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.
|Journal||Biosensors and Bioelectronics|
|Publication status||Published - 2022 Aug 1|
Bibliographical noteFunding Information:
This work was supported by National R&D Programs through National Research Foundation (NRF) of Korea funded by the Ministry of Science and ICT (MSIT) of Korea [ NRF-2020R1A2C1010453 , NRF-2021M3E5E3080844 , NRF-2021M3H4A1A02051048 , NRF-2021M3E5E3080379 , NRF-2018M3A9E2022821 , NRF-2018M3A9E2022828 , NRF-2019H1A2A1073468 , NRF-2021R1A2B5B03001739 , and NRF-2017M3A7B4041975 ]; Global Frontier Program through Center for BioNano Health-Guard funded by MSIT of Korea [ H-GUARD_2014M3A6B2060507 and H-GUARD_2013M3A6B2078950 ]; Technology Development Program for Biological Hazards Management in Indoor Air through Korea Environment Industry & Technology Institute (KEITI) funded by Ministry of Environment (ME) of Korea [ 2021003370003 ]; Industrial Technology Alchemist Program funded by the Ministry of Trade, Industry, and Energy (MOTIE) of Korea [ 20012435 ]; Nanomedical Devices Development Program of National Nano Fab Center [ CSM2105M101 ]; and KRIBB Research Initiative Program [ 1711134081 ].
© 2022 The Authors
All Science Journal Classification (ASJC) codes
- Biomedical Engineering