A NOVEL APPROACH TO SOLID PHASE CHEMICAL SYNTHESIS OF OLIGONUCLEOTIDE mRNA CAP ANALOGS

A novel approach for the synthesis of 5′-capped 2′-O-methyloligoribonucleotides on a disulfide-tethered solid support is described. The key step of the synthesis is ZnCl₂ promoted coupling of m⁷GDP imidazolide to a fully deprotected oligonucleotide 5′-phosphate on-support. By this methodology m⁷G⁵′pppm²′Apm²′Upm^2′Ap has been prepared.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

cDNA cloning and sequencing of tarantula hemocyanin subunits

Summary Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.     

人体小卫星DNA探针的制备

根据人体小卫星DNA核心顺序,化学合成长23碱基寡核苷酸探针,筛选人体基因组文库,旨在获得能用作遗传分析探针的小卫星顺序。结果得到15个含小卫星的阳性重组子。随机取其一(C_(35.9))作探针,试做群体分析。所有个体均可检出多条杂交带。其中某些带具有多态性。在一定检测条件下,检出的DNA图谱在有限的个体内具有个体特异性。结果表明筛选文库得到的小卫星顺序可用于小卫星多态性的检测。其它小卫星探针的筛选和应用性研究正在进行。     

经体外缺失诱变组建人杂交干扰素αA/γ

使用寡核苷酸指导的定点突变方法,将人αA型干扰素的完整基因与γ干扰素C端16个氨基酸的编码序列融合,在噬菌体λP_L启动子控制下,合成了一个杂交蛋白质。此蛋白质经抗人α干扰素单克隆抗体纯化后,在MDBK细胞上具有抗病毒活性,并像γ干扰素一样,可被依赖于cAMP的蛋白激酶磷酸化。[γ-~(32)P]杂交蛋白与MDBK细胞的结合,可被αA型干扰素大大抑制(70％)。     

用改进的寡核苷酸诱导突变法改造人胰岛素前体基因

我们用改进了的寡聚核苷酸诱导突变法,将两个单一限制性内切酶位点引入人胰岛素前体B链与C链连接处附近,及C链与A铁连接处附近。用含U模板法将B链第30个残基密码子ACC改成ACG,从而引入MluⅠ位点。用修改了的缺口双链DNA突变法,将A链第4个残基密码于GAG改成CTG,引入了一个PstⅠ位点。突变效率的为17％-36％。这个突变体在人胰岛素前体结构及蛋白质折叠的研究中,将有利于更换不同的C-肽。     

Limiting concentrations of activated mononucleotides necessary for poly(C)-directed elongation of oligoguanylates

Summary Selected imidazolide-activated nucleotides have been subjected to hydrolysis under conditions similar to those that favor their template-directed oligomerization. Rate constants of hydrolysis of the P–N bond in guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-monophosphate imidazolide (ImpG), k_h, have been determined in the presence/absence of magnesium ion as a function of temperature and polycytidylate [poly(C)] concentration. Using the rate constant of hydrolysis of 2-MeImpG and the rate constant of elongation, i.e., the reaction of an oligoguanylate with 2-MeImpG in the presence of poly(C) acting as template, the limiting concentration of 2-MeImpG necessary for oligonucleotide elongation to compete with hydrolysis can be calculated. The limiting concentration is defined as the initial concentration of monomer that results in its equal consumption by hydrolysis and by elongation. These limiting concentrations of 2-MeImpG are found to be 1.7 mM at 37°C and 0.36 mM at 1°C. Boundary conditions in the form of limiting concentration of activated nucleotide may be used to evaluate a prebiotic model for chemical synthesis of biopolymers. For instance, the limiting concentration of monomer can be used as a basis of comparison among catalytic, but nonenzymatic, RNA-type systems.We also determined the rate constant of dimerization of 2-MeImpG, k₂=0.45±0.06 M^–1 h^–1 in the absence of poly(C), and 0.45±0.06k₂0.97±0.13 M^–1 h^–1 in its presence at 37°C and pH 7.95. This dimerization, as well as the trimerization of 2-MeImpG, which represent the first steps in the oligomerization reaction, are markedly slower than the elongation of longer oligoguanylates, (pG) _n n>6. This means that in the presence of low concentrations of 2-MeImpG (1.7 mM) the system directs the elongation of longer oligomers more efficiently than the formation of short oligomers such as dimers and trimers. These results will be discussed as a possible example of chemical selection in template-directed reactions of nucleotides.     

几株杆状病毒包涵体蛋白双向高压电泳指纹图谱比较

用蔗糖密度梯度离心提纯的6种昆虫病毒包涵体(BsNPV1,BsNPV2,BtNPV,EpNPV,PrGV,PxGV)经DAS碱解,粗提纯的包涵体蛋白经Sepharose-6B柱层析和Caaalco-prep-disc法进一步纯化后,在SDS-PAGE中显示为一条分子量约28,000d的带。用常规双向免疫扩散法检查各种包涵体蛋白之间的差异,结果表明血清学BsNPV与BtNPV,PrGV与PxGV之间都是不可分辩的。6种包涵体蛋白的双向高压指纹图谱表明,它们的指纹图谱都不同程度地存在着一些相同或十分相似的肽点。就每种病毒的指纹图谱又都是独特的,NPV间相似性低于GV间相似性。我们认为NPV和GV的包涵体蛋白在结构上存在着一些相同的或相似的保守区域,但不同种之间在整个一级结构上是有差异的。利用包涵体蛋白的指纹图谱鉴定杆状病毒是个灵敏可行的方法。