Department of Chemistry, U niversity of California-Irvine, Irvine, California 92697, USA.
J Am Chem Soc. 2013 Feb 13;135(6):2072-5. doi: 10.1021/ja311367t. Epub 2013 Jan 31.
A novel method to quantitatively measure the binding of proteins to single-stranded DNA (ssDNA) aptamers that employs the inhibition of the DNAzyme hydrolysis of aptamer monolayers is described. A 28-base DNAzyme was designed to specifically bind to and cleave a 29-base ssDNA sequence that can fold into a G-quartet aptamer and bind the protein thrombin. The binding strength of the DNAzyme to the aptamer sequence was designed to be less than the binding strength of the thrombin to the aptamer (ΔG° = -43.1 and -51.8 kJ/mol, respectively). Formation of the thrombin-aptamer complex was found to block DNAzyme cleavage activity both in solution and in an ssDNA aptamer monolayer. We denote this method for detecting protein-aptamer complexation as "DNAzyme footprinting" in analogy to the process of DNase footprinting for the detection of protein-DNA interactions. By attaching a 40-base reporter sequence to the ssDNA aptamer monolayer, the detection of any protein-aptamer complexes remaining on the surface after DNAzyme activity can be greatly enhanced (down to one thrombin-aptamer complex per 10,000 ssDNA molecules corresponding to 100 fM thrombin in solution) by a subsequent surface RNA transcription amplification reaction followed by RNA detection with nanoparticle-enhanced SPR imaging. In addition to RNA transcription, DNAzyme footprinting can be coupled to a wide variety of other nucleic acid surface amplification schemes and thus is a powerful new route for the enzymatically amplified detection of proteins via protein-aptamer complex formation.
一种用于定量测量蛋白质与单链 DNA (ssDNA) 适体结合的新方法，该方法采用抑制适体单层中 DNA 酶水解的方法。设计了一种 28 碱基的 DNA 酶，以特异性结合并切割 29 碱基的 ssDNA 序列，该序列可以折叠成 G-四联体适体并结合蛋白质凝血酶。DNA 酶与适体序列的结合强度设计为小于凝血酶与适体的结合强度（ΔG°分别为-43.1 和-51.8 kJ/mol）。发现凝血酶-适体复合物的形成会阻断 DNA 酶在溶液中和 ssDNA 适体单层中的切割活性。我们将这种检测蛋白质-适体复合物形成的方法称为“DNA 酶足迹法”，类似于用于检测蛋白质-DNA 相互作用的 DNase 足迹法。通过将 40 碱基的报告序列连接到 ssDNA 适体单层上，可以大大增强表面上任何剩余的蛋白质-适体复合物的检测（在溶液中每 10,000 个 ssDNA 分子中只有一个凝血酶-适体复合物，对应于 100 fM 凝血酶），随后进行表面 RNA 转录扩增反应，然后用纳米粒子增强 SPR 成像检测 RNA。除了 RNA 转录外，DNA 酶足迹法还可以与各种其他核酸表面扩增方案相结合，因此是通过蛋白质-适体复合物形成酶促放大检测蛋白质的强大新途径。
