Ligation-free isothermal nucleic acid amplification

Jeong Moon; Jayeon Song; Hyowon Jang; Hyunju Kang; Yong Min Huh; Hye Young Son; Hyun Wook Rho; Mirae Park; Chandana S. Talwar; Kwang Hyun Park; Euijeon Woo; Jaewoo Lim; Eun Kyung Lim; Juyeon Jung; Yongwon Jung; Hyun Gyu Park; Taejoon Kang

doi:10.1016/j.bios.2022.114256

Ligation-free isothermal nucleic acid amplification

Jeong Moon, Jayeon Song, Hyowon Jang, Hyunju Kang, Yong Min Huh, Hye Young Son, Hyun Wook Rho, Mirae Park, Chandana S. Talwar, Kwang Hyun Park, Euijeon Woo, Jaewoo Lim, Eun Kyung Lim, Juyeon Jung, Yongwon Jung, Hyun Gyu Park, Taejoon Kang

Department of Radiology

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

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.

Original language	English
Article number	114256
Journal	Biosensors and Bioelectronics
Volume	209
DOIs	https://doi.org/10.1016/j.bios.2022.114256
Publication status	Published - 2022 Aug 1

Bibliographical note

Funding 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 ].

Publisher Copyright:
© 2022 The Authors

All Science Journal Classification (ASJC) codes

Biotechnology
Biophysics
Biomedical Engineering
Electrochemistry

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.bios.2022.114256

Cite this

@article{0a347ad51c6948789510dbdef1f30b34,

title = "Ligation-free isothermal nucleic acid amplification",

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.",

author = "Jeong Moon and Jayeon Song and Hyowon Jang and Hyunju Kang and Huh, {Yong Min} and Son, {Hye Young} and Rho, {Hyun Wook} and Mirae Park and Talwar, {Chandana S.} and Park, {Kwang Hyun} and Euijeon Woo and Jaewoo Lim and Lim, {Eun Kyung} and Juyeon Jung and Yongwon Jung and Park, {Hyun Gyu} and Taejoon Kang",

note = "Funding 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 ]. Publisher Copyright: {\textcopyright} 2022 The Authors",

year = "2022",

month = aug,

day = "1",

doi = "10.1016/j.bios.2022.114256",

language = "English",

volume = "209",

journal = "Biosensors and Bioelectronics",

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T1 - Ligation-free isothermal nucleic acid amplification

AU - Moon, Jeong

AU - Song, Jayeon

AU - Jang, Hyowon

AU - Kang, Hyunju

AU - Huh, Yong Min

AU - Son, Hye Young

AU - Rho, Hyun Wook

AU - Park, Mirae

AU - Talwar, Chandana S.

AU - Park, Kwang Hyun

AU - Woo, Euijeon

AU - Lim, Jaewoo

AU - Lim, Eun Kyung

AU - Jung, Juyeon

AU - Jung, Yongwon

AU - Park, Hyun Gyu

AU - Kang, Taejoon

N1 - Funding 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 ]. Publisher Copyright: © 2022 The Authors

PY - 2022/8/1

Y1 - 2022/8/1

N2 - 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.

AB - 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.

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DO - 10.1016/j.bios.2022.114256

M3 - Article

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AN - SCOPUS:85128210468

SN - 0956-5663

VL - 209

JO - Biosensors and Bioelectronics

JF - Biosensors and Bioelectronics

M1 - 114256

ER -

Ligation-free isothermal nucleic acid amplification

Abstract

Bibliographical note

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UN SDGs

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