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Biosens. Bioelectron., Available online 16 January 2023 | https://doi.org/10.1016/j.bios.2023.115085 Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube

Jayeon Song^a,1, Younseong Song^b,c,1, Hyowon Jang^a, Jeong Moon^a,b, Hyunju Kang^a,d, Yong-Min Huh^e,f,g,h, Hye Young Son^e,g, Hyun Wook Rho^e, Mirae Park^e, Eun-Kyung Lim^a,i,j, Juyeon Jung^a,j, Yongwon Jung^d, Hyun Gyu Park^b, Kyoung G. Lee^c, Sung Gap Im^b, Taejoon Kang^a,j

^aBionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
^bDepartment of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
^cDivision of Nano-Bio Sensors/Chips Development, National NanoFab Center (NNFC), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
^dDepartment of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
^eDepartment of Radiology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
^fDepartment of Biochemistry & Molecular Biology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
^gSeverance Biomedical Science Institute, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
^hYUHS-KRIBB Medical Convergence Research Institute, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
ⁱDepartment of Nanobiotechnology, KRIBB School of Biotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea
^jSchool of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do, 16419, Republic of Korea

¹These authors contributed equally to this work.
Corresponding authors:Kyoung G. Lee, Sung Gap Im, Taejoon Kang

Abstract

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

논문정보

형식Research article
게재일2023년 01월 (BRIC 등록일 2023-01-26)
연구진국내(교신)+국외 연구진
분야 바이오·의료융합 > 바이오센싱 및 나노바이오물질

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