Terminal Protection of Small Molecule-Linked DNA for Small Molecule–Protein Interaction Assays

Methods for the detection of specific interactions between diverse proteins and various small-molecule ligands are of significant importance in understanding the mechanisms of many critical physiological processes of organisms. The techniques also represent a major avenue to drug screening, molecular diagnostics, and public safety monitoring. Terminal protection assay of small molecule-linked DNA is a demonstrated novel methodology which has exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of small molecule–protein interactions. Herein, we review the basic principle of terminal protection assay, the development of associated methods, and the signal amplification strategies adopted for performance improving in small molecule–protein interaction assay.     

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity

This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca²⁺-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca²⁺ at 22–65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκβα and Bcl-2. In contrast, rMbovNase^Δ181–342 without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.     

裂解基因E和核酸酶基因串联表达载体的构建及大肠杆菌菌影的制备

目的构建噬菌体裂解基因E和核酸酶基因串联表达载体,制备高质量的大肠杆菌菌影。方法自行设计一对引物,PCR扩增PhiX174噬菌体裂解基因E,将该基因亚克隆至原核表达载体pGEX-6P-1,构建大肠杆菌菌影的表达载体pGEX-E。在E基因基础上与辅助基因葡萄球菌核酸酶A(SN)基因串联,并插入pGEX-6P-1载体,构建双基因串联高效表达载体pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,采用CaCl2法将其转入大肠杆菌BL21(DE3),经IPTG诱导,制备大肠杆菌菌影。结果裂解基因E单基因及串联基因已成功插入融合表达载体pGEX-6P-1,所构建的大肠杆菌菌影表达载体pGEX-E、pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,诱导后经透射电镜及活菌计数显示大肠杆菌已发生不同程度裂解,制成了大肠杆菌菌影。电镜下菌影形态完整,内容物已释放到胞外。结论已成功构建了噬菌体裂解基因E单基因及裂解基因和核酸酶基因串联基因表达载体,并制成了大肠杆菌菌影,为进一步研究菌影这一新型的疫苗及佐剂形式奠定了基础。     

Copper(II) Complexes Containing Natural Flavonoid Pomiferin Show Considerable In Vitro Cytotoxicity and Anti-inflammatory Effects

A series of new heteroleptic copper(II) complexes of the composition [Cu(L)(bpy)]NO₃·2MeOH (1), [Cu(L)(dimebpy)]NO₃·2H₂O (2), [Cu(L)(phen)]NO₃·2MeOH (3), [Cu(L)(bphen)]NO₃·MeOH (4), [Cu(L)(dppz)]NO₃·MeOH (5) was prepared, where HL = 3-(3,4-dihydroxyphenyl)-5-hydroxy-8,8-dimethyl-6-(3-methylbut-2-ene-1-yl)-4H,8H-benzo[1,2-b:3,4-b′]dipyran-4-one, (pomiferin) and bpy = 2,2′-bipyridine, dimebpy = 4,4′-dimethyl-2,2′-bipyridine, phen = 1,10-phenanthroline, bphen = 4,7-diphenyl-1,10-phenanthroline, and dppz = dipyrido[3,2-a:2′,3′-c]phenazine. The complexes were characterized using elemental analysis, infrared and UV/Vis spectroscopies, mass spectrometry, thermal analysis and conductivity measurements. The in vitro cytotoxicity, screened against eight human cancer cell lines (breast adenocarcinoma (MCF-7), osteosarcoma (HOS), lung adenocarcinoma (A549), prostate adenocarcinoma (PC-3), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), colorectal adenocarcinoma (Caco-2) and monocytic leukemia (THP-1), revealed the complexes as effective antiproliferative agents, with the IC₅₀ values of 2.2–13.0 μM for the best performing complexes 3 and 5. All the complexes 1–5 showed the best activity against the A2780R cells (IC₅₀ = 2.2–6.6 μM), and moreover, the complexes demonstrated relatively low toxicity on healthy human hepatocytes, with IC₅₀ > 100 μM. The complexes were evaluated by the Annexin V/propidium iodide apoptosis assay, induction of cell cycle modifications in A2780 cells, production of reactive oxygen species (ROS), perturbation of mitochondrial membrane potential, inhibition of apoptosis and inflammation-related signaling pathways (NF-κB/AP-1 activity, NF-κB translocation, TNF-α secretion), and tested for nuclease mimicking activity. The obtained results revealed the corresponding complexes to be effective antiproliferative and anti-inflammatory agents.     

Chemical Approach to Biological Safety: Molecular‐Level Control of an Integrated Zinc Finger Nuclease

Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off‐target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy, and nano‐electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator, DNA‐binding, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.     

氨基树脂表面活化提高固定化核酸酶P1连续催化性能

考察了固定化酶常用载体-氨基树脂几何结构及表面活化过程对固定化核酸酶P₁性能的影响,并进行了动力学和连续催化稳定性研究。FESEM、BET及FTIR等表征发现,氨基树脂具有大量酶可利用的孔,在固定化过程中核酸酶P₁主要利用的孔径范围为4～30 nm。相对游离的核酸酶P₁而言,所得固定酶耐酸、耐热性增强;米氏动力学研究表明各组固定酶对底物的亲和力下降,最大反应速率下降;后交联组的重复利用性相对物理吸附、化学交联组明显增强。优化的固定化条件为：酶和载体比例为3：20（质量比）,酶浓度为0.8 g·L^-1,酶液pH为6.0,固定时间为10 h,在优化条件下所得固定酶的单位载体酶活为10013 U·g^-1。同时设计及优化柱连续反应器操作条件,确定反应温度为65℃,进料流量为0.75 ml·min^-1,使得产品的核苷酸浓度维持30 g·L^-1（水解率为60%）以上的累计时长达120 h,有利于核酸酶P₁连续生产核苷酸的工业化应用。     

应用生物核酸酶提高原油采收率实验研究

实验考察了生物核酸酶溶液的酸碱性、与注入水和地层水的配伍性、对界面张力和驱油效率的影响,对地层的伤害性、钢的腐蚀性以及原油凝固点的影响。结果表明,生物核酸酶与注入水和地层水配伍性好,配制的溶液呈弱碱性,能降低界面张力,注入0．5PV的0．5％生物核酸酶溶液可提高驱油效率17．60％,对岩心有一定伤害性,能改善或缓解注入水对钢表面的腐蚀性,可使原油凝固点降f氐4．5％一11％。