Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles

Abstract

Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.

Keywords: FRET; RNA and DNA nanoparticles; RNA and DNA nanotechnology; RNAi; RNA−DNA hybrids reassociation.

Figure 1

Activation of RNAi with RNA nanocubes 3′-side functionalized with six Dicer substrate RNAs. (a) Schematics of nanocube formation and release of siRNAs through dicing. (b) Cellular uptake of fluorescently labeled cubes (10 nM) and duplexes (60 nM) was assessed with confocal microscopy and analyzed by flow cytometry. (c) Localization of fluorescently labeled functional nanocubes (10 nM) with commonly used markers for endosomal compartments EEA1 and Rab7. (d) GFP knockdown assays for human breast cancer cells (MDA-MB-231/GFP) which stably express GFP. At 3, 5, 6, 7, 12, and 14 days after the transfection of cells with nanocubes, eGFP expression was statistically analyzed with flow cytometry experiments. As the control, siRNA duplexes against eGFP were used for all time points. gMFI corresponds to the geometric mean fluorescence intensity. Error bars denote SEM. The images were taken 3 days post-transfection.

Figure 2

Assembly of RNA nanocubes functionalized with six different Dicer substrate RNAs against HIV-1. Virus produced from cells transfected with a mixture of siRNAs or an RNA–RNA nanocube targeting HIV-1 RNA was assessed for infectivity. Error bars denote SD; N = 3.

Figure 3

Activation of split functionalities during reassociation of RNA nanocubes 3′-side decorated with six RNA–DNA (in blue) hybrids (carrying six Dicer substrate RNA antisense) with six cognate hybrids (carrying Dicer substrate RNA senses). (a) Schematics of reassociation and activation of FRET and RNAi. (b) Assembled RNA cubes were analyzed by total SYBR Gold staining native PAGE and DLS experiments. (c) FRET time traces during reassociation of fluorescently labeled cubes and hybrids labeled with Alexa 488 and Alexa 546. (d) GFP knockdown was quantified through flow cytometry. Please note that the individual hybrids and RNA nanocubes decorated with hybrids cause no decrease in eGFP production.

Figure 4

Activation of different split functionalities during reassociation of DNA nanocubes (in blue) 3′-side decorated with six RNA–DNA hybrids (carrying six DS RNA antisenses, in red) with six cognate hybrids (carrying DS RNA senses). (a) Schematics of reassociation and activation of FRET and RNAi. (b) The formation of DNA cubes was confirmed by total SYBR Gold staining native PAGE and DLS experiments. (c) FRET time traces during reassociation of fluorescently labeled cubes and hybrids labeled with Alexa 546 and Alexa 488. (d) FRET experiments: cells were cotransfected with cubes and cognate hybrids labeled with Alexa 546 and Alexa 488 and images were taken on the next day. (e) GFP knockdown was measured via flow cytometry. Please note that the individual hybrids and DNA nanocubes decorated with hybrids cause no decrease in eGFP expression. Image numbers in (d) correspond to differential interference contrast (DIC) images (1), Alexa 488 emission (2), Alexa 546 emission (3), bleed-through corrected FRET image (4), 3D chart representation of zoomed fragment indicated by a white box of bleed-through corrected FRET image with the white dot indicating the correspondence (5).

Figure 5

Induction of type I interferon and pro-inflammatory cytokine in human peripheral blood mononuclear cells in vitro. Human PBMC from 2 healthy donor volunteers was incubated with control and test samples for 24 h. Cell culture supernatants were analyzed by conventional ELISA to detect IFNb (a) or IL-1b (b). Error bars denote SD; N = 2 donors. Variability of response within each individual donor was low (% CV < 20). NC is negative control (PBS); PC is positive control in (a) 5 μg/mL of ODN2216, in (b) 20 ng/mL of ultrapure K12 Escherichia coli LPS. Lipofectamine 2000 (L2K) at the same concentration as used to deliver constructs and 5 μg/mL polyIC were used as additional controls.