Thermodynamic Melting Studies on Oligonucleotide-Peptide Conjugates

Abstract

A small library of oligonucleotide-peptide conjugates has been prepared and studied to explore the influence of the various peptide side chain (cationic, anionic or hydrophobic) on the hybridation properties of the DNA.     

Galactose-Containing Polymer-DOX Conjugates for Targeting Drug Delivery

A novel multifunctional drug delivery system was fabricated by conjugating galactose-based polymer, methoxy-poly(ethylene glycol)-block-poly(6-O-methacryloyl-D-galactopyranose) (mPEG-b-PMAGP) with doxorubicin (DOX) via an acid-labile carbamate linkage. The mPEG-b-PMAGP-co-DOX nanoparticles were spherical in shape, and the diameter determined by dynamic light scattering (DLS) was 54.84?±?0.58 nm, larger than that characterized by transmission electron microscopy (TEM). The in vitro drug release profiles were studied, and the release of DOX from the nanoparticles was pH-responsive. The cellular uptake behavior of free-DOX and mPEG-b-PMAGP-co-DOX nanoparticles by asialoglycoprotein (ASGP) receptor-positive cancer cell line (HepG2) and ASGP receptor-negative cancer cell lines (MCF-7 and A549 cells) was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM), respectively. The mPEG-b-PMAGP-co-DOX nanoparticles which contain galactose functional groups exhibited higher cellular uptake behavior via ASGP receptor-mediated endocytosis in HepG2 cells than in other two cancer cells. The in vitro cytotoxicity assay manifested that the mPEG-b-PMAGP-co-DOX nanoparticles exhibited higher anticancer efficacy against HepG2 cells than MCF-7 cells. These results indicated that the multifunctional mPEG-b-PMAGP-co-DOX nanoparticles possessing pH-responsible and hepatoma-targeting function have great potential to be used as a targeting drug delivery system for hepatoma therapy.     

缩微芯片实验室

生物芯片技术是近年发展起来的新型实用技术 ,已成为高效、大规模获取相关生物信息的重要手段。所谓生物芯片就是缩小了的生物化学分析器 ,通过芯片上微加工获得的微米结构和生物化学处理结合 ,将成千上万个与生命相关的信息集成在一块厘米见方的氧化硅、玻璃或塑料等材质制成的芯片上。目前该技术的应用领域主要有基因表达谱分析、新基因发现、基因突变及多态性分析、基因组文库作图、疾病诊断、药物筛选、基因测序等。从 80年代初ＳＢＨ (ｓｅｑｕｅｎｃｉｎｇｂｙｈｙ ｂｒｉｄｉｚａｔｉｏｎ)概念的提出 ,到 90年代初以美国为主开始进行…     

抗肿瘤抗体-药物偶联物的临床研究进展

个体化靶向治疗已成为肿瘤临床治疗的新趋势.抗肿瘤靶向药物与传统的细胞毒性化疗药物相比具有特异性高、选择性强和非细胞毒性等优点,近年来发展迅速.抗体-药物偶联物(ADCs)属于抗肿瘤靶向药物,由抗体、“弹头”药物(细胞毒性药物)通过链分子连接而成.ADCs将抗体的靶向性与细胞毒性药物的抗肿瘤作用相结合,可以降低细胞毒性抗肿瘤药物的不良反应,提高肿瘤治疗的选择性,还能更好地应对靶向单抗的耐药性问题.目前,FDA已批准2种ADC药物上市,即Mylotarg和Adcetris,有多种ADCs处于Ⅰ～Ⅲ期临床试验阶段,取得了显著的临床效果.本文概述了以美登素,卡奇霉素、Auristantin等三种细胞毒性药物为“弹头”药物的ADCs药物的临床研究状况及临床试验结果,为ADCs的研究和应用提供参考.     

Addressing challenges for large-scale trophic rewilding

The climate and biodiversity crises are inextricably linked and curbing both requires large-scale ecosystem restoration to be put into practice. In this context, trophic rewilding, which focusses on the restoration of megafauna and their ecological roles, remains a particularly major challenge. Many landscapes across the globe currently have low densities of megafauna or have lost these species altogether. Although some megafauna species have recovered in some places, through both passive and active rewilding, they are often confined to small areas. There is an increasing recognition that protected areas alone may not suffice to retain and recover high trophic complexity over large spatial extents in most of the world. This raises a clear need to scale up rewilding initiatives. Here, we discuss major challenges and potential solutions for such a scaling up of trophic rewilding, including land availability, supportive policies, the supply of animals for translocations and reintroductions, and funding. We illustrate these challenges and opportunities for two cases, the steppes of Kazakhstan and the Mediterranean rangelands. We highlight that ongoing structural change and agricultural abandonment offers opportunities for trophic rewilding in different world regions. Making use of these opportunities would require mainstreaming land-use planning that supports rewilding regardless of the current protection status of landscapes, and a reorientation of subsidies for agricultural activities in marginal lands to supporting restoration efforts. The supply of animals for reintroduction and population reinforcement projects represents another key challenge. This could be supported through a transition from farming to wildlife ranching, combined with ambitious breeding programs for keystone megafauna. Upscaling restoration efforts has recently been agreed upon as a global conservation target, and we here highlight the challenges and opportunities for restoring megafauna and their key role in ecosystems.     

Study on the Methods for Synthesis of GM-CSF Conjugates with Alendronic Acid

Russian Journal of Bioorganic Chemistry - In order to develop a target drug we studied the process of conjugation of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and...     

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.     

Oligonucleotide Conjugates Designed for Discriminative Hybridization at Physiological Temperature

Abstract

We report the synthesis of oligonucleotide conjugates engineered to allow discriminative hybridization at temperatures around physiological. Two types of structural modifications were introduced: 1) internal oligomethylene and oligoethylene glycol spacers, and 2) terminal phenazinium residues. The thermal denaturation behaviour of the complexes formed by these oligonucleotide conjugates with a target sequence is compared to that of natural duplexes. We observed a lowering of the T_m of the duplexes formed by the internal modified oligonucleotides, whilst the terminal phenazinium residues enhance their stability. The effect of the spacers is modulated by their length and hydrophobic or hydrophilic nature. Alkylating substituents, which modify the target DNA strand on hybridization, were introduced on all conjugates, and the target cleavage obtained after piperidine treatment used as a further indicator of hybridization.     

Metal-Free DNA Linearized Nuclease Based on PASP-Polyamine Conjugates

Genome manipulation controlled by small metal complexes has attracted extensive interest because of their potential application in the fields of molecular biotechnology and drug development. However, their medicinal application is still limited due to the distinct toxicity of the free radicals generated by partial metal complexes based on oxidative cleaving processes. Thus, it is still a challenge for us to use metal free agent to cleave DNA. In this work, we showed that a family of polyamine-grafted PASP (poly(aspartic acid)) conjugates is able to rapidly induce DNA cleavage in the absence of metal ions, and obtain a high-yield linearization product via a hydrolytic path. From the results of detailed control experiments, it was revealed that the formation of polyamine cation/phosphate anion pair and free ungrafted nucleophilic groups would be the key factors to improve DNA linearization. Constructing polyamine conjugates based on short peptide such as polyamine-grafted PASP, as achieved here, could provide an attractive strategy for developing mild and efficient artificial nucleases as well as researching catalytic mechanisms on DNA chemistry.     

Rapid Fluorescein and Protein Assay Method for Fluorescent-Antibody Conjugates

A method is presented for the rapid determination of the fluorescein content, protein content, and fluorescein-to-protein ratio for immune globulin conjugates with fluorescein isothiocyanate as the fluor. This method is based on the absorbance of the fluorescent antibody at those wavelengths primarily associated with the fluorescein and gamma-globulin fractions, and permits these materials to be determined by a single nondestructive analytical procedure. A small sample of the fluorescent antibody, in many cases 0.1 ml or less, is adequate for the above determinations. A nomograph is presented which allows simultaneous determination of the materials from the observed absorbance at the two wavelengths. The method is sufficiently accurate for most applications of the fluorescent-antibody techniques. Although this procedure has been developed primarily for fluorescent-antibody conjugates prepared from rabbit gamma-globulin, it can be used directly for antibodies prepared from other animals provided the gamma-globulin is relatively free from albumin.     

Chip devices for miniaturized biotechnology

Chip devices were introduced in chemistry and molecular biology to improve the read-out of information from molecular systems by efficient analytical procedures and to organize automated experiments. Biochips and chip reactor systems are of interest for cellular processes, too, and can be regarded as components in interfaces for the information exchange between living nature and digital electronic systems. In this minireview, different types of chip reactors for biotechnological applications like nanotiterplates, chip thermocyclers and devices for segmented flow operations are discussed. Finally, an outlook is given on the application of chip reactor systems, which are promising tools for automated experiments with highly parallelized screening procedures, for artificial microcompartmentation, cell analogue systems, micro-ecological studies, investigations on modulated morphogenesis, and for a bioanalogue molecular nanotechnology.     

膜芯片检测A组轮状病毒的初步研究

建立表面带正电荷的尼龙膜为基片的基因芯片,采用RT-semi-nested PCR方法,对A组轮状病毒RNA实现高度扩增,并通过5'端带地高辛标记的上游引物实现扩增产物的标记.通过同膜芯片上探针的杂交和免疫显色,实现对A组轮状病毒的检测.结果表明,膜芯片对探针的固定效果、杂交吸脱和检测结果可靠,空白对照和阴性对照均为阴性,探针均显示为阳性信号,并且信号强弱与探针浓度关系不大,而主要与探针本身同互补链的结合相关.以上结果说明已经初步建立了快速检测A组轮状病毒的膜基因芯片检测技术.     

Reagents for the Preparation of Chromophorically Labeled Polyethylene Glycol-Protein Conjugates

We have developed a new class of reagents (2) for the covalent attachment of polyethylene glycol to proteins. These reagents (2) are the monomethoxypolyethylene glycol esters of 4-fluoro-3-nitrobenzoic acid. The reaction of 2 with lysine ε-amino groups produces a chromophore which can be used to quantitate the polyethylene glycol to protein molar ratio. Bovine (Zn, Cu) superoxide dismutase was used as a model protein for conjugation with 2. When monomethoxypolyethylene glycol of average molecular weight 2105 was used, a conjugate was obtained with a polyethylene glycol to protein molar ratio of 8.88 retaining 100% of native enzymatic activity; monomethoxypolyethylene glycol of average molecular weight 5210 yielded a conjugate with a polyethylene glycol to protein molar ratio of 9.96 retaining 73% of native enzymatic activity.     

Evaluation of Commercial Conjugates for Fluorescent Antibody Detection of Salmonellae

Fluorescent antibody (FA) reagents for Salmonella produced by Difco, Sylvana, and Clinical Sciences, Inc., were evaluated for physicochemical and performance characteristics. The Difco panvalent (A through 064) and the Difco polyvalent (A through S) were similar in physicochemical characteristics. They had less than 60% gamma globulin with 3% albumin and had fluorescein to protein (F/P) ratios of less than 10. The Sylvana conjugate had 81% gamma globulin with less than 1% albumin. Its F/P was 33.9. The Clinical Sciences reagent contained 75% unlabeled albumin as packaged in the Fluoro-kit. Analysis of the original conjugate showed 86.5% gamma globulin with only 0.5% albumin. The (F/P) was 32.8. The performance characteristics were determined by using a variety of Enterobacteriaceae and food and feed samples. All conjugates stained the homologous Salmonella strains. The majority of cross-reactions were limited primarily to the Arizona, Citrobacter, and Escherichia coli groups. The Difco panvalent was more reactive with heterologous organisms. It stained 89% of the Arizona compared with 42% stained by the Difco polyvalent (A through S) and 39% stained by the Sylvana and Clinical Sciences reagents. We found 90% agreement between FA and culture when the Difco polyvalent was used to examine food and feed samples and 94% agreement when the Clinical Sciences Fluoro-kit was used on another group of samples.     

Evolution of Bombesin Conjugates for Targeted PET Imaging of Tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel ^64/67Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC₅₀: 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle¹⁴ by Met, and deletion of D-Tyr⁶, further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6^th, 11^th, and 14^th position of BN induced only slight influences on affinity to mouse GRPr. [Cu^II]-CPTA-[βAla¹¹] BN(7–14) ([Cu^II]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [^64/67Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [^64/67Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [^64/67Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.     

口蹄疫等5种动物病毒基因芯片检测技术的研究

用分子克隆方法获得口蹄疫病毒、水泡性口炎病毒、蓝舌病病毒、鹿流行性出血热病毒和赤羽病病毒各一段高度保守的基因片段,用芯片点样仪点样到包被过的玻璃片上,制备成检测芯片。提取样品中的RNA,进行反转录和荧光标记后滴加到芯片上进行特异性杂交,对杂交结果进行扫描检测,可同时诊断上述5种动物传染病,此方法不但快速、准确、敏感,而且可同时进行多种病毒的检测,达到大批动物高通量检疫的目的。     

Conjugates of polyacrylamide with oligonucleotides and their mimetics for diagnostic purposes

Approaches to preparing acrylamide and polyacrylamide conjugates with oligonucleotides and some peptide nucleic acid-related DNA mimics are considered. Their physicochemical properties and application to the nucleic acid analysis are discussed.