Moon Jeong, Lim Jaewoo, Lee Seoyoung, Son Hye Young, Rho Hyun Wook, Kim Hongki, Kang Hyunju, Jeong Jinyoung, Lim Eun-Kyung, Jung Juyeon, Huh Yong-Min, Park Hyun Gyu, Kang Taejoon
Bionanotechnology Research Center, KRIBB, Daejeon, 34141, Republic of Korea; Department of Chemical and Biomolecular Engineering (BK 21+ Program), KAIST, Daejeon, 34141, Republic of Korea.
Bionanotechnology Research Center, KRIBB, Daejeon, 34141, Republic of Korea; Department of Nanobiotechnology, KRIBB School of Biotechnology, UST, Daejeon, 34113, Republic of Korea.
Biosens Bioelectron. 2020 Nov 1;167:112474. doi: 10.1016/j.bios.2020.112474. Epub 2020 Aug 5.
Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.
外泌体信使核糖核酸（mRNA）因其在体液中的稳定性及其生物调节功能，已成为基于液体活检的疾病诊断和预后的重要生物标志物。在此，我们报告了一种基于三向连接（3WJ）形成的快速一步等温基因扩增反应，并成功检测了荷瘤小鼠尿液中的外泌体mRNA。在靶RNA存在的情况下，3WJ探针（3WJ模板和3WJ引物）可形成3WJ结构。3WJ结构形成后，3WJ引物通过DNA聚合酶和切口内切酶的组合反复延伸和切割，产生多个信号引物。随后，信号引物促进一个专门设计的网络反应途径，在等温条件下产生G-四链体探针。最后，G-四链体结构与硫黄素T结合时产生高度增强的荧光信号。该方法对靶RNA具有高选择性，检测限为1.23 pM（24.6 amol）。更重要的是，该方法可用于检测各种mRNA，包括乳腺癌细胞mRNA、乳腺癌外泌体mRNA，甚至乳腺癌小鼠尿液中的外泌体mRNA。我们预计，所开发的RNA检测方法可用于各种生物医学应用，如疾病诊断、预后和治疗监测。
