采用毛细管电泳技术筛选特异识别蓖麻毒素适配子的研究

采用指数富集配基的系统进化(SELEX)技术从随机寡核苷酸文库中筛选获得特异识别蓖麻毒素靶分子的适配子. 将毛细管电泳技术作为分离手段引入到SELEX筛选中, 利用毛细管电泳高效的分离能力使得筛选周期大大缩短. 酶联免疫和斑点杂交实验结果表明, 仅经4轮筛选即可获得特异识别蓖麻毒素蛋白的寡核苷酸适配子.     

应用微阵列芯片检测与2型糖尿病相关的单核苷酸多态性

基于三维(3D)寡核苷酸微阵列芯片的荧光检测法, 研制了一种用于筛选能检测2型糖尿病的特定寡核苷酸探针. 使用第4代(G4)聚(酰胺-胺)(PAMAM)树枝状大分子修饰的载玻片为基底, 以氨基修饰的寡核苷酸为固定探针构建3D寡核苷酸微阵列芯片. 采用荧光化合物Cy5修饰的寡核苷酸为检测探针获得荧光信号. 以2型糖尿病易感基因TCF7L2的rs7903146位点为研究对象, 通过对含有16种(8对)寡核苷酸的寡核苷酸文库的筛选, 获得了1对能用于2型糖尿病检测的寡核苷酸探针. 通过单核苷酸多态性和等位基因分析证明, 该寡核苷酸探针对靶标寡核苷酸检测具有高特异性, 并能准确检测低至2%的等位基因频率.     

亲和色谱中配基的筛选与应用

亲和配基的选择与筛选是发展新的亲和色谱填料或构建一个新的亲和色谱体系所必须解决的首要问题。该文结合作者所在实验室的工作,对配基的选择、筛选与应用方面的一些进展进行了简要评述。作者所在实验室针对特定蛋白质和多肽的多肽亲和配基的筛选,开展了反义肽简并性的研究,发展了基于反义肽的组合化学筛选新方法。与常规的组合合成法相比,该方法简单、快捷、有效,极大地减小了合成和筛选的工作量,降低了筛选后亲和组分结构鉴定的难度。所建立的筛选策略已应用于流感病毒、严重急性呼吸道综合征(SARS)病毒亲和抑制剂的筛选和用于人β-干扰素测定的石英晶体微天平（QCM）生物传感器的构建,均取得了有意义的结果。     

基于Cell-SELEX的核酸适配体在生化分析与生物成像中的应用

基于Cell-SELEX的核酸适配体是指以活细胞为靶标物,通过指数富集的配基系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从人工合成的DNA/RNA文库中筛选得到的单链寡核苷酸.它能够与靶标细胞高亲和性、高特异性结合,具有分子量低、合成简单、化学稳定性好、免疫原性低、易于功能化修饰等优点,已广泛应用于生命科学研究领域.本文综述了基于Cell-SELEX技术筛选的核酸适配体在肿瘤细胞检测、分析和成像方面的研究进展,并对核酸适配体研究的发展前景和趋势进行了展望.     

激光诱导荧光毛细管电泳法优化随机寡核苷酸文库聚合酶链反应扩增的条件

寡核苷酸文库的聚合酶链反应(PCR)扩增是指数富集系统配基进化技术(SELEX)筛选适配子技术中的重要步骤.本研究采用毛细管电泳技术结合激光诱导荧光检测器(CE-LIF),通过对双链产物、PCR副产物以及剩余引物的考察,研究了寡核苷酸文库PCR扩增时的影响因素,并对其进行优化.结果表明,随机寡核苷酸文库的扩增与传统均一模板的扩增显著不同,其在扩增十几个循环时产物就达到最大值;此外,初始模板数、退火温度、DNA聚合酶浓度等条件也有所不同.因此, 在进行SELEX筛选之前必须对文库PCR条件进行详细优化.本研究中N39文库优化后的PCR扩增条件为:初始模板的量为105个分子,DNA聚合酶浓度0.05 U/μL,退火温度70 ℃,18个循环.本研究为利用SELEX技术有效筛选适配子,降低筛选的假阳性及提高特异性提供了参考依据.     

亲和层析研究进展

亲和层析具有高选择性,高活性回收率和高纯度等特点,已成为纯化蛋白质等生物大分子最有效的技术之一,本文综述了亲和层析的类型,配基的种类,选择方法以及亲和层技术的最新研究进展,重点介绍了多肽作为亲和配基的制备及筛选方法及其在生物大分了职的应用,并比较了不同亲和层析方法的优缺点及其发展趋势。     

ω-芋螺毒素的定量构效关系与虚拟筛选

ω-芋螺毒素属于海洋生物活性多肽,由24-31个氨基酸残基组成.特异性作用于电压敏感的钙离子通道(VGCCs),能够直接开发成药物或作为先导化合物进行新药开发.本文应用新型氨基酸残基结构描述符cscales和遗传偏最小二乘算法,对ω-芋螺毒素进行定量构效关系(QSAR)研究,并设计、构建了容量为2244个化合物的N-型和P/Q-型VGCC拮抗剂虚拟组合多肽库,然后分别采用QSAR模型预测和相似性搜索方法对组合多肽库进行了虚拟筛选.研究结果表明,建立的N-型和P/Q-型VGCC拮抗剂QSAR模型均具有较好的预测能力,交叉验证相关系数(CV-r2)均大于0.89.主成分分析和聚类分析结果表明,虚拟组合多肽库中化合物具有较好的结构多样性和差异性.通过虚拟筛选,得到了具有高预测活性的6个N-型和19个P/Q-型钙离子通道拮抗剂,为进一步的合成和活性评价奠定了理论基础.同时,本文建立的多肽QSAR预测模型和虚拟筛选策略,为其它多肽类化合物的定量构效关系研究和虚拟筛选提供了参考.     

ω-芋螺毒素的定量构效关系与虚拟筛选

ω-芋螺毒素属于海洋生物活性多肽, 由24-31 个氨基酸残基组成. 特异性作用于电压敏感的钙离子通道(VGCCs), 能够直接开发成药物或作为先导化合物进行新药开发. 本文应用新型氨基酸残基结构描述符cscales和遗传偏最小二乘算法, 对ω-芋螺毒素进行定量构效关系(QSAR)研究, 并设计、构建了容量为2244 个化合物的N-型和P/Q-型VGCC拮抗剂虚拟组合多肽库, 然后分别采用QSAR模型预测和相似性搜索方法对组合多肽库进行了虚拟筛选. 研究结果表明, 建立的N-型和P/Q-型VGCC拮抗剂QSAR模型均具有较好的预测能力, 交叉验证相关系数(CV-r²)均大于0.89. 主成分分析和聚类分析结果表明, 虚拟组合多肽库中化合物具有较好的结构多样性和差异性. 通过虚拟筛选, 得到了具有高预测活性的6 个N-型和19 个P/Q-型钙离子通道拮抗剂, 为进一步的合成和活性评价奠定了理论基础. 同时, 本文建立的多肽QSAR预测模型和虚拟筛选策略, 为其它多肽类化合物的定量构效关系研究和虚拟筛选提供了参考.     

毛细管电泳在核酸适配体研究及核酸适配体筛选中的应用

核酸适配体是指通过指数富集配体系统进化(SELEX)技术从随机寡核苷酸文库中筛选得到的高亲和性与特异性的寡核苷酸序列配体。毛细管电泳是高效、快速、低成本的微量分离分析技术。应用毛细管电泳高效、快速筛选核酸适配体是近几年出现的新方法。本文介绍了核酸适配体筛选过程中的主要分离方法如亲和色谱、醋酸纤维素膜、凝胶电泳和磁性分离等方法,并对近年来毛细管电泳在核酸适配体中的亲和作用研究以及用于核酸适配体筛选(CE-SELEX)的主要方法(ECEEM,NECEEM,Non-SELEX和三者比较)和研究进展进行了综述。     

胰蛋白酶定向固定化亲和配基的理性设计

在胰蛋白酶三维(3D)结构的基础上, 首先利用分子对接从ZINC 数据库中筛选获得了与胰蛋白酶具有较高亲和性的小分子配基2-硝基苯基-β-D-葡糖苷, 并分析了该配基与蛋白质之间的相互作用力主要为范德华和氢键相互作用. 并利用分子动力学模拟进一步验证了2-硝基苯基-β-D-葡糖苷与胰蛋白酶之间具有较强的亲和作用. 分子动力学(MD)模拟结果表明, 配基-目标蛋白质之间形成稳定的复合物且它们之间的距离基本没有变化. 此外, 一个水分子通过氢键在配基和目标蛋白质的结合腔之间架桥. 最后制备了偶联有该配基的亲和载体, 进行了胰蛋白酶的定向固定化, 并考察了该固定化酶的活性. 研究结果表明, 利用修饰2-硝基苯基-β-D-葡糖苷配基的亲和载体固定化胰蛋白酶的酶活达到340.8 U·g^-1, 比活达到300.3 U·mg^-1, 分别是未修饰亲和配基载体的10倍和5倍, 具有明显的优势. 上述结论证明了结合分子对接和分子动力学模拟理性设计定向固定化亲和配基的方法是可行的, 具有一定的理论和实用价值.     

Total chemical synthesis by native chemical ligation of the all-D immunoglobulin-like domain 2 of Axl

Axl is a tyrosine kinases receptor playing crucial role in several cellular responses. The deregulation of Axl signaling has been associated to many high impact diseases ranging from cancer to multiple sclerosis. We report the successful procedure for the chemical synthesis of the Ig2 domain of Axl, one of the key extracellular regions of the receptor involved in ligand binding. The protein was synthesized in its d-enantiomeric form (D-Axl-2), opening the way to the selection of D-peptides selectively targeting Axl receptor through the mirror-image phage display peptide library screening approach.     

Conformational structure of N-ethylacetamide and N-isopropylacetamide. NMR shift reagent study

The structure of the stable conformers of N-ethylacetamide and N-isopropylacetamide was determined by NMR spectroscopy with lanthanide shift reagents. N-ethylacetamide was found to exist in the form of two mirror-image isomers, with the ethyl group twisted out of the plane of the amide bond by the angle ψ XXX ± 105°. Similarly N-isopropylacetamide exists in the form of two mirror-image isomers, with the isopropyl methyl groups in positions with ψ₁ XXX ± 90° and ψ₂ XXX ? 150°.     

Biomolecular screening with novel organosilica microspheres

Organosilica microspheres synthesised via a novel surfactant-free emulsion-based method show applicability towards optical encoding, solid-phase synthesis and high-throughput screening of bound oligonucleotide and peptide sequences.     

Towards single-spot multianalyte molecular beacon biosensors

The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. Herein, we will discuss several successful efforts to develop oligonucleotide sensors based on the surface immobilization of functionalized DNA hairpins. We also will discuss the development of prototypical single-spot multianalyte “Molecular Beacon” biosensors. Importantly, we show that organic fluorophores will likely be inadequate in moving this technology forward and new approaches, such as the use of nanotechnology, will be needed.     

High-density synthetic peptide microarrays: emerging tools for functional genomics and proteomics

New approaches for manufacturing and application of peptide arrays on planar surfaces are emerging, thereby opening advanced opportunities to probe the expression and function of the proteome. In complementing DNA and protein array analyses, peptide fragment screening directly addresses functional protein interaction sites, leading to a detailed insight into the discovered molecular recognition events, placing them in the context of the whole genome, and even allowing rapid determination of the chemical nature of these interactions. This information can then be transferred into powerful small peptide tools that interfere with these interactions in vivo and help to link targets with phenotypes. With the spreading of new peptide array tools, peptide screening will extend its impact on modern genome-driven molecular biology. This will advance the systematic discovery and validation of new pharmaceutical targets as well as the development of potent molecular diagnostics for medical and ecological monitoring.     

A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.     

Current development of bicyclic peptides

Bicyclic peptides, a class of polypeptides with two loops within their structure, have emerged as powerful tools in the development of new peptide drugs. They have the potential to bind to challenged drug targets, with antibody-like affinity and selectivity. Meanwhile, bicyclic peptides possess small molecule-like access to chemical synthesis, which is conducive to large-scale synthesis and screening. In the last five years, bicyclic peptide technology has been increasingly developed, and researchers have carried out a variety of studies to elucidate the potential functions of bicyclic peptides. With the continuous development of synthetic methods and the advances of new technology to build bicyclic peptide libraries, bicyclic peptides are now becoming widely used in the development of new drugs for various diseases. This perspective provides an overview of the structure types, synthesis and applications of bicyclic peptides in current drug development, and our own views on future challenges of bicyclic peptides.     

Maleimide-dimethylfuran exo adducts: effective maleimide protection in the synthesis of oligonucleotide conjugates

The reaction of maleimide-containing compounds with 2,5-dimethylfuran gives a mixture of exo and endo isomers from which the exo cycloadduct can be easily isolated taking advantage of its stability in concentrated aqueous ammonia. Bifunctional compounds incorporating a dimethylfuran-protected maleimide (exo adduct) have been attached to resin-linked oligonucleotide chains. Removal of protecting groups masking oligonucleotide functionalities followed by retro-Diels-Alder maleimide deprotection affords maleimido-oligonucleotides suitable for conjugation, as assessed by their reaction with different thiols.     

(-)-Epigallocatechin gallate inhibits hepatitis C virus (HCV) viral protein NS5B

In this study, we elucidated a small molecule inhibitor on viral protein NS5B identified through a high-throughput screening strategy using optical nanoparticle-based RNA oligonucleotide. We have previously shown that quantum dots (QDs)-RNA oligonucleotide can specifically recognize the HCV viral proteins. We have also demonstrated that conjugated QDs-RNA oligonucleotide can specifically and sensitively interact with designed biochips [1] and [2]. Among the flavonoids examined, (−)-epigallocatechin gallate (EGCG) demonstrated a remarkable inhibition activity on HCV viral protein, NS5B. (−)-Epigallocatechin gallate, at 0.005 μg mL⁻¹ or more, concentration-dependently attenuated the binding affinity on a designed biochip as evidenced by QDs-RNA oligonucleotide. At a concentration of 0.1 μg mL⁻¹, (−)-epigallocatechin gallate showed a 50% inhibition activity on QDs-RNA oligonucleotide biochip assay. We screened a small molecule inhibitor on the viral protein, NS5B, identified through a high-throughput screening strategy using on-chip optical nanoparticle-based RNA oligonucleotide on chip. In this designed strategy, the convenient and efficient screening and development of an on-chip viral protein inhibitor using a QDs-RNA oligonucleotide assay is achievable with high sensitivity and simplicity. In addition, this platform is expected to be applicable toward the inhibitor screening of other types of diseases.     

Creation of chiral adsorbed structures using external inputs: results from lattice Monte Carlo simulations

Patterning of solid surfaces with organic molecules has been recognized as a promising method to create functional 2D matrices with tunable structure and properties. In this work we use the lattice Monte Carlo simulations to study chiral pattern formation in adsorbed systems comprising simple molecular building blocks differing in shape. To that end we consider five-membered rigid isomers whose composite segments can occupy vertices of a triangular lattice and interact with short-range (nearest neighbors) forces. Our main focus is on those molecules which are prochiral, that is they can adopt mirror-image planar configurations when adsorbed. Moreover, the effect of orientational in-plane confinement of the molecules, which reflects their coupling with an external directional field, on the structure formation and chiral resolution in 2D is explored. The obtained results demonstrate that the confinement imposed on the surface enantiomers can induce their resolution and formation of extended homochiral domains. However, it is also shown that for certain molecular shapes the confinement can produce mixed racemic crystals instead of the homochiral assemblies. The insights from our simulation studies can be helpful in preliminary screening of molecular libraries to select optimal building blocks able to self-assembly into chiral 2D patterns with predefined architecture.