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DNA-Wrapped CNT Sensor for Small Nucleic Acid Detection: Influence of Short Complementary Sequence

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Abstract

Carbon nanotubes (CNTs) are versatile materials that act as natural fluorescence quenchers and double scaffolds for DNA that can wrap around them based on π–π stacking. We exploited these properties of CNTs to develop a hybridization-based sensor for the detection of microRNA. We designed a fluorescein amidite (FAM)-labeled single-stranded oligonucleotide containing a CNT binding region (Poly T) followed by a sequence complementary to the target nucleic acid (probe sequence). As the DNA wraps around a CNT, FAM fluorescence is quenched in the absence of the target, whereas in the presence of the target, fluorescence emission is obtained. Experimentally, we found that one of the major issues with this sensor is its compromised sensitivity due to competition for adsorption of the probe DNA onto the CNT versus that of hybridization with the target DNA. To overcome this, we introduced a short complementary sequence (SCS) that binds to the probe sequence and found that it significantly improved the limit of detection of the sensor approximately 25-fold. To gain further insights into the mechanism of SCS in improving the sensor performance, we performed molecular dynamics (MD)-based simulations. Based on hybridization energy calculations performed using MM-GBSA, we found that the position of the SCS is key to shaping the binding affinity of the probe to the CNT. The MD-based calculations were validated using experimental results by comparing the sensor’s experimental limit of detection and the hybridization energy obtained computationally. Finally, we demonstrated the applicability of this sensor for the experimental detection of the cervical cancer-related biomarker miR-21-5p in human serum.

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Acknowledgements

We acknowledge the support received from the National Research Foundation (NRF) of Korea for the Basic Science Research Program (NRF-2019R1A6A1A03033215) and the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant (2020R1A6C103B101) funded by the Ministry of Education. This research was also supported by the National Research Foundation (NRF) of Korea for Bio & Medical Technology Development Program (NRF-2022M3A9G8017220) funded by the Ministry of Science & ICT. The authors are grateful to the Ministry of Human Resource Development, Government of India, for financial support through the Scheme for Promotion of Academic and Research Collaboration (SPARC) project “SPARC/2018-19/P402/SL”. Shrute Kannappan appreciates Sungkyunkwan University for the SKKU Research Matters Fellowship.

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  1. Shrute Kannappan and Junhyuck Chang have contributed equally to this work.

Authors and Affiliations

  1. Department of Precision Medicine, School of Medicine, Sungkyunkwan University (SKKU), Suwon, 16419, Korea

    Shrute Kannappan & Kyeong Kyu Kim

  2. Research Center for Advanced Materials Technology, Core Research Institute, Suwon, 16419, Korea

    Shrute Kannappan, Jun Hyuk Heo & Jung Heon Lee

  3. School of Advanced Materials Science and Engineering, Sungkyunkwan University (SKKU), Suwon, 16419, Korea

    Junhyuck Chang, Priyannth Ramasami Sundharbaabu, Jun Hyuk Heo, Jae Chul Ro & Jung Heon Lee

  4. School of Electrical and Electronics Engineering, SASTRA Deemed University, Thanjavur, 613401, India

    John Bosco Balaguru Rayappan

  5. Centre for Nanotechnology and Advanced Biomaterials (CeNTAB), SASTRA Deemed University, Thanjavur, 613401, India

    John Bosco Balaguru Rayappan

  6. PNG Biomed, Youngin-Si, 16950, Republic of Korea

    Won-kee Sung

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  1. Shrute Kannappan
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Kannappan, S., Chang, J., Sundharbaabu, P.R. et al. DNA-Wrapped CNT Sensor for Small Nucleic Acid Detection: Influence of Short Complementary Sequence. BioChip J 16, 490–500 (2022). https://doi.org/10.1007/s13206-022-00088-7

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