Eulberg Dirk, Buchner Klaus, Maasch Christian, Klussmann Sven
NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.
Nucleic Acids Res. 2005 Mar 3;33(4):e45. doi: 10.1093/nar/gni044.
We have developed an automated SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process that allows the execution of in vitro selection cycles without any direct manual intervention steps. The automated selection protocol is designed to provide for high flexibility and versatility in terms of choice of buffers and reagents as well as stringency of selection conditions. Employing the automated SELEX process, we have identified RNA aptamers to the mirror-image configuration (d-peptide) of substance P. The peptide substance P belongs to the tachykinin family and exerts various biologically important functions, such as peripheral vasodilation, smooth muscle contraction and pain transmission. The aptamer that was identified most frequently was truncated to the 44mer SUP-A-004. The mirror-image configuration of SUP-A-004, the so-called Spiegelmer, has been shown to bind to naturally occurring l-substance P displaying a K(d) of 40 nM and to inhibit (IC50 of 45 nM) l-substance P-mediated Ca2+ release in a cell culture assay.
我们开发了一种自动化的指数富集配体系统进化（SELEX）方法，该方法无需任何直接的人工干预步骤即可进行体外筛选循环。自动化筛选方案旨在在缓冲液和试剂的选择以及筛选条件的严格性方面提供高度的灵活性和通用性。利用自动化SELEX方法，我们鉴定出了针对P物质镜像构型（d-肽）的RNA适配体。肽类物质P属于速激肽家族，具有多种重要的生物学功能，如外周血管舒张、平滑肌收缩和疼痛传递。最常鉴定出的适配体被截短为44聚体SUP-A-004。SUP-A-004的镜像构型，即所谓的镜像体，已被证明能与天然存在的l-物质P结合，解离常数（K(d)）为40 nM，并在细胞培养试验中抑制（半数抑制浓度（IC50）为45 nM）l-物质P介导的Ca2+释放。
