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Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection

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RNA Nanostructures

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1632))

Abstract

MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce “contaminative” nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide–PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.

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Acknowledgment

We appreciate Amy Gu and Michael Pennella who contributed to the nanopore experiments as highschool student research trainees. This work was supported through initial NSF CAREER award 0546165 and NIH R01-GM079613, and current NIH R01-GM114204. This investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program Grant C06-RR-016489-01 from the National Center for Research Resources, National Institutes of Health.

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Authors and Affiliations

  1. Department of Bioengineering, University of Missouri, 134 Research Park, Columbia, MO, 65211, USA

    Kai Tian, Ruicheng Shi, Amy Gu, Michael Pennella & Li-Qun Gu

  2. Dalton Cardiovascular Research Center, University of Missouri, 134 Research Park, Columbia, MO, 65211, USA

    Kai Tian, Ruicheng Shi, Amy Gu, Michael Pennella & Li-Qun Gu

Authors
  1. Kai Tian
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  2. Ruicheng Shi
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  3. Amy Gu
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  4. Michael Pennella
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  5. Li-Qun Gu
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Corresponding author

Correspondence to Li-Qun Gu .

Editor information

Editors and Affiliations

  1. Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Frederick, Maryland, USA

    Eckart Bindewald

  2. RNA Structure and Design Section, RNA Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA

    Bruce A. Shapiro

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Tian, K., Shi, R., Gu, A., Pennella, M., Gu, LQ. (2017). Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection. In: Bindewald, E., Shapiro, B. (eds) RNA Nanostructures . Methods in Molecular Biology, vol 1632. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7138-1_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7138-1_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7137-4

  • Online ISBN: 978-1-4939-7138-1

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