以完整细胞为靶子的SELEX技术研究进展

指数富集的配体系统进化（SELEX）是一种从大容量寡核苷酸文库中经反复分离扩增步骤得到针对靶分子的高亲和力、高特异性核酸配基——适配体的体外筛选技术。自1990年以来,SELEX技术得到了迅猛发展,筛选的靶分子已由最初的单一物质发展到完整的动物细胞、细菌病原体等复杂靶子。以完整细胞为靶子的SELEX技术有其独特的技术优势,可以在筛选细胞上特定靶分子未知的情况下进行筛选,为药物筛选、临床诊断、疾病治疗和基础医学研究等带来了新的思路和方法。随着对适配体研究的深入,尤其是纳米材料与其相结合应用,该技术将在肿瘤诊断治疗及微生物检测领域具有更为广泛的应用前景。     

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia  coli -expressed glutathione- S -transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.     

核糖开关的研究及应用

核糖开关是一类自然界中天然存在的适配子,通过结合小分子代谢物调控基因的表达。它位于特定的mRNA非编码区,可以不依赖任何蛋白质因子而直接结合代谢物并发生构象变化,在转录和翻译水平上参与调控生物的基本代谢途径。目前已知核糖开关不仅广泛存在于细菌的代谢相关基因中,还存在于某些真菌和植物中。对核糖开关的深入研究将为基因功能研究、生物传感器研发以及新型抗菌药物开发等提供新的途径。     

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).     

Aptamer-based and DNAzyme-linked colorimetric detection of cancer cells

This paper reports a novel method to detect human leukemic lymphoblasts (CCRF-CEM cells). While the aptamer of the cancer cells was employed as the recognition element to target cancer cells, peroxidase-active DNAzyme was used as the sensing element to produce catalysis-induced colorimetric signals. The elegant architecture integrating the aptamer and DNAzyme made it feasible to detect cancer cells easily and rapidly by the color change of the substrate for DNAzyme. Experimental results showed that 500 cells can well indicate the cancer, while as control, 250,000 Islet Island Beta cells only show tiny signals, suggesting that the method proposed in this paper has considerable sensitivity and selectivity. Furthermore, since it does not require expensive apparatus, or modification or label of DNA chains, the method we present here is also cost-effective and conveniently operated, implying potential applications in future cancer diagnosis.     

多肽配体技术在植物功能基因组学中的应用前景

人工智能配体或适配体(Aptamer)技术是近年来兴起的一项特异性极强的基因干扰技术。通过人工合成特异的智能配体结合靶基因的蛋白产物, 达到特异干扰靶基因的生物学功能, 这是人工智能配体技术的基本设想。文章综述了多肽配体(Peptide aptamer)技术在基因功能验证中的主要进展, 着重阐明它在植物基因功能验证和作物抗病毒育种中的应用前景, 并提出克服该技术主要风险对策。     

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.     

RNA-based gene therapy for the treatment and prevention of HIV: from bench to bedside

Gene therapy is considered a feasible approach for the treatment and prevention of HIV/AIDS. Targeting both viral genes and host dependency factors can interfere with the viral lifecycle and prevent viral replication. A number of approaches have been taken to target these genes, including ribozymes, aptamers, and RNAi based therapies. A number of these therapies are now beginning to make their way into clinical trials and providing proof of principle that gene therapy is a safe and realistic option for treating HIV. Here, we focus on those therapies that have progressed along the pipeline to preclinical and clinical testing.     

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and differential scanning calorimetry (DSC) to study the properties of novel bioengineered DNA aptamer dimers sensitive to fibrinogen (F) and heparin (H) binding sites of thrombin and compared them with canonical single stranded aptamer sensitive to fibrinogen binding site of thrombin (Fibri). The homodimer (FF) and heterodimer (FH) aptamers were constructed based on hybridization of their supported parts. CD results showed that both FF and FH dimers form stable guanine quadruplexes in the presence of potassium ions like those in Fibri. The thermal stability of aptamer dimers was slightly lower compared to those of canonical aptamers, but sufficient for practical applications. Both FF and FH aptamer dimers exhibited a potassium-dependent inhibitory effect on thrombin-mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.     

G-四链体核酸的生物学功能

G-四链体是由富含鸟嘌呤的DNA或RNA折叠形成的高级结构。可形成G-四链体的序列在人基因组中广泛分布,涉及DNA复制、端粒维持、基因表达与调控以及遗传不稳定性等过程。研究发现有些化学合成的G-四链体序列也具有生物活性,如核仁素的核酸适体AS1411具有抑制恶性肿瘤增殖活性。G-四链体的生物学功能研究对于恶性疾病的发病机理和靶向治疗,以及设计开发核酸类抗癌药物有重要意义。