱氧核酶DRz164, DRz256体外具有定点切割近日钟基因per1mRNA组分的活性, 使转染细胞per1 mRNA 和Per1蛋白表达下降.     

核酶对人巨细胞病毒mRNA片段的体外切割

引导序列GSs(GuideSequences)是能与mRNA互补,引导核酶RNaseP催化核心M1RNA对互补区域特异切割的小片段游离RNA。针对人巨细胞病毒HCMV(humancytomegalovirus)DNA聚合酶mRNA序列设计GS,共价结合到大肠杆菌来源M1RNA中,构建成M1GST7核酶。通过对巨细胞病毒DNA聚合酶亚克隆片段转录产物体外切割实验,表明该核酶具备对DNA聚合酶mRNA片段的特异切割能力 。     

Dominance and independent segregation of metabolic cooperation-competence and pluripotency in an embryonal carcinoma cell hybrid

We report the isolation of a fusion hybrid, PR3, from a pluripotent embryonal carcinoma (EC) cell line, PSA4, which is metabolic cooperation-competent, and an EC line R5/3OA which has a reduced capacity for metabolic cooperation and a restricted developmental capacity. PR3 resembles its pluripotent parent PSA4 in its capacity for gap-junction-mediated transfer of uridine nucleotides and in its pluripotency both in embryoid bodies in vitro and in tumors in vivo. This enabled the relationship between pluripotency and metabolic cooperation to be examined by the selection of segregant lines. Cooperation-deficient lines were isolated from a thioguanine-resistant intermediate line (PR3Tg12) using "Kiss of Death" selection. A novel method was devised for the selection of differentiation-deficient segregants using feeder cell-conditioned medium which partially inhibits in vitro differentiation. It was found that communication-competence and in vitro pluripotency segregated independently, demonstrating that the loss of developmental capacity in R5/3OA cannot be attributed to its communication-deficiency.     

SERF: in vitro election of random RNA fragments to identify protein binding sites within large RNAs

In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant (SERF, selection of random RNA fragments) that is useful for the identification of short RNA fragments originating from large RNAs that bind specifically to a protein. A pool of randomly fragmented RNA is constructed from a large RNA, which is the natural binding partner for a protein. Such a pool contains all the potential binding sites and is therefore used as starting material for affinity selection with the purified protein to find its natural target. Here we provide a detailed experimental protocol of the method. SERF has been developed for ribosomal systems and is a general approach providing a basis for functional and structural characterization of RNA-protein interactions in large ribonucleoprotein particles.     

Construction of dopamine sensors by using fluorescent ribonucleopeptide complexes

A facile strategy of stepwise molding of a ribonucleopeptide (RNP) complex affords fluorescent RNP sensors with selective dopamine recognition. In vitro selection of a RNA-derived RNP library, a complex of the Rev peptide and its binding site Rev Responsive Element (RRE) RNA appended with random nucleotides in variable lengths, afforded RNP receptors specific for dopamine. The modular structure of the RNP receptor enables conversion of dopamine-binding RNP receptors to fluorescent dopamine sensors. Application of conditional selection schemes, such as the variation of salt concentrations and application of a counter-selection step by using a competitor ligand norepinephrine resulted in isolation of RNP receptors with defined dopamine-binding characteristics. Increasing the salt condition at the in vitro selection stage afforded RNP receptors with higher dopamine affinity, while addition of norepinephrine in the in vitro selection milieu at the counter-selection step reinforced the selectivity of RNP receptors to dopamine against norepinephrine. Thermodynamic analyses and circular dichroismic studies of the dopamine-RNP complexes suggest that the dopamine-binding RNP with higher selectivity against norepinephrine forms a pre-organized binding pocket and that the dopamine-binding RNP with higher affinity binds dopamine through the induced-fit mechanism. These results indicate that the selection condition controls the ligand-binding mechanism of RNP receptors.     

In vitro selection at cellular level for red rot resistance in sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.)

In the present study, in vitro selection technique using pathogen culture filtrate of Colletotrichum falcatum Went was employed with the aim to identify associations (if any), between selection at the cellular and plant level for red rot resistance in sugarcane (Saccharum sp.). Five to eight months old sugarcane calli of genotypes CoJ 88 and CoJ 64 were screened in vitro against pathogen culture filtrate for two selection cycles. Effect of pathogen culture filtrate on callus survival and/or proliferation was observed to be directly related to its concentration in the selection media. Calli survived and exhibited further proliferation at 5, 10 and 15% v/v pathogen culture filtrate concentrations whereas, at higher concentrations (20 and 25% v/v) proliferation was completely inhibited. Shoot regeneration percent was higher in calli selected on 5% pathogen culture filtrate concentration than those selected on 10 and 15% concentrations. In vivo screening of field transferred somaclones against two pathtypes (Cf 03 and Cf 08) showed considerable variation for red rot resistance. Somaclones regenerated from resistant and/or tolerant calli exhibited better resistance than the parental genotypes. The results indicated that in vitro selection for red rot resistance was effective and expressed when somaclones were screened in the field. This indicated a positive association between in vitro and in vivo methods of selection for disease resistance in sugarcane.     

Selection of proteins with desired properties from natural proteome libraries using mRNA display

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca(2+)-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display-based selection can be accomplished in ~7 d.     

核糖体展示及体外分子选择与进化

核糖体展示是20世纪90年代中期发展起来的一种简便而有效的体外分子选择与进化技术。它也是第一种完全在体外进行蛋白质或多肽分子选择与进化的方法。本主要概述了体外核糖体展示技术的建立基础、基本原理和技术特点等,并跟踪了目前该领域的最新研究进展和发展前景。     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

A general method to select for M13 clones carrying base pair substitution mutants constructed in vitro.

In this paper we describe a method to select base pair substitution mutants constructed in vitro. The mutagenesis is performed by forcing mistakes during in vitro synthesis from a primer annealed to a single stranded DNA template. The selection is based on the fact that, following transformation, the progeny of the strand elongated in vitro and the template strand have different phenotypes. The method is general and applicable to any DNA segment; the type of base pair substitution and its position can be chosen at will. The combined efficiency of mutagenesis and selection allows for 85% frequency of mutants in all analyzed clones.