Research ArticleGENETICS

Cellular microRNA detection with miRacles: microRNA- activated conditional looping of engineered switches

See allHide authors and affiliations

Science Advances  13 Mar 2019:
Vol. 5, no. 3, eaau9443
DOI: 10.1126/sciadv.aau9443
  • Fig. 1 Concept and workflow of the miRacles assay.

    (A) Workflow of the miRacles assay: Customized DNA nanoswitches are mixed with target miRNA sample, incubated, and run on an agarose gel for detection. (B) DNA nanoswitches undergo a conformational change from a linear “off” state to a looped “on” state when bound to a target miRNA. Inset: The nanoswitch is composed of a single-stranded M13 scaffold, backbone oligonucleotides, and single-stranded extensions (detectors) complementary to the target miRNA. Intercalating dyes intrinsic to the electrophoresis process provide the signal to visualize the nanoswitches. (C) The two conformations are resolvable in a standard agarose gel.

  • Fig. 2 Validation of the miRacles assay.

    (A) Specificity of the DNA nanoswitches with detectors designed for let-7b. As low as 1-nt mismatch between the detectors and the target miRNA eliminates the signal. (B) Limit of detection of the assay. NC, negative control. AU, arbitrary units. (C) Time course of the assay for a low-concentration target. (D) Dynamic range of the assay at different reaction times. Data points and error bars represent the means and SD, respectively, from triplicate measurements.

  • Fig. 3 miRNA detection from differentiating myoblast cells.

    (A) Schematic showing myoblast cells, harvested while growing in GM and on differentiation days 1 and 4, processed to yield total and small RNA fractions. An early myogenic differentiation marker, Myog, and a late myogenic differentiation marker, MHC, were measured by (B) Western blotting and (C) by immunocytochemistry to confirm differentiation. Both Myog and MHC were up-regulated in DM1 and DM4. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) served as a control in (B). (D) We detected miR-206 in the differentiated samples with 50 ng of small RNAs and (E) with 500 ng of total RNAs. Quantification of gel intensities shows a sharp progressive up-regulation during differentiation, similar in both (F) small RNA and (G) total RNA samples. From DM4 samples, we note detection from as little as (H) 200 pg of small RNAs and (I) 500 pg of total RNAs. Error bars represent SD from triplicate measurements.

  • Fig. 4 Five-channel multiplexing.

    (A) Multiplexing enables the detection of different miRNAs with different loop sizes. (B) A multiplexed nanoswitch mixture shows five bands with similar intensity in a positive control consisting of all five target miRNAs. In 50 ng of DM4 small RNAs, four different miRNAs are detected at various expression levels, with miR-39 (a C. elegans–specific miRNA) not being detected.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaau9443/DC1

    Fig. S1. Design and construction of the DNA nanoswitch.

    Fig. S2. Programmable design of the DNA nanoswitch.

    Fig. S3. Concept validation using miRNA let-7b.

    Fig. S4. Specificity and sensitivity in other miRNAs.

    Fig. S5. Mismatch detection in small RNA samples.

    Fig. S6. Protocol modification for total RNA detection.

    Fig. S7. Sensitivity of miR-206 detection from RNA extracts.

    Fig. S8. MicroRNA detection in different cell lines.

    Fig. S9. Stability of the nanoswitch after drying.

    Table S1. Comparison of miRacles assay with currently existing miRNA detection methods.

    Movie S1. Time-lapse movie of miRNA detection in 1 hour using miRacles assay.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Design and construction of the DNA nanoswitch.
    • Fig. S2. Programmable design of the DNA nanoswitch.
    • Fig. S3. Concept validation using miRNA let-7b.
    • Fig. S4. Specificity and sensitivity in other miRNAs.
    • Fig. S5. Mismatch detection in small RNA samples.
    • Fig. S6. Protocol modification for total RNA detection.
    • Fig. S7. Sensitivity of miR-206 detection from RNA extracts.
    • Fig. S8. MicroRNA detection in different cell lines.
    • Fig. S9. Stability of the nanoswitch after drying.
    • Table S1. Comparison of miRacles assay with currently existing miRNA detection methods.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Time-lapse movie of miRNA detection in 1 hour using miRacles assay.

    Files in this Data Supplement:

Navigate This Article