Peptide Conjugates of Oligonucleotides As Enhanced Antisense Agents

The use of synthetic oligonucleotides and their analogs to block gene expression by binding the complementary RNA sequences in cells, the antisense principle, has been limited by poor uptake of the agents by cells in culture. This review describes attempts to harness by chemical conjugation the ability of certain peptides that may cross membranes to enhance the cellular uptake of oligonucleotides. These include fusogenic and hydrophobic peptides, nuclear localization signals, receptor targeting and translocating peptides, and various combinations. We also outline briefly some popular methods of peptide–oligonucleotide conjugation. Finally, we review the use of noncovalent peptide additives and the recent studies of conjugates of peptide nucleic acid (PNA) with peptides.     

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: An organochlorine pesticide

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL⁻¹ to 1000 ng mL⁻¹ to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL⁻¹ with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.     

Substrate complexation and aggregation influence the cyclodextrin glycosyltransferase (CGTase) catalyzed synthesis of alkyl glycosides

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) was used to convert dodecyl-β-maltoside (DDM) to dodecyl-β-maltooctaoside (DDMO) using α-cyclodextrin (α-CD) or starch as glycosyl donors. At 300 mM α-CD, varied DDM concentration and 60 °C, the reaction obeyed Michaelis-Menten kinetics with a K_m value of 18 mM and a V_max value of 100 U/mg enzyme. However, at 25 mM α-CD the reaction rate decreased with increasing DDM concentration (5-50 mM), and when the α-CD concentration was varied at fixed DDM concentration an S shaped curve was obtained. The deviations from Michaelis-Menten kinetics were interpreted as being caused by formation of inclusion complexes between α-CD and DDM and by micellation of DDM. To achieve a high reaction rate, a high concentration of free α-CD is necessary, since α-CD in the form of a complex has low reactivity. When starch is used as glycosyl donor in the CGTase catalyzed alkyl glycoside elongation reaction, it is thus important to choose reaction conditions under which the cyclization of starch to α-CD is efficient.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Mutual conversion of substrate specificities of Thermoactinomyces vulgaris R-47 alpha-amylases TVAI and TVAII by site-directed mutagenesis

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVAI and TVAII, differing in substrate specificity from each other. TVAI favors high-molecular-weight substrates like starch, and scarcely hydrolyzes cyclomaltooligosaccharides (cyclodextrins) with a small cavity. TVAII favors low-molecular-weight substrates like oligosaccharides, and can efficiently hydrolyze cyclodextrins with various sized cavities. To understand the relationship between the structure and substrate specificity of these enzymes, we precisely examined the roles of key residues for substrate recognition by X-ray structural and kinetic parameter analyses of mutant enzymes and successfully obtained mutants in which the substrate specificity of each enzyme is partially converted into that of another.     

Antibody-drug conjugates: Intellectual property considerations

Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs.     

A novel homogeneous bioluminescence resonance energy transfer element for biomolecular detection with CCD camera or CMOS device

A novel optical signal element based on homogeneous bioluminescence resonance energy transfer (BRET) was developed for biomolecular detection. A fluorescent dye and alkaline phosphatase (AP) conjugate was used as a reporter and light‐generation element for imaging detection platforms that use a CCD camera or CMOS chip‐based devices. In the presence of a luminescence substrate, the energy from the first light emission of a bioluminescence enzymatic reaction was transferred to fluorescent dyes which were conjugated to an enzyme. This resulted in a second light emission with a shorter wavelength. The second light was localized at the position of target molecules without the diffusion problems present in current technology. To optimize energy transfer efficiency, the ratio of enzyme to fluorophore in the conjugates, the fluorescent dyes used in the conjugates and the luminescence substrates used for BRET were investigated. BRET was demonstrated by using both a CCD camera and a CMOS imaging device. Image spatial resolution was greatly improved compared with conventional chemiluminescence detection. This new signal element opens a door for the direct measurement of fluorescent signals on an imaging chip without an external light source and portable instrumentation normally required for the fluorescent detection of biomolecules. Copyright © 2007 John Wiley & Sons, Ltd.     

ADC药物的抗体组成及其作用靶点研究进展*

抗体药物偶联物(antibody-drug conjugates,ADC)是一类由单克隆抗体和小分子细胞毒性药物通过连接子偶联而成的新型生物治疗药物。与传统的细胞毒药物相比,ADC具有靶向性强、毒副作用小等优势,在临床上展现较好的治疗潜力。其中,抗体部分通过与肿瘤细胞表面的靶向抗原结合,精准地将小分子细胞毒性药物递送至肿瘤部位,从而实现肿瘤特异性杀伤效果,是影响ADC疗效的核心要素之一。对近年来ADC药物中抗体的组成及其作用靶点的研究进展进行了综述。     

Enhanced chemiluminescence of 6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one by attachment of a cyclomaltooligosaccharide (cyclodextrin). Attachment of cyclomaltononaose (delta-cyclodextrin)

2-Methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one (MCLA) is an oxygen-induced chemiluminescent compound. It has been shown that the chemiluminescence can be enhanced by forming a cyclomaltooligosaccharide (cyclodextrin)-bound MCLA, and therefore, in continuation of the survey of the types of cyclodextrins, in this study, MCLA was attached to the secondary hydroxyl face of delta-cyclodextrin, which consists of nine D-glucose units. Although the oxygen-induced chemiluminescence efficiency of delta-cyclodextrin-bound MCLA in a pH 8.0 aqueous phosphate buffer was 12 times greater than that of MCLA, the efficiency was markedly lower than that of gamma-cyclodextrin-bound MCLA, which exhibited the highest chemiluminescence efficiency in the previous investigation. Although fluorescence efficiency and light-emitter formation efficiency for delta-cyclodextrin-bound MCLA were similar to those for gamma-cyclodextrin-bound MCLA, singlet-excited state formation efficiency for delta-cyclodextrin-bound MCLA was lower than that for gamma-cyclodextrin-bound MCLA. This study distinctly indicated the optimum cyclodextrin for construction of greatly luminescent cyclodextrin-bound MCLA is gamma-cyclodextrin.     

The Solution Synthesis of Antisense Oligonucleotide‐Peptide Conjugates Directly Linked via Phosphoramide Bond by Using a Fragment Coupling Approach

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell‐penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1‐hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2′‐dithiopyridine and 4‐dimethylaminopyridine in organic media. Several oligonucleotides, including a 18‐mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.