核酶的研究进展

核酶是一类具有催化功能的核酸分子.对天然核酶的改造和人工核酶的筛选使核酶的催化反应谱得到了扩展,也为核酶作为基因治疗药物和核酸探针提供了多种选择.现综述核酶在基因治疗和靶基因识别等方面的研究进展,并提出目前核酶研究中存在问题及相关对策.针对各种致病基因,小型核酶和普适性脱氧核酶“10-23”在细胞水平显示了诱人的前景.随着有效的转运和表达方法的建立及核酸化学的进步,核酶将成为基因治疗方法之一.     

基因治疗新策略——酶性DNA的设计和应用

本文综述了能够剪切RNA分子的酶性DNA的种类、结构、作用机制、动力学特征及其在用作新型基因治疗药物方面的优势、设计要点、应用现状与前景等最新研究进展。     

icl mRNA特异性10-23DRz在小鼠体内抗结核分枝杆菌感染的实验研究

目的探讨iclmRNA特异性10-23脱氧核酶(deoxyribozyme,DRz)对结核分枝杆菌(Mycobacterium tuberculosis,Mtb)在小鼠体内感染的影响,及其单独或与异烟肼(isoniazidum,INH)联合应用治疗小鼠结核病的效果。方法分别用DZ4、INH、INH+DZ4-S或INH+DZ4对Mtb进行预处理,再用上述经过预处理的Mtb感染BALB/c小鼠,一定时间后进行小鼠肺组织Mtb培养及病理学改变观察。利用DZ4-FITC溶液对BALB/c小鼠进行滴鼻处理,检测滴鼻途径给药的效果。采用尾静脉注射法建立小鼠结核病模型,将模型小鼠随机分为5组(n=10),分别给予生理盐水、DZ4、INH、INH+DZ4、INH+DZ4-polyG治疗,于治疗完成2周后进行小鼠肺组织Mtb荷菌量检测和病理学改变观察。结果Mtb感染后2周,各组小鼠的肺组织荷菌量均无显著性差异(P>0.05)。感染进行至4～12周时,INH+DZ4预处理组Mtb感染小鼠的肺组织荷菌量较其它各组均显著偏低(P<0.05),肺组织的病理改变较其他各组也明显轻微。经滴鼻途径给予DZ4-FITC溶液4h后,小鼠肺组织冰...     

10-23 脱氧核酶的生物催化功能研究进展

10-23脱氧核酶是利用体外筛选技术得到的一种具有催化功能的单链DNA片段,具有高效的催化活性及特异的序列识别能力,能催化RNA特定部位的切割反应,从mRNA水平上抑制基因表达,具有潜在的治疗应用价值。     

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na⁺), has remained underdeveloped, even though Na⁺ is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na⁺-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (k_obs) ∼0.1 min⁻¹], and the transformation of this DNAzyme into a fluorescent sensor for Na⁺ by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na⁺ catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na⁺ over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na⁺-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na⁺ in living cells.Metal ions play crucial roles in a variety of biochemical processes. As a result, the concentrations of cellular metal ions have to be highly regulated in different parts of cells, as both deficiency and surplus of metal ions can disrupt normal functions (1–4). To better understand the functions of metal ions in biology, it is important to detect metal ions selectively in living cells; such an endeavor will not only result in better understanding of cellular processes but also novel ways to reprogram these processes to achieve novel functions for biotechnological applications.Among the metal ions in cells, sodium (Na⁺) serves particularly important functions, as changes in its concentrations influence the cellular processes of numerous living organisms and cells (5–8), such as epithelial and other excitable cells (9). As one of the most abundant metal ions in intracellular fluid (10), Na⁺ affects cellular processes by triggering the activation of many signal transduction pathways, as well as influencing the actions of hormones (11). Therefore, it is important to carefully monitor the concentrations of Na⁺ in cells. Toward this goal, instrumental analyses by atomic absorption spectroscopy (12), X-ray fluorescence microscopy (13), and ²³Na NMR (14) have been used to detect the concentration of intracellular Na⁺. However, it is difficult to use these methods to obtain real-time dynamics of Na⁺ distribution in living cells. Fluorescent sensors provide an excellent choice to overcome this difficulty, as they can provide sensitive detection with high spatial and temporal resolution. However, despite significant efforts in developing fluorescent metal ion sensors, such as those based on either genetically encoded probes or small molecular sensors, most fluorescent sensors reported so far can detect divalent metal ions such as Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ (15–21). Among the limited number of Na⁺ sensors, such as sodium-binding benzofuran isophthalate (22), Sodium Green (23), CoroNa Green/Red (24, 25), and Asante NaTRIUM Green-1/2 (26), most of them are not selective for Na⁺ over K⁺ (22–25, 27, 28) or have a low binding affinity for Na⁺ (with a K_d higher than 100 mM) (25, 27–31). Furthermore, the presence of organic solvents is frequently required to achieve the desired sensitivity and selectivity for many of the Na⁺ probes (32–34), making it difficult to study Na⁺ under physiological conditions. Therefore, it is still a major challenge to design fluorescent sensors with strong affinity for Na⁺ and high selectivity over other monovalent and multivalent metal ions that work under physiological conditions.To meet this challenge, our group and others have taken advantage of an emerging class of metalloenzymes called DNAzymes (deoxyribozymes or catalytic DNA) and turned them into metal ion probes. DNAzymes were first discovered in 1994 through a combinatorial process called in vitro selection (35). Since then, many DNAzymes have been isolated via this selection process. Among them, RNA-cleaving DNAzymes are of particular interest for metal ion sensing, due to their fast reaction rate and because the cleavage, which is catalyzed by a metal ion cofactor, can easily be converted into a detectable signal (36–38). Unlike the rational design of either small-molecule or genetically encoded protein sensors, DNAzymes with desired sensitivity and specificity for a metal ion of interest can be selected from a large library of DNA molecules, containing up to 10¹⁵ different sequences (35, 39). A major advantage of DNAzymes as metal ion sensors is that metal-selective DNAzymes can be obtained without prior knowledge of necessary metal ion binding sites or specific metal–DNA interaction (40, 41). In addition, through the in vitro selection process, metal ion binding affinity and selectivity can be improved by tuning the stringency of selection pressure and introducing negative selection against competing metal ions (39, 40). Finally, DNA is easily synthesized with a variety of useful modifications and its biocompatibility makes DNAzyme-based sensors excellent tools for live-cell imaging of metal ions. As a result, several metal-specific DNAzymes have been isolated and converted into sensors for their respective metal ion cofactors, including Pb²⁺ (35, 42, 43), Cu²⁺ (44, 45), Zn²⁺ (46), UO₂²⁺ (47), and Hg²⁺ (48). They have recently been delivered into cells for monitoring UO₂²⁺ (41, 49), Pb²⁺ (50), Zn²⁺ (51), and histidine (52) in living cells.However, in contrast to the previously reported DNAzymes with divalent metal ion selectivity, no DNAzymes have been reported to have high selectivity toward a specific monovalent metal ion. Although DNAzymes that are independent of divalent metal ions have been obtained (53–55), including those using modified nucleosides with protein-like functionalities (i.e., guanidinium and imidazole) (56–58), no DNAzyme has been found to be selective for a specific monovalent metal ion over other monovalent metal ions. For example, the DNAzyme with the highest reported selectivity for Na⁺ still binds Na⁺ over K⁺ with only 1.3-fold selectivity (54). More importantly, those DNAzymes require very high concentrations of monovalent ions (molar ranges) to function and display very slow catalytic rates (e.g., 10⁻³ min⁻¹) (53–55). The poor selectivity, sensitivity, and slow catalytic rate render these DNAzymes unsuitable for cellular detection of Na⁺, due to interference from other monovalent ions such as K⁺ (which is present in concentrations about 10-fold higher than Na⁺), and the need to image the Na⁺ rapidly.In this study, we report the in vitro selection and characterization of an RNA-cleaving DNAzyme with exceptionally high selectivity (>10,000-fold) for Na⁺ over other competing metal ions, with a dynamic range covering the physiological Na⁺ concentration range (0.135–50 mM) and a fast catalytic rate (k_obs, ∼0.1 min⁻¹). This Na⁺-specific DNAzyme was transformed into a DNAzyme-based fluorescent sensor for imaging intracellular Na⁺ in living cells, by adopting an efficient DNAzyme delivery method using a cationic polypeptide, together with a photocaging strategy to allow controllable activation of the probe inside cells.     

10-23脱氧核酶对新生大鼠心肌细胞ET-1mRNA表达的影响

目的:将体外筛选具有切割内皮素-1(endothelin-1,ET-1)mRNA的10-23脱氧核酶转染原代培养新生大鼠心肌细胞,观察其对心肌细胞ET-1 mRNA的影响.方法:体外转录ET-1全长RNA底物,设计并合成5条ET-1 10-23脱氧核酶(DZ1～DZ5),其5‘及3‘端各有2个核苷酸硫代修饰,体外切割ET-1 RNA底物,筛选有效脱氧核酶;5‘标记荧光素FAM的10-23脱氧核酶瞬间转染新生大鼠心肌细胞以观察对10-23脱氧核酶的摄取;采用半定量RT-PCR检测ET-1基因的表达.结果:DZ2、DZ3、DZ4及DZ5在体外均可切割RNA底物,其中DZ4两侧结合区结合自由能之差最大,其切割效率最高,达83.5%,而DZ3两侧结合区结合自由能之差最小,其切割效率亦最低(47.3%);瞬间转染新生大鼠心肌细胞24h,心肌细胞内可见10-23脱氧核酶的分布,半定量RT-PCR结果显示转染24h后的DZ4(0.2μmol/L)可减少血清诱导肥大心肌细胞ET-1mRNA及细胞总蛋白,降低细胞活力与蛋白质合成速率(P＜0.05).结论:本研究设计的10-23脱氧核酶能切割体外转录的ET-1全长RNA底物,转染心肌细胞后,可抑制ET-1 mRNA的表达,减缓血清诱导心肌细胞肥大,10-23脱氧核酶两侧结合区自由能的差值与切割效率有关.     

计算机辅助设计抗MRSA耐药基因mecR1的10-23型脱氧核酶

应用Primer Premier 5.0和RNA structure 4.6两种软件设计和筛选有效抗mecR1的10-23型脱氧核酶（DRz）,采用电转化的方法将其导入细菌体内,实时PCR的方法定量检测其对mecR1转录的影响,并通过平板克隆形成实验观察脱氧核酶抑制细菌生长的情况。结果表明,计算机辅助设计合成的5条抗MRSA耐药调控基因mecR1的10-23型脱氧核酶,它们能不同程度的降低mecR1的转录水平,有效抑制临床耐药菌MRSA080309的生长,其中DRz6抑制效果最显著。说明联合应用这两种计算机软件设计抗mecR1的10-23型脱氧核酶,是一种经济实用而有效的方法,能够大大缩短有效反义药物的筛选时间。     

“10～23”型脱氧核酶的研究进展

脱氧核酶是利用体外分子进化技术获得的一种具有酶活性的单链DNA分子。迄今为止，利用该技术已筛选出多种具有催化功能的脱氧核酶分子，其中研究最多的是具有RNA切割活性的脱氧核酶，尤其是10～23型脱氧核酶，该酶能催化RNA特定部位的切割反应，从mRNA水平使基因灭活，从而调控蛋白质的表达，在抗肿瘤、抗病毒等基因治疗领域具有广阔的应用前景。     

二级结构模拟预测HBV脱氧核酶有效作用靶点

目的:通过二级结构模拟预测作用靶点,观察HBV特异性脱氧核酶的体外切割活性,探讨其用于抗病毒制剂的可能性。方法:以HBVX基因mRNA序列为靶设计脱氧核酶DRz-X29,对其特异的切割活性进行体外实验。结果:特定反应条件下,DRz-X29成功对二级结构模拟预测的底物靶点进行了特异切割。结论:底物二级结构模拟能够有效预测HBV脱氧核酶作用靶点,为其发展成一种新型抗病毒制剂奠定了实验基础。