Selection and Identification of Aptamers Against B Cell Activating Factor

Systemic lupus erythematosus (SLE) is the most common autoimmune disease in China. B cell activating factor (BAFF) is an important target for the treatment and detection of SLE. It is of great significance to develop novel molecular recognition elements with high affinity for BAFF. In this study, artificial nucleic acid aptamers against BAFF were screened from a 78 nt single-stranded DNA random library by systematic evolution of ligands exponential enrichment (SELEX) in vitro based on several selection and amplification steps. Through ten rounds of selection, the aptamers with high specificity and affinity for BAFF were identified. After high-throughput sequencing, several aptamers were selected and further examined for binding affinity and specificity. The investigation by dot blotting, Eastern blotting analyses and enzyme-linked oligonucleotide assay (ELONA) showed that the aptamers Apt 7 and Apt 12 with dissociation constants of 241.00±19.75 nmol/L and 413.51±46.94 nmol/L were able to recognize BAFF specifically. After molecular docking analysis, Apt 7 was truncated to Apt 7～1, and the dissociation constant was 192.10±28.61 nmol/L. A sandwich ELONA using Apt 7～1 and BAFF antibodies was established to detect BAFF. The detection limit was estimated to be 0.227 nmol/L. This study provides new molecular recognition elements for the detection of BAFF and the study of antagonists.     

In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

Kuwanon W,a natural Diels—Alder type adduct from the root bark of Morus lhou

A natural Diels—Alder type adduct, named kuwanon W, was isolated from ethyl acetate extracts of the root bark of cultivated mulberry tree, and its structure was determined on the basis of spectral and chemical evidence. Kuwanon W is regarded biogenetically as a Diels—Alder adduct of a chalcone derivative and dehydroprenyl-flavone.     

抑制RANKL诱导破骨细胞分化的DNA适配子的筛选

目的:筛选能高特异性、高亲和力结合RANKL蛋白并有效抑制RANKL对破骨细胞诱导分化作用的DNA适配子。方法:首先,采用原核系统表达并纯化RANKL蛋白,通过SELEX(Systematic evolution of ligands by exponential)技术从人工合成的单链随机寡核苷酸文库中筛选能高特异性、高亲和力结合RANKL蛋白的DNA适配子。然后,用RNAfolding sever software分析适配子空间结构,以ELISA检测DNA适配子和RANKL亲和力大小并筛选出亲和力最高的一组DNA适配子用以验证DNA适配子对RANKL诱导破骨细胞分化的抑制作用。结果:(1)成功在原核系统表达并纯化RANKL蛋白;(2)筛选出能高特异性、高亲和力结合RANKL蛋白的12个DNA适配子。(3)与对照组相比,不同浓度DNA适配子能明显抑制TRAP阳性破骨细胞数量(P0.05),且浓度越高抑制效果越明显。结论:成功筛选出的DNA适配子能特异性结合RANKL蛋白并有效抑制RANKL对破骨细胞的诱导分化功能。     

Development of DNA aptamers for cytochemical detection of acetylcholine

This report describes a novel approach to the detection of acetylcholine using DNA aptamers. Aptamers were developed by eight rounds of acetylcholine affinity column chromatography and polymerase chain reaction (PCR) amplification. Sequences from rounds 5 and 8 were screened by colorimetric enzyme-based microtiter plate assays and found to bind acetylcholine and related compounds, but not unrelated compounds. One of the highest affinity aptamers, designated ACh 6R, was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. Using Neuro-2a murine neuroblastoma cells induced to differentiate in the presence of 1 μM all-trans-retinoic acid for 5–7 d, ACh 6R detected cholinergic cells by both the peroxidase and fluorescence methods. Unrelated DNA aptamers did not stain the cells using either method. Fixation with cold 2% paraformaldehyde was compared to cold alkaline allyl alcohol plus glutaraldehyde for immobilization of acetylcholine in situ and appeared to enable detection of greater numbers of cholinergic cells, although differences in levels of differentiation may have been a factor as well. Acetylcholine generally appeared to be distributed throughout the differentiated Neuro-2a cell bodies. However, in some cells, punctate staining along neurite outgrowths and near the termini of cellular processes suggested detection of acetylcholine in discrete vesicles.     

A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.     

Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system.     

Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer

A selective kanamycin-binding single-strand DNA (ssDNA) aptamer (TGGGGGTTGAGGCTAAGCCGA) was discovered through in vitro selection using affinity chromatography with kanamycin-immobilized sepharose beads. The selected aptamer has a high affinity for kanamycin and also for kanamycin derivatives such as kanamycin B and tobramycin. The dissociation constants (K_d [kanamycin] = 78.8 nM, K_d [kanamycin B] = 84.5 nM, and K_d [tobramycin] = 103 nM) of the new aptamer were determined by fluorescence intensity analysis using 5′-fluorescein amidite (FAM) modification. Using this aptamer, kanamycin was detected down to 25 nM by the gold nanoparticle-based colorimetric method. Because the designed colorimetric method is simple, easy, and visible to the naked eye, it has advantages that make it useful for the detection of kanamycin. Furthermore, the selected new aptamer has many potential applications as a bioprobe for the detection of kanamycin, kanamycin B, and tobramycin in pharmaceutical preparations and food products.