抗体和寡核苷酸双标记纳米金生物探针的制备及生物学特性

纳米金通过静电吸附抗体, 与寡核苷酸共价结合制备双标记纳米金生物探针, 比较了双标记纳米金生物探针和单标记抗体IgG或ss-DNA的稳定性和反应性. 结果表明, 在水溶液中纳米金由于ss-DNA的结合使IgG抗体的吸附能力明显改善, IgG的吸附也影响二硫苏糖醇(DDT)对ss-DNA的解离作用. 双标记纳米粒上覆盖(50±15)条ss-DNA和(10±2)条IgG, 较单标记ss-DNA纳米金上的(70±15)条要少. 斑点免疫和杂交实验证明, 纳米金表面标记的IgG和ss-DNA具有良好生物学活性. 双标记纳米金生物探针在超微量蛋白质的检测中具有应用价值.     

非放射性多碘标记-偶合催化反应的极谱免疫分析研究

提出非放射性多碘标记-偶合催化反应的极谱免疫分析新体系,该体系以人白蛋白(HSA)作模型抗原,用含4个非放射性碘有机化合物藻红B(EB)经一步化学反应标记HSA.该标记方法简便,条件温和.所得标记物中EB:HSA摩尔比为12:1.用本方法获得的标记蛋白质含碘量远高于其它现有方法.利用标记的含碘抗原对As(Ⅲ)-Ce(Ⅳ)反应的催化作用,用极谱法检测底物中 As(Ⅲ)量变化,建立了异相竞争极谱免疫分析新方法.HSA可测浓度范围为1～200μg/mL,检测限为0.66μg/mL.提出的新体系也适用于其他蛋白质的免疫分析.     

荧光标记寡核苷酸的反相离子对色谱分析

建立荧光标记寡核苷酸反相离子对色谱分析方法,优化了流动相醋酸三乙胺浓度(0～0.15 mol/L), pH 4.5～7.0和洗脱强度等色谱条件.对5-mer, 10-mer和15-mer非标记和5'-羧基荧光素(5'FAM)标记寡核苷酸的保留进行比较分析,研究荧光标记寡核苷酸的保留机理,并分离TaqMan~(TM)探针等多种常用荧光标记寡核苷酸.结果显示,不同长度荧光标记寡核苷酸在0.01 mol/L醋酸三乙胺,pH 7.0的条件下获得最大分离.荧光标记寡核苷酸的保留与非标记寡核苷酸有明显差异,两者可完全分离.在一定长度范围内非标记寡核苷酸随长度的增加,保留时间增长;相反,荧光标记寡核苷酸的长度增加,保留时间减短.荧光染料疏水性对其标记的寡核苷酸在反相柱中的保留有较大影响,荧光染料疏水性越强,其标记寡核苷酸保留时间越长.但疏水性的影响程度随标记寡核苷酸长度增加而逐渐变小.     

稳定同位素标记15N-N,N-二甲基苯胺的合成

以15NH4Cl为同位素标记前体,经与苯甲酰氯胺化、霍夫曼降解得到15N标记苯胺,15N标记苯胺再与对甲苯磺酸酯进行甲基化反应,合成得到一种重要的同位素标记基础试剂15N-N,N-二甲基苯胺.以消耗的15NH4Cl计,三步反应总收率大于58.3%.产品结构经红外(IR)、核磁(NMR)、高效液相(HPLC)、质谱(MS)等表征进行确定,其化学纯度大于99.0%,同位素丰度大于98.4%15N.     

目视化乙肝病毒基因芯片

优化了基于纳米金标记探针的目视化检测方法,将其用于检测从阳性血清中提取的乙肝病毒(HBV)基因,并与基于荧光素异硫氰酸酯(FITC)标记探针的荧光检测方法进行了比较.结果表明,目视化乙肝病毒基因芯片诊断方法操作简单,成本低廉.此类基因芯片在病毒基因检测领域将会有广泛用途.     

新型核素~(64)Cu标记D-脱氧葡萄糖及其小动物正电子发射断层扫描显像

为拓展脱氧葡萄糖(DG)在肿瘤代谢显像中的应用,以新型核素64Cu标记葡萄糖胺-1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(DOTA-DG).通过优化反应条件,于25℃反应30 min后得到高放化纯度和高比活度的标记化合物64Cu-DOTA-DG,标记产物经放射性高效液相色谱(Radio-HPLC)检测.体外稳定性实验结果表明,64Cu-DOTA-DG有良好的稳定性.将64Cu-DOTA-DG通过尾静脉注射入荷瘤肝癌细胞(Hep-G2)裸鼠体内,分别于注射后1和3 h进行小动物正电子发射断层扫描(Micro-PET)显像.结果表明,其在肿瘤部位有所富集.64Cu-DOTA-DG的合成及分子显像研究拓宽了以18F-氟代脱氧葡萄糖为代表的肿瘤代谢类显像剂的应用范围,为新型核素标记肿瘤代谢显像剂提供一种新途径.     

偶合标记铜离子催化放大作用的极谱免疫分析法研究

提出了一种基于标记金属离子催化放大底物转化和极谱检测生成产物的极谱免疫分析新体系.在该体系中,用铜离子代替天然酶作底物转化的催化剂,通过双功能螯合剂二乙三胺五乙酸(DTPA)将其标记于模型抗原白喉类毒素(DT).在竞争免疫反应后,标记铜离子催化底物o-邻苯二胺(OPD)的氧化反应,生成电活性产物2,3-二氨基吩嗪(DAP),然后用单扫描极谱法测定DAP.其方法可测定10～1000ng/mL DT,灵敏度比以往直接检测标记金属离子的方法提高了两个数量级,可应用于多种蛋白质的免疫分析测定.     

基于电喷雾电离质谱(ESI-MS)的苯胺稳定同位素标记对还原性寡糖(链)的定性定量分析方法

建立了一种基于电喷雾电离质谱的苯胺稳定同位素标记对还原性寡糖链进行定性及相对定量分析的研究方法. 用苯胺标记乳糖标准品, 优化了影响标记效率的各种因素, 在弱酸性环境下, 选择糖链/苯胺/硼氢氰化钠的摩尔比为1∶1.2∶10, 于70 ℃反应15 min即可标记完全; 同时考察了4对d0/d5苯胺标记的麦芽糊精寡糖在电喷雾电离质谱中的线性、动态范围以及重现性. 结果表明, 在15倍动态范围内, 相对定量方法呈良好的线性关系(R=0.9986)和重现性(CV=10.20%). 为进一步验证定量方法的可靠性, 将其应用于人奶中游离寡糖(HMOs)和牛奶中游离寡糖(BMOs)的分析. 研究结果表明, 人奶中的乳糖含量高于牛奶, 人奶游离寡糖比牛奶游离寡糖种类复杂, 且岩藻糖基化程度高. 该方法成本低廉, 标记效率高且后处理方法简单方便, 适于微量样品通量化分析, 对差异糖组的研究有重要意义.     

小硅藻光合作用脂13 C稳定同位素定量标记分析

以小硅藻( Nitzschia closterium f. minutissima)作为研究对象,利用超高压液相色谱联用四极杆-静电场轨道阱高分辨质谱技术,研究了处于平台期的小硅藻中的光合作用脂被13 C标记的程度和速度。结果显示：在平台期13 C人工标记的10天内,所有4类光合作用脂均被13 C明显标记,其中二酰甘油双半乳糖脂( DG-DG)、磷脂酰甘油( PG)、二酰甘油硫代糖脂( SQDG)被13 C标记的总量,从刚进入平台期到平台期第8天逐渐增加,此后出现下降趋势,在第8天分别达到最大值(173±24) ng/mg,(473±41) ng/mg 及(1224±21) ng/mg,标记率分别达到56.3%,38.4%和62.6%;而二酰甘油单半乳糖脂(MGDG)被13C标记的总量在整个平台期呈现持续上升趋势,并在第10天达到(956±51) ng/mg,标记率为87.3%。可见,不同脂质在藻体内合成的先后时间有差别,为了清晰地了解生物摄食过程中对微藻中特定脂类的同化机理,需要藻体内被13 C标记的每类脂质含量的最大化,针对DGDG、PG、SQDG,应选取标记了8天的微藻来投喂生物,而针对MGDG,则应选取标记了10天的微藻投喂生物。     

基于磁性分离-硫化铜纳米粒子标记的DNA流动注射化学发光检测

本文基于磁性粒子(MB)良好的分离、富集能力,研究了硫化铜纳米粒子标记的流动注射-化学发光(FI-CL)DNA检测体系.通过硫化铜标记的探针1与目标DNA及连有磁球的探针2形成三明治结构,实现对目标DNA的捕获、分离与标记;通过其溶解释放出CuS标记颗粒的铜离子,引起化学发光信号增强,实现了目标DNA序列的定性定量检测.该方法对完全互补单链DNA(ssDNA)检测的线性范围为1.0×10-11～1.6×10-9 mol/L,检出限为3.0×10-12 mol/L,对1.0×10-9 mol/L目标DNA测定的相对标准偏差为3.2%(n=11),对目标碱基序列具有良好的识别能力.     

Contribution of telomere G-quadruplex stabilization to the inhibition of telomerase-mediated telomere extension by chemical ligands

Inhibition of telomerase activity through stabilizing telomere G-quadruplex with small chemical ligands is emerging as a novel strategy for cancer therapy. For the large number of ligands that have been reported to inhibit telomerase activity, it is difficult to validate the contribution of G-quadruplex stabilization to the overall inhibition. Using a modified telomere repeat amplification protocol (TRAP) method to differentiate the telomere G-quadruplex independent effect from dependent ones, we analyzed several ligands that have high affinity and/or selectivity to telomere G-quadruplex. Our results show that these ligands effectively inhibited telomerase activity in the absence of telomere G-quadruplex. The expected G-quadruplex-dependent inhibition was only obvious for the cationic ligands at low K(+) concentration, but it dramatically decreased at physiological concentration of K(+). These observations demonstrate that the ligands are much more than G-quadruplex stabilizers with a strong G-quadruplex-irrelevant off-target effect. They inhibit telomerase via multiple pathways in which stabilization of telomere G-quadruplex may only make a minor or neglectable contribution under physiologically relevant conditions depending on the stability of telomere G-quadruplex under ligand-free conditions.     

Disubstituted 2-phenyl-benzopyranopyrimidine derivatives as a new type of highly selective ligands for telomeric G-quadruplex DNA

A series of 2-phenyl-benzopyranopyrimidine (PBPP) derivatives with alkylamino side chains were synthesized and found to be a new type of highly selective ligand to bind with telomeric G-quadruplex DNA, and their biological properties were reported for the first time. Their interactions with telomeric G-quadruplex DNA were studied with FRET melting, surface plasmon resonance, CD spectroscopy, and molecular modeling. Our results showed that the disubstituted PBPP derivatives could strongly bind to and effectively stabilize the telomeric G-quadruplex structure, and had significant selectivity for G-quadruplex over duplex DNA. In comparison, the mono substituted derivatives had much less effect on the G-quadruplex, suggesting that the disubstitution of PBPP is essential for its interaction with the G-quadruplex. Furthermore, telomerase inhibition of the PBPP derivatives and their cellular effects were studied, and compound 11b was found to be the most promising compound as a telomerase inhibitor and telomeric G-quadruplex binding ligand for further development for cancer treatment.     

比较蛋白质组学研究中的稳定同位素标记技术

比较蛋白质组学是指在蛋白质组学水平上研究正常和病理情况下细胞或组织中蛋白质表达变化,以期发现具有重要功能的生物标识物,为疾病的早期诊断提供依据。近年来它正成为蛋白质组学研究的热点和发展趋势。比较蛋白质组学的研究方法和策略有多种,本文就最近几年来稳定同位素标记技术(体内代谢标记技术和体外化学标记技术)在比较蛋白质组学研究中的进展进行综述。     

Intercalation of a Heterocyclic Ligand between Quartets in a G-Rich Tetrahelical Structure

YES G-rich oligonucleotide VK2 folds into an AGCGA-quadruplex tetrahelical structure distinct and significantly different from G-quadruplexes, even though it contains four G₃ tracts. Herein, a bis-quinolinium ligand 360A with high affinity for G-quadruplex structures and selective telomerase inhibition is shown to strongly bind to VK2. Upon binding, 360A does not induce a conformational switch from VK2 to an expected G-quadruplex. In contrast, NMR structural study revealed formation of a well-defined VK2–360A complex with a 1:1 binding stoichiometry, in which 360A intercalates between GAGA- and GCGC-quartets in the central cavity of VK2. This is the first high-resolution structure of a G-quadruplex ligand intercalating into a G-rich tetrahelical fold. This unique mode of ligand binding into tetrahelical DNA architecture offers insights into the stabilization of an AGCGA-quadruplex by a heterocyclic ligand and provides guidelines for rational design of novel VK2 binding molecules with selectivity for different DNA secondary structures.     

Specific Binding of Anionic Porphyrin and Phthalocyanine to the G-Quadruplex with a Variety of <em>i</em><em>n Vitro</em> and<em> </em><em>i</em><em>n Vivo</em> Applications

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property.     

Inhibition of translation by small RNA-stabilized mRNA structures in human cells

RNA-mediated gene regulation and expression are critically dependent on both nucleic acid architecture and recognition. We present a novel mechanism for the regulation of gene expression through direct RNA-RNA interactions between small RNA and mRNA in human cells. Using mRNA reporters containing G-rich sequences in the 5'-untranslated region (5'-UTR), in the coding region, or both, we showed that G-rich small RNAs bind to the reporter mRNAs and form an intermolecular RNA G-quadruplex that can inhibit gene translation in living cells. Using a combination of circular dichroism (CD) and RNase footprinting in vitro, we found that the intermolecular G-quadruplexes show a parallel G-quadruplex structure. We next investigated whether the intermolecular G-quadruplex is present in living cells. Employing the fluorophore-labeled probes, we found that two G-rich RNA molecules form an intermolecular G-quadruplex structure in living cells. These results extend the concept of small RNA-mediated expression and suggest an important role for such RNA structures in the inhibition of mRNA translation.     

Biosynthetic Studies on Antibiotics

The intricacies of the synthetic chemistry executed by living cells in the assembly of complex organic natural products can be probed in great detail by stable-isotope labeling in conjunction with sophisticated 2D and multiple-quantum NMR methods of analysis. Mechanistic details are revealed by unraveling the “cryptic” stereochemistry of biosynthetic reactions with the aid of stereospecific isotope labeling. Analysis of blocked mutants and cloning of biosynthetic genes provides new tools for the study of biosynthetic pathways and for the exploitation of the chemical capabilities of living organisms.     

A site-specific low-enrichment (15)N,(13)C isotope-labeling approach to unambiguous NMR spectral assignments in nucleic acids

We demonstrate that base and sugar protons within a DNA oligomer can be assigned unambiguously following site-specific 15N,13C isotope incorporation at levels as low as 1% enrichment. This simple and cost-effective methodology is demonstrated on the d(GGGTTCAGG) DNA sequence, which forms a dimeric G-quadruplex containing G.G.G.G tetrads sandwiched between G.(C-A) triads.     

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity**

A facile imide coupling strategy for the one-step preparation of G-quadruplex ligands with varied core chemistries is described. The G-quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV-Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G-quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3 , which was based on a perylene scaffold and exhibited 40-fold selectivity for a telomeric G-quadruplex over duplex DNA. Surprisingly, a tetra-substituted flexible core, compound 11 , also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78-fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.