Research ArticlePLANT SCIENCES

Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown

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Science Advances  24 Jun 2020:
Vol. 6, no. 26, eaaz0495
DOI: 10.1126/sciadv.aaz0495
  • Fig. 1 siRNA-SWNT preparation and characterization.

    (A) Two sets of siRNA sequences targeting the GFP gene of transgenic mGFP5 Nb were separately tested in this study. Sequences on the left were chosen from Tang et al. (46), and sequences on the right were designed specifically for this study. (B) Suspension of pristine SWNTs with sense and antisense ssRNA sequences via probe-tip sonication. (C) Absorbance spectra of all RNA-SWNT suspensions. (D) nIR spectra of all RNA-SWNT suspensions. a.u., arbitrary units.

  • Fig. 2 ssRNA-SWNT internalization into transgenic mGFP5 N. benthamiana leaves.

    (A) Schematic showing samples tested for internalization into mGFP5 Nb leaves (Cy3-tagged RNA-SWNTs and Cy3-tagged free RNA as a control) and samples subsequently tested for silencing of a constitutively expressed GFP gene (RNA-SWNTs and free siRNA as a control). (B) Representative confocal images of Cy3-RNA-SWNT– and Cy3-RNA–infiltrated Nb leaves; intracellular GFP (green), Cy3 (red), and colocalization (white) channels. All scale bars, 20 μm.

  • Fig. 3 Thermodynamic analysis of RNA desorption from SWNTs: Hybridization in extracellular and intracellular conditions and proposed gene silencing mechanism.

    (A) An equimolar mixture of sense-SWNT and antisense-SWNT suspensions are infiltrated into transgenic Nb leaves with a needleless syringe. In the extracellular area of leaf tissue, RNA desorption and hybridization are not thermodynamically favorable because of the high free-energy cost of bare SWNTs. (B) Inside cells, RNA desorption from SWNTs and hybridization are thermodynamically favorable because molecules can occupy the bare SWNT surface and lower the RNA desorption free-energy cost. Upon desorption from SWNTs, double-stranded active siRNA assembles with the gene silencing complex and complexes with target mRNA for cleavage and gene silencing.

  • Fig. 4 GFP gene silencing with RNA-SWNTs at the mRNA transcript and protein level.

    (A) Representative confocal microscopy images of nontreated and s-RNA-SWNT–, free siRNA–, a-siRNA-SWNT–, and b-siRNA-SWNT–infiltrated transgenic Nb leaves 2 days after infiltration. Scale bars, 100 μm. (B) Quantitative fluorescence intensity analysis of confocal images for s-RNA-SWNT and a-siRNA-SWNT at 1, 2, 3, and 7 days after infiltration. ****P < 0.0001 in one-way ANOVA. Error bars, SD (n = 10). (C) Representative Western blot for GFP extracted from s-RNA-SWNT– and a-siRNA-SWNT–infiltrated Nb leaves 1 and 2 days after infiltration and quantification of GFP protein. ***P = 0.0001 in one-way ANOVA; error bars, SEM (n = 3). (D) qPCR analysis for GFP mRNA fold change at days 1 and 7 after infiltration for all samples tested. **P = 0.0016; ***P = 0.0008; ****P < 0.0001 in two-way ANOVA (n.s., nonsignificant). All error bars, SEM (n = 3). (E) qPCR analysis for GFP mRNA fold change at days 1, 3, and 7 and day 7 with reinfiltration at day 5 for a-siRNA-SWNT–treated Nb leaf sample. ****P < 0.0001 in one-way ANOVA; all error bars, SEM (n = 3). All qPCR data for GFP expression are normalized with respect to housekeeping gene elongation factor 1 (EF1) and a control sample of a nontreated leaf.

  • Fig. 5 RNA protection from enzymatic degradation and SWNT biocompatibility analysis.

    (A) smTIRF microscopy of Cy3-labeled RNA and Cy3-labeled RNA-SWNTs before and after incubation with RNase A. Scale bars, 5 μm. (B) Quantification of percent decrease in number of intact RNA molecules upon RNase A treatment. Error bars, SEM (n = 3). ****P < 0.0001 in two-tailed unpaired t test. (C) Agarose gel electrophoresis of free RNA and RNA-SWNTs incubated in plant cell lysate for 1, 3, 6, 12, and 24 hours. (D) Quantification of intact RNA from the agarose gel in (C). (E) Quantification of percent RNA degradation from the agarose gel in (C). (F) qPCR analysis of NbrbohB following a 3-hour exposure to samples. ****P < 0.0001 in one-way ANOVA; error bars, SEM (n = 3).

Supplementary Materials

  • Supplementary Materials

    Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown

    Gozde S. Demirer, Huan Zhang, Natalie S. Goh, Rebecca L. Pinals, Roger Chang, Markita P. Landry

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    The PDF file includes:

    • Thermodynamic analysis of RNA desorption and hybridization
    • Extracellular thermodynamics analysis
    • Intracellular thermodynamics analysis
    • Tables S1 to S3
    • Figs. S1 to S11
    • References

    Other Supplementary Material for this manuscript includes the following:

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