한빛사논문
- 조회389
Jeong Moon a,b, Jayeon Song a, Hyowon Jang a, Hyunju Kang a,c, Yong-Min Huh d,e,f,g, Hye Young Son d,g, Hyun Wook Rho d, Mirae Park d, Chandana S. Talwar h,i, Kwang-Hyun Park h, Euijeon Woo h,i, Jaewoo Lim a,j, Eun-Kyung Lim a,j, Juyeon Jung a, Yongwon Jung c, Hyun Gyu Park b,*, Taejoon Kang a,*
a Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea b 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 c Department of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea d Department of Radiology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea e YUHS-KRIBB Medical Convergence Research Institute, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea f Department of Biochemistry & Molecular Biology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea g Severance Biomedical Science Institute, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea h Disease Target Structure Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea i Department of Biomolecular Science, KRIBB School of Bioscience, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea j Department of Nanobiotechnology, KRIBB School of Biotechnology, UST, 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea
* Corresponding author.
Abstract
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.
논문정보
- Research article
- 2022년 04월 (BRIC 등록일 2022-05-26)
- 국내 연구진
- 바이오·의료융합 > 바이오센싱 및 나노바이오물질
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