Yang Litao, Fung Christine W, Cho Eun Jeong, Ellington Andrew D
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Anal Chem. 2007 May 1;79(9):3320-9. doi: 10.1021/ac062186b. Epub 2007 Mar 23.
Real-time nucleic acid amplification methods can be extremely useful for the identification and quantitation of nucleic acid analytes, but are more difficult to adapt to protein or other analytes. To facilitate the development of real-time rolling circle amplification (RCA) for protein targets, we have developed a novel type of conformation-switching aptamer that can be circularized upon interaction with its protein target, the platelet-derived growth factor (PDGF). Using the structure-switching aptamer, real-time RCA can be used to specifically quantitate PDGF down to the low-nanomolar range (limit of detection, 0.4 nM), even against a background of cellular lysate. The aptamer can also be adapted to RCA on surfaces, although quantitation proved to be more difficult. One of the great advantages of the method described herein is that it can be immediately adapted to almost any aptamer and does not require two or more affinity reagents as do sandwich or proximity assays.
实时核酸扩增方法对于核酸分析物的鉴定和定量可能极其有用，但更难适用于蛋白质或其他分析物。为了促进针对蛋白质靶标的实时滚环扩增（RCA）的发展，我们开发了一种新型的构象转换适体，它在与其蛋白质靶标血小板衍生生长因子（PDGF）相互作用时可以环化。使用这种结构转换适体，实时RCA可用于特异性定量低至纳摩尔范围（检测限为0.4 nM）的PDGF，即使在细胞裂解液背景下也是如此。该适体也可适用于表面RCA，尽管定量证明更困难。本文所述方法的一大优点是它几乎可以立即适用于任何适体，并且不像夹心或邻近分析那样需要两种或更多种亲和试剂。
