Improvement of RNA aptamer activity against myasthenic autoantibodies by extended sequence selection.

Myasthenia gravis (MG) is mainly engendered by autoantibodies directed against acetylcholine receptors (AChRs) located in the postsynaptic muscle cell membrane. Previously, we isolated an RNA aptamer with 2'-amino pyrimidines using in vitro selection techniques that acted as a decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and patient autoantibodies with MG (1). However, low affinity of this RNA to mAb198 relative to that of AChR might limit potential of the RNA as an inhibitor of the autoantibodies. To improve decoy activity of the RNA aptamer against autoantibodies, here we employed in vitro selection methods with RNA libraries containing extra random nucleotides extended to the 3' end of previously selected RNA sequences. RNAs isolated in this study showed significant increases in the binding affinities to mAb198 as well as bioactivities protecting AChRs on human cells from both mAb198 and patient autoantibodies, compared with the previous RNA aptamers. These results have important implications for the development of antigen-specific modulation of autoimmune diseases including MG.     

In vitro selection of sialic acid specific RNA aptamer and its application to the rapid sensing of sialic acid modified sugars

Sialic acids (SAs) are located on the terminal positions of glycan on a cell surface, which play important role in the spread and metastasis of cancer cells and infection of pathogen. For their detection and diagnosis, the finding of SA specific ligand is an essential prerequisite. Here, RNA aptamer for N‐acetylneuraminic acid (Neu5Ac), a representative of SAs, with the high affinity of 1.35 nM and the selectivity was screened by in vitro selection method. The strong binding of the screened aptamer was enough to protect the hydrolysis of Neu5Ac by neuraminidase with the stoichiometry of 1:1 molar ratio. For the rapid detection of SAs, the RNA aptamer was further engineered to the aptazyme sensor by conjugating with a ribozyme following the characterization of selected aptamer by RNase footprinting assay. Without additional desialylation, modification, or/and purification processes, the aptazyme indicated high catalytic activities in the presence of Neu5Ac over 20 µM in several minutes. Also, we observed that the aptazyme sensor shows high sensitivities to Neu5Ac‐conjugated sugars as well as Neu5Ac monomer, but not in non‐Neu5Ac modified sugars. The aptamer for Neu5Ac can support valuable tools in a wide range of bioanalytical applications as well as biosensors. Biotechnol. Bioeng. 2013; 110: 905–913. © 2012 Wiley Periodicals, Inc.     

In vitro selection of RNA aptamers that selectively bind danofloxacin

Danofloxacin is a synthetic fluoroquinolone with broad spectrum antibacterial activity that is used for the treatment of respiratory diseases in animal husbandry. However, danofloxacin has many adverse reactions and is toxic to humans. Especially, it detrimentally affects muscle, central nerve system, peripheral nerve system, liver, and skin in those who ingest foods in which danofloxacin has accumulated. Prescreening and determination of the level of danofloxacin in foods or food products is necessary for human health. Aptamers are composing of oligonucleotides that specifically interact with target molecules. They are emerging as detection/diagnostic ligands. Here, we used the SELEX in vitro selection technology to identify specific and high-affinity RNA aptamers with 2′-fluoro-2′-deoxyribonucleotide modified pyrimidine nucleotides against danofloxacin. Selected RNA aptamers bound specifically to danofloxacin, but not to tetracycline. Truncation of RNA aptamer up to 36 mer did not comprise specificity and affinity. The truncated RNA aptamer specifically bound to target chemical, allowing the discrimination of danofloxacin from other fluoroquinolones. The isolated specific aptamer could be a potential agent used for the rapid and cost-effective detection and sensing of danofloxacin, replacing instrumental methods including the more expensive and time-consuming methods of high performance liquid chromatography and liquid chromatography/mass spectrometry.     

Structure specific chromatographic selection in targeted proteomics

The whole proteome of any organism is too complicated to be analyzed in a simple one-step process and direct attempts for the entire proteome analysis normally lead to considerable loss of information. A practical approach is the targeting of the specific structural feature of interest using chromatography. This approach simplifies the proteome while preserving most of the vital information necessary for analysis. Selection of peptides with specific amino acids (cysteine, histidine and methionine) or N- or C-terminal peptides is an accepted procedure for proteome simplification when general analysis is desired. While selection of enzymatically and non-enzymatically modified proteins and peptides is used when post-translational modifications are targeted. Protein interaction with small molecules as well as other proteins also has been studied using chromatographic selection methods.     

Measurement of concurrent selection episodes

Current methods for measuring selection with longitudinal data have been developed with the assumption that episodes of selection are sequential. However, a number of empirical examinations have demonstrated that natural and sexual selection may act concurrently and in opposing directions. Other recent work has highlighted the difficulty of assigning fitness values for survival when reproduction and mortality within a population temporally overlap. I treat these as facets of a single problem; how to analyze selection where mortality and reproduction are concurrent. To address this problem, I formalize a method to estimate total fitness of individuals over a period of time utilizing longitudinal data. I then show how the fitness may be partitioned to provide two separate estimates of fitness for reproductive opportunity and reproductive success. In addition, another total fitness estimate for the period can be obtained from the two partitioned estimates. This procedure will allow calculation of total fitness where there are some missing datapoints for reproductive success of an individual. A simulation indicates that bias is generally low for the various fitness estimates. These methods should expand our ability to understand the interaction of different selection episodes.     

酿酒酵母组氨酸转运蛋白Hip1p的泛素化水平对组氨酸利用的影响

【目的】为了研究泛素化对组氨酸转运及调控的影响。【方法】应用泛素化位点预测及定点突变等技术手段,Hip1p的3个潜在的泛素化位点K30、K42和K52被突变。这些突变的Hip1p被克隆到泛素化检测质粒中,检测泛素化位点突变对Hip1p的泛素化水平的影响。同时这些突变对细胞生长及组氨酸利用的影响也进一步做了检测。【结果】Hip1p的3个赖氨酸位点K30、K42和K52突变能有效降低其泛素化水平。同时,双重突变对其泛素化水平有明显的协同作用,并进一步影响了细胞生长和组氨酸利用。【结论】泛素化水平调控能有效调节组氨酸代谢,引起细胞对组氨酸利用的改变,为进一步研究氨基酸转运蛋白的调控机制提供了重要的依据。     

Chiral phase partitioning by enantioselective complexation: Characterisation by the semi‐empirical application of regular solution theory

The thermodynamics underlying enantioselective complexation and partitioning behaviour are poorly understood. This paper presents a model that decouples the effects of enantioselective complexation and subsequent diastereomer partitioning. Regular solution theory is applied in a semi‐empirical manner to describe the diastereomer partitioning process, which is reported to be governed by hydrophobic interactions. The model was shown to give a good fit to experimental partitioning for the enantioselective extraction of phenylalanine isomers by two chiral extractants; a modified amino acid [copper (II) N‐decyl‐(L)‐hydroxyproline] and a chiral crown ether [(S)‐bis(phenylnaphtho)‐20‐crown‐6]. A variety of aliphatic and aromatic solvents were tested. The predicted and observed experimental enantioselectivities were found to vary exponentially with the difference in the solubility parameters of the aqueous and organic phases and with those of the two diastereomeric complexes formed. This model provides the basis for a better understanding of enantioselective partitioning effects. Chirality 11:241–248, 1999. © 1999 Wiley‐Liss, Inc.     

Application of Microchip Electrophoresis in the Analysis of RNA Aptamer-Protein Interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (< 3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Relative contributions of nine genes in the pathway of histidine biosynthesis to the control of free histidine concentrations in Arabidopsis thaliana

Despite the functional importance of histidine (His) as an essential amino acid in proteins and as a metal-coordinating ligand, comparatively little is known about the regulation of its biosynthesis in plants and the potential for metabolic engineering of this pathway. To investigate the contribution of different steps in the pathway to overall control of His biosynthesis, nine His biosynthetic genes were individually over-expressed in Arabidopsis thaliana to determine their effects on free amino acid pools. Constitutive, CaMV 35S -driven over-expression of the cDNAs encoding either isoform of ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme in the pathway, was sufficient to increase the pool of free His by up to 42-fold in shoot tissue of Arabidopsis , with negligible effect on any other amino acid. In contrast, over-expression of cDNAs for seven other enzymes in the biosynthetic pathway had no effect on His content, suggesting that control of the pool of free His resides largely with ATP-PRT activity. Over-expression of ATP-PRT and increased His content had a negative pleiotropic effect on plant biomass production in 35S:PRT1 lines, but this effect was not observed in 35S:PRT2 lines. In the presence of 100 µM Ni, which was inhibitory to wild-type plants, a strong positive correlation was observed between free His content and biomass production, indicating that the metabolic cost of His overproduction was outweighed by the benefit of increased tolerance to Ni. His-overproducing plants also displayed somewhat elevated tolerance to Co and Zn, but not to Cd or Cu, indicating chemical selectivity in intracellular metal binding by His.     

乙内酰脲酶及其在氨基酸手性合成中的应用

乙内酰脲水解酶、氨甲酰化酶和乙内酰脲消旋酶构成的酶系能够以5-取代乙内酰脲类化合物为原料合成天然和非天然D-或L-氨基酸,用于各种手性氧基酸的生产。近来的研究重点在分离新酶或提高原酶的活性,包括定向突变、三维结构解析与结构功能关系研究、酶固定化、蛋白融合和构建完整细胞生物催化剂等。     

An RNA aptamer that recognizes a specific conformation of the protein calsenilin

The generation of molecules that selectively recognize specific conformations of a protein is an important component of the elucidation protein function. We have used SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology to produce aptamers that bind in a conformationally selective manner to calsenilin, which involved in Ca²⁺-mediated apoptotic signaling. Since the conformations of calsenilin are quite different in the presence and absence of Ca²⁺, aptamers were selected against the dimeric protein both under calcium-bound and calcium-free conditions. We have found that aptamer-12 selectively binds to the dimeric form of the protein in the presence of calcium ion, while the binding of aptamer-2 does not discriminate between the Ca²⁺ bound and unbound protein. Data obtained from biochemical and biophysical experiments suggest that a dominant conformation of calcium-bound calsenilin exists in one dominant conformation and that one aptamer can be generated to recognize this conformation. In addition, observation made in this effort that aptamers selected against the two different conformations of calsenilin have different characteristics suggest that aptamers can serve as a plausible tool for recognizing various conformations of proteins, even those caused by interactions with small molecules or ions such as Ca²⁺.     

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.     

Computational approaches toward the design of pools for the in vitro selection of complex aptamers

It is well known that using random RNA/DNA sequences for SELEX experiments will generally yield low-complexity structures. Early experimental results suggest that having a structurally diverse library, which, for instance, includes high-order junctions, may prove useful in finding new functional motifs. Here, we develop two computational methods to generate sequences that exhibit higher structural complexity and can be used to increase the overall structural diversity of initial pools for in vitro selection experiments. Random Filtering selectively increases the number of five-way junctions in RNA/DNA pools, and Genetic Filtering designs RNA/DNA pools to a specified structure distribution, whether uniform or otherwise. We show that using our computationally designed DNA pool greatly improves access to highly complex sequence structures for SELEX experiments (without losing our ability to select for common one-way and two-way junction sequences).     

Specific modulation of the anti-DNA autoantibody-nucleic acids interaction by the high affinity RNA aptamer

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody-nucleic acid interaction.     

肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定

目的获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体（asialoglycoprotein receptor,ASGPR）的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。方法合成一个长度为115nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,以肝脏ASGPR大亚基为靶蛋白,采用SELEX（systematic evolution of ligands by exponential enrichment）技术筛选具有高亲和力的AsGPR特异性RNA适配子;通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力。结果经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子。结论成功地筛选出了具有离亲和力的肝脏ASGPR特异性RNA适配子库。     

AN RNA APTAMER CONTAINING TWO BINDING SITES AGAINST THE HCV MINUS-IRES DOMAIN I

The higher order structure of HCV (?)IRES containing five stem-loop structures (domain I) is essential for HCV replication because the viral RNA-dependent RNA polymerase, NS5B, recognizes it as the initiation site for plus-strand synthesis. To inhibit a de novo synthesis of plus-strand RNA molecules, in vitro selection against (?)IRES domain I was performed. One of the obtained aptamers, AP30, contained two consensus sequences within a random sequence region. Two consensus sequences form two apical loops and mutational analysis showed that both sequences were essential for binding to the target and for inhibiting NS5B-mediated RNA synthesis in vitro.