适体酶的筛选及应用

适体酶也称为别构效应核酶或别构效应脱氧核酶,是一种新的人工合成酶,它兼具适体的靶物质特异性结合与核酶或脱氧核酶的催化活性优点。这种酶是从大量随机序列库中针对各种效应分子筛选获得的目的酶。适体酶为效应分子的定量分析提供了新的思路。适体酶不仅在基因组学和蛋白质组学研究中得到应用,而且在生物传感器和DNA分子逻辑研究中也具有潜在的应用前景。     

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.     

RNA适体研究及其在医药上的应用

核酸适体（nucleic acid aptamer）是从人工合成的随机单链核酸库中筛选出的特异性与靶物质高度亲和的核酸分子,包括DNA适体和RNA适体. 体外获得核酸适体的方法称为指数富集配体系统进化技术,即SELEX（systematic evolution of ligands by exponential enrichment）. 在SELEX技术获得的核酸适体中,RNA适体因其结构的多样性而具有靶分子广、亲和力高、特异性强等特点. 同时,相比传统抗体,RNA适体分子量小、易改造修饰、制备方便且无免疫原性. 因此,RNA适体在基础研究、临床诊断、药物研制等方面展现了广阔的应用前景. 本文综述了RNA适体的产生、特点、作用方式、优势与局限性,并详细介绍了其在医药研究领域的应用.     

茶碱激活型RNA分子开关的构建及在枯草芽胞杆菌中调节外源基因表达的性能

枯草芽胞杆菌Bacillussubtilis在工业生物技术以及合成生物学领域作为一种重要的微生物可广泛用作代谢工程、重组蛋白表达以及新型基因电路的底盘。在B. subtilis中构建基于非编码RNA的高精准调节元件,能够实现不依赖蛋白质因子的基因表达调控,丰富B.subtilis基因表达通用工具。通过基因工程手段,设计了基于茶碱适体域的核糖开关E和适体核酶AZ调节元件,并与不同的B.subtilis内源组成型启动子适配,构建出茶碱激活型基因表达控制元件。测定这两种调节元件与6种组成型启动子组合匹配下报告基因GFP的荧光强度,鉴定并分析各调控元件的工作性能。并进一步以红色荧光蛋白mCherry和普鲁兰酶两种不同的异源蛋白验证核糖开关或适体核酶与启动子的最优组合。结果表明,同一种RNA调节元件与不同启动子组合呈现不同水平的调控效率。在核糖开关与启动子的组合中,启动子PsigW和核糖开关E组合(sigWE)对GFP表达的诱导率最高,达到16.8。在适体核酶与启动子的组合中,AZ与启动子P43、PrpoB组合(P43AZ和rpoBAZ)的诱导率最高,分别达到了6.1和6.2。进一步验证结果显示,sigWE调控mCherry的诱导率最高(9.2),而P43E调控普鲁兰酶的诱导率最高(32.8),产酶水平达到了81U/mL。核糖开关和适体核酶对GFP、mCherry、普鲁兰酶均能实现调控,但是不同元件组合的调控性能有所差异,对不同基因的调控效果也不尽相同。     

适体的临床应用

适体(aptamer)因其与靶分子结合的高亲合力及强特异性,而具有广泛的应用价值。近年来对适体功能的研究多集中在分子和细胞水平,要将适体应用于临床治疗必须进一步对适体在动物体内及人体内的活性功能进行研究。本文对已处于动物实验或临床实验阶段的适体进行综述。     

以分子信标为报告分子的凝血酶检测新方法

以分子信标为报告分子,核酸适体为识别分子,发展了一种新的凝血酶检测方法.含有分子信标互补序列的核酸适体探针与凝血酶结合后,分子信标的荧光信号下降,从而得到凝血酶的浓度信息.该方法快速、灵敏,核酸适体探针无需荧光标记、设计简单,检测限达到0.83nmol/L.     

信号适体的设计策略及应用

信号适体兼具有分子识别和信号转导的功能.从随机寡核苷酸库中筛选出的适体,要经过合理设计和筛选后修饰,才具备信号转导功能.信号适体可分为标记和非标记两大类.本文着重介绍荧光标记信号适体的设计策略,包括基于荧光偏振分析标记一个荧光基团,及基于荧光共振能量转移同时标记荧光基团、淬灭基团,或两个荧光基团的信号适体(包括分子信标适体、结构转换和原位标记信号适体).非标记信号适体的设计,有嵌合法、置换法、光转换复合物法,及适体－多聚物偶联法.此外,亦可直接从体外筛选出信号适体.信号适体的诸多优点利于其用于生物传感器及均相液相中实时蛋白识别与定量分析.     

DNA生物催化功能研究进展

近年来发现 ,不少结构特殊的DNA分子分别具有剪切RNA分子或DNA分子、T4聚核苷酸激酶样活性、DNA连接酶样活性以及催化卟啉金属离子化等多种生物催化功能 ,这些DNA分子被称为脱氧核酶或酶性DNA .它们在用作RNA和DNA工具酶、基因分析和诊断手段以及基因治疗药物等方面的潜力引人注目 .综述这些DNA分子的种类、结构特征、催化活性及应用现状和前景等方面的最新研究进展     

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。     

SELEX技术在病毒感染诊断和治疗中的应用

指数富集的配体系统进化（SELEX）技术是一种新的组合化学技术,它利用人工合成的随机寡核苷酸文库,通过体外多轮筛选与扩增,获得能与靶物质特异性结合的寡核苷酸适体。适体的靶分子广泛,包括病毒代谢相关产物,且与靶物质结合的亲和力高、特异性强,在体内代谢及稳定性等方面优于抗体。在细胞和动物模型中,适体显示出很多优于抗体的特性,而且已经有适体作为药物进入临床试验阶段。这种体外筛选技术是一种较成熟的技术,由此产生的适体具有较好的理化特征,可以抑制病毒复制感染的各个阶段,而且在病毒感染所引发的相关疾病诊断和治疗等方面具有较好的应用前景。     

Simultaneous detection of diverse analytes with an aptazyme ligase array

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.     

Combined kinetic mechanism describing activation and inhibition of muscle glycogen phosphorylase b by adenosine 5'-monophosphate

The kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of concentrations of AMP under the saturation of the enzyme by glycogen. The applicability of 23 different variants of the kinetic model involving the interaction of AMP and glucose 1-phosphate binding sites in the dimeric enzyme molecule is considered. A kinetic model has been proposed which assumes: (i) the independent binding of one molecule of glucose 1-phosphate in the catalytic site on the one hand, and AMP in both allosteric effector sites and both nucleoside inhibitor sites of the dimeric enzyme molecule bound by glycogen on the other hand; (ii) the binding of AMP in one of the allosteric effector sites results in an increase in the affinity of other allosteric effector site to AMP; (iii) the independent binding of AMP to the nucleoside inhibitor sites of the dimeric enzyme molecule; (iv) the exclusive binding of the second molecule of glucose 1-phosphate in the catalytic site of glycogen phosphorylase b containing two molecules of AMP occupying both allosteric effector sites; and (v) the catalytic act occurs exclusively in the complex of the enzyme with glycogen, two molecules of AMP occupying both allosteric effector sites, and two molecules of glucose 1-phosphate occupying both catalytic sites.     

CYP2E1 substrate inhibition. Mechanistic interpretation through an effector site for monocyclic compounds

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min(-1)) with relatively low K(m) (24 microM); nevertheless, at concentrations of >100 microM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K(ss) = 260 microm), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K(i) for the catalytic site was 2.0 microM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K(ss). Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K(i) = 0.12 microM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.     

Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.     

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.     

Structure of the E.coli aspartate transcarbamoylase trapped in the middle of the catalytic cycle

Snapshots of the catalytic cycle of the allosteric enzyme aspartate transcarbamoylase have been obtained via X-ray crystallography. The enzyme in the high-activity high-affinity R state contains two catalytic chains in the asymmetric unit that are different. The active site in one chain is empty, while the active site in the other chain contains an analog of the first substrate to bind in the ordered mechanism of the reaction. Small angle X-ray scattering shows that once the enzyme is converted to the R state, by substrate binding, the enzyme remains in the R state until substrates are exhausted. Thus, this structure represents the active form of the enzyme trapped at two different stages in the catalytic cycle, before the substrates bind (or after the products are released), and after the first substrate binds. Opening and closing of the catalytic chain domains explains how the catalytic cycle occurs while the enzyme remains globally in the R-quaternary structure.     

Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase.

The allosteric effectors of aspartate transcarbamoylase from Escherichia coli, CTP and ATP, associate with both the regulatory and the catalytic moieties of the enzyme. Studies with isolated, active subunits yield one binding site per regulatory dimer and one per catalytic trimer. Investigations of effector association with hybrid enzymes, containing either the three regulatory dimers or the two catalytic trimers in inactivated forms, indicate that the data obtained with isolated subunits can be used to analyze the binding patterns of these ligands to the native hexamer. Thus, the nonlinear Scatchard plots, characteristic of the binding of CTP and ATP to the native enzyme, can be interpreted in terms of three effector molecules associating with the regulatory subunits, and two binding to the catalytic moiety of the enzyme. Results with native protein in the presence of saturating concentrations of active site ligands support these assignments. The differences between the binding isotherms of CTP and ATP to the enzyme are due to their different affinities to the two types of subunits. The apparent half-of-the-site saturation of the regulatory moiety of aspartate transcarbamoylase supports the concept that this protein has a tendency to exist in an asymmetric state.     

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general.