Nitsche Andreas, Kurth Andreas, Dunkhorst Anna, Pänke Oliver, Sielaff Hendrik, Junge Wolfgang, Muth Doreen, Scheller Frieder, Stöcklein Walter, Dahmen Claudia, Pauli Georg, Kage Andreas
Centre for Biological Safety 1, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany.
BMC Biotechnol. 2007 Aug 15;7:48. doi: 10.1186/1472-6750-7-48.
As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.
The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.
The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.
作为一类新型的治疗和诊断试剂，RNA和DNA适配体在十五年多前就被鉴定为可与众多小分子化合物、蛋白质结合，甚至很少能与完整的病原体颗粒结合的分子。大多数适配体是通过一种称为SELEX的过程从合成核酸的复杂文库中分离出来的，该过程基于几个筛选和扩增步骤。在此，我们报告了一种新的一步筛选方法（MonoLEX）的应用，以获得与用作复杂靶标结构模型生物的痘苗病毒结合的高亲和力DNA适配体。
针对完整痘苗病毒颗粒的筛选产生了一种64个碱基的DNA适配体，经斑点印迹分析、表面等离子体共振、荧光相关光谱和实时PCR验证，该适配体特异性结合正痘病毒，随后进行适配体印迹分析。相同的寡核苷酸显示出以浓度依赖的方式抑制痘苗病毒和其他正痘病毒体外感染的能力。
如本文所示，MonoLEX方法是一种直接的程序，用于鉴定与痘苗病毒结合的高亲和力DNA适配体。MonoLEX包括单个亲和色谱步骤, 随后对亲和树脂进行物理分割以及对结合的适配体进行单个最终PCR扩增步骤。因此，该程序通过减少PCR步骤中不同亲和力适配体之间的竞争来改进高亲和力适配体的筛选，这表明单轮MonoLEX方法具有优势。
