School of Life Sciences, Central South University, Changsha 410013, China.
Anal Methods. 2021 May 14;13(18):2107-2113. doi: 10.1039/d1ay00309g. Epub 2021 Apr 19.
MicroRNA (miRNA) expression is perturbed in various diseases. Herein, we have aimed to develop a novel and rapid fluorescence-based assay for detecting microRNA-21 (miR-21) activity based on FAM molecular signal amplification and graphene oxide (GO) quenching. In this system, a single stranded DNA (ssDNA) with a phosphate group at the 5'-end is labeled with a FAM molecular label at the 3'-end. In the presence of miR-21, this ssDNA forms a DNA/RNA duplex, which is cleaved by λ exonuclease (λ-exo), releasing FAM and resulting in fluorescence signal amplification at 530 nm. However, the DNA/RNA duplex is not generated in the absence of miR-21, which impedes λ-exo cleavage; subsequently, GO quenches the fluorescence intensity. The results show a detection limit of 0.02 nM and a wide linear range of 0.02-5 nM. The high sensitivity and easy operability of this assay can be applied for detecting miR-21 during clinical diagnosis of certain diseases and in biological research.
微 RNA (miRNA) 的表达在各种疾病中受到干扰。在此，我们旨在开发一种新颖、快速的基于荧光的测定法，用于检测 microRNA-21 (miR-21) 的活性，该方法基于 FAM 分子信号放大和氧化石墨烯 (GO) 猝灭。在该系统中，5' 端带有磷酸基团的单链 DNA (ssDNA) 在 3' 端用 FAM 分子标记标记。在 miR-21 的存在下，该 ssDNA 形成 DNA/RNA 双链体，被 λ 外切酶 (λ-exo) 切割，释放 FAM，并在 530nm 处产生荧光信号放大。然而，在没有 miR-21 的情况下，不会生成 DNA/RNA 双链体，从而阻止 λ-exo 切割；随后，GO 猝灭荧光强度。结果表明，检测限为 0.02nM，线性范围为 0.02-5nM。该测定法具有高灵敏度和易于操作的特点，可应用于某些疾病的临床诊断和生物研究中检测 miR-21。
