Aptamer-Based Sensitive Detection of Target Molecules via RT-PCR Signal Amplification

作者：Liao, Shiqi^*; Liu, Yuanqiang; Zeng, Jiayu; Li, Xiaoxu; Shao, Ningsheng; Mao, Aihong; Wang, Li; Ma, Jin; Cen, Ha; Wang, Yunpu; Zhang, Xueli; Zhang, Ruitang; Wei, Zhengli; Wang, Xiaohui

来源：Bioconjugate Chemistry, 2010, 21(12): 2183-2189.

DOI：10.1021/bc100032v

• AFFINITY RNA LIGANDS
• NUCLEIC-ACID
• SYSTEMATIC EVOLUTION
• PROTEIN
• BEACONS
• RECOGNITION
• SELECTION
• PROBE
• SELEX

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores. we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative OCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fe fragment of mouse IgG were selected and subjected to coupling with the OCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the OCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative OCR (A-PCR) was conducted to validate the application for such template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-OCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal. and the feasibility of the OCR template tethered aptamer as a facile, specific. and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.

• 出版日期2010-12
• 单位甘肃省医学科学研究院; 西北师范大学