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Rapid and ultrasensitive miRNA detection by combining endonuclease reactions in a rolling circle amplification (RCA)–based hairpin DNA fluorescent assay

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Abstract

MicroRNA (miRNA) sensing strategies employing rolling circle amplification (RCA) coupled with the hairpin DNA (HD) probe–mediated FRET assay have shown promise, but achieving rapid, sensitive, and specific detection of target miRNA remains a challenge in clinical diagnostics. Herein, we incorporate PstI endonuclease cleavage (PEC) into a conventional RCA-based HD probe FRET assay to develop an effective and feasible method. Long single-stranded RCA products are synthesized from miRNA-21 loaded on a circular dumbbell DNA, and the resultant RCA products self-assemble to generate long HD structures with double-stranded stem regions that are specifically recognized and cleaved by PstI endonucleases when incubated with PstI enzymes. This releases large amounts of short single-stranded DNA fragments that hybridize and open to the complementary loop-stem regions of HD probes labeled with FAM at one end and BHQ-1 at the other, resulting in a reduction in FRET efficiency. This assay achieves a 39.7 aM detection limit for target miRNA-21, approximately 37-fold higher than that of the conventional assay (1.5 fM). Moreover, quantitative detection is possible in a wide range from 1 aM to 1 pM within 90 min with high sequence specificity. We demonstrate the assay with the detection of target miRNA-21 in total RNA extracted from MCF-7 cancer cells.

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Funding

This work was supported by a Yeungnam University Research Grant (219A580020).

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  1. Yun Jin Lee and Ji Yun Jeong contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Yeungnam University, 280 Daehak-Ro, Gyeongsangbuk-Do 38541, Gyeongsan-Si, Republic of Korea

    Yun Jin Lee, Ji Yun Jeong, Ji Yoon Do & Cheol Am Hong

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  1. Yun Jin Lee
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  2. Ji Yun Jeong
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Contributions

Yun Jin Lee and Ji Yun Jeong: conceptualization; methodology; investigation; validation; and writing—original draft preparation. Ji Yoon Do: methodology, validation, and resources. Cheol Am Hong: supervision; project administration; conceptualization; methodology; funding acquisition; and writing—reviewing and editing.

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Correspondence to Cheol Am Hong.

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Lee, Y.J., Jeong, J.Y., Do, J.Y. et al. Rapid and ultrasensitive miRNA detection by combining endonuclease reactions in a rolling circle amplification (RCA)–based hairpin DNA fluorescent assay. Anal Bioanal Chem 415, 1991–1999 (2023). https://doi.org/10.1007/s00216-023-04618-6

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