Moon Jeong, Song Jayeon, Jang Hyowon, Kang Hyunju, Huh Yong-Min, Son Hye Young, Rho Hyun Wook, Park Mirae, Talwar Chandana S, Park Kwang-Hyun, Woo Euijeon, Lim Jaewoo, Lim Eun-Kyung, Jung Juyeon, Jung Yongwon, Park Hyun Gyu, Kang Taejoon
Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Chemical and Biomolecular Engineering (BK21+ Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Biosens Bioelectron. 2022 Aug 1;209:114256. doi: 10.1016/j.bios.2022.114256. Epub 2022 Apr 11.
In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.
在本研究中，我们发现了一种在两个相邻的片段化探针中进行无连接DNA延伸的方法，这两个探针与靶RNA杂交，用于开发一种无连接核酸扩增反应。在该反应中，无论连接与否，在靶RNA存在的情况下，DNA从正向探针延伸至硫代磷酸化发夹探针。然后，第二次DNA延伸在硫代磷酸化探针的切口位点和自引发区域同时发生。因此，成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）12a的结合位点被反复扩增，在CRISPR-Cas12a存在的情况下诱导荧光信号。这种无连接等温基因扩增方法能够以49.2 fM的灵敏度检测靶RNA。此外，两种类型的mRNA检测都是可行的，因此证明了该方法在癌症伴随诊断中的潜力。值得注意的是，所提出的方法在应用于检测从人细胞、荷瘤小鼠组织和尿液样本中提取的mRNA时也显示出有效性。因此，这种新开发的无连接等温核酸扩增系统有望在各种基因检测平台中得到广泛应用。
