Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing
The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome...
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MDPI AG
2024-12-01
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| author | Shurui Tao Yi Long Guozhen Liu |
| author_facet | Shurui Tao Yi Long Guozhen Liu |
| author_sort | Shurui Tao |
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| description | The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay’s working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon’s loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies. |
| format | Article |
| id | doaj-art-a8afdf6130a94118bcf63a0d6ae0eb64 |
| institution | Kabale University |
| issn | 2079-6374 |
| language | English |
| publishDate | 2024-12-01 |
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| series | Biosensors |
| spelling | doaj-art-a8afdf6130a94118bcf63a0d6ae0eb642024-12-27T14:14:17ZengMDPI AGBiosensors2079-63742024-12-01141261810.3390/bios14120618Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA BiosensingShurui Tao0Yi Long1Guozhen Liu2CUHKSZ-Boyalife Regenerative Medicine Engineering Joint Laboratory, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, ChinaCUHKSZ-Boyalife Regenerative Medicine Engineering Joint Laboratory, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, ChinaCUHKSZ-Boyalife Regenerative Medicine Engineering Joint Laboratory, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, ChinaThe Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay’s working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon’s loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies.https://www.mdpi.com/2079-6374/14/12/618rolling circle amplificationminimum secondary structured RCAmolecular beaconHPV detectionsensitivity |
| spellingShingle | Shurui Tao Yi Long Guozhen Liu Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing Biosensors rolling circle amplification minimum secondary structured RCA molecular beacon HPV detection sensitivity |
| title | Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing |
| title_full | Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing |
| title_fullStr | Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing |
| title_full_unstemmed | Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing |
| title_short | Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing |
| title_sort | entropy driven molecular beacon assisted special rca assay with enhanced sensitivity for room temperature dna biosensing |
| topic | rolling circle amplification minimum secondary structured RCA molecular beacon HPV detection sensitivity |
| url | https://www.mdpi.com/2079-6374/14/12/618 |
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