Multifunctional RNA nanoparticles

Abstract

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Keywords: RNA interference; RNA nanoparticles; RNA nanotechnology; RNA−DNA hybrid reassociation; aptamers.

Figure 1

Schematic representation of assemblies leading to the formation of RNA nanorings functionalized with (a) Dicer substrate RNAs, (b) malachite green (MG) aptamers for in vitro visualization, (c) J18 aptamers for cell targeting and phycoerythrin for visualization in vivo, (d) Dicer substrate RNAs introduced via the toehold interactions, and (e) RNA–DNA hybrids with split functionalities (RNAi and FRET). Functional siRNAs can be released by Dicer nuclease. KLs stand for kissing loops.

Figure 2

Structural characterization by cryo-EM of RNA nanorings functionalized with six DS RNAs. (a) A typical cryo-EM image of the DS RNA nanoring particles (left panel). Class averages for each DS RNA nanoring as observed by cryo-EM (central panel), with corresponding projections of the reconstructed three-dimensional structure (right panel). (b) Single particle reconstruction of functionalized RNA nanorings. Different views of the model fit with the electron density volume are shown. The volume map is thresholded at the minimum level at which all the atoms of the model can be fit inside the volume. The resolution is 16 Å.

Figure 3

Cell uptake, endosomal colocalization, silencing, and RNA aptamer mediated binding efficiencies of functional nanorings. (a) Transfection efficiencies using human breast cancer cells (MDA-MB-231). DS RNAs (60 nM final) covalently labeled with one Alexa 546 per duplex were compared to the functionalized nanorings (10 nM final) labeled with six Alexa 546 dyes. One day after the transfection, the efficiencies were analyzed by confocal fluorescence microscopy and flow cytometry experiments. (b) Studying the localization of nanorings with commonly used markers for endosomal compartments Early Endosome Antigen 1 (EEA1) and Rab7. (c) GFP knockdown assays using human breast cancer cells (MDA-MB-231/GFP) which stably express enhanced GFP (eGFP). Fluorescence microscopy (left panel) and statistical analysis (30000 cells per sample) of flow cytometry experiments (right panel) of eGFP expression 3 days after the transfection of cells with DS RNA duplexes and nanorings functionalized with six DS RNAs against eGFP. The ratio of DS RNA duplexes to DS RNA functionalized nanorings was 6:1. (d) Nanorings labeled with phycoerythrin (PE) and containing different numbers of the EGFR-specific J18 aptamer selected to specifically bind EGFR expressed on A431 cells were tested for relative binding efficiencies using FACS. The J18 RNA aptamer model is a conceptual cartoon, based on the minimum free energy secondary structure (MFE). Image numbers in (b) correspond to differential interference contrast (DIC) images (1), Alexa546 emission (2), EAA1 antibody staining (3), and Rab7 antibody staining (4). Images (1 + 2 + 3) and (1 + 2 + 4) are superpositions of three different images.

Figure 4

Activation of different functionalities by RNA–DNA hybrids. (a) Scheme showing an activation of multiple functionalities (RNAi, FRET) upon reassociation of nonfunctional nanorings decorated with RNA–DNA hybrids and six nonfunctional cognate RNA–DNA hybrids. (b) FRET time traces during reassociation of hybrid nanorings labeled with Alexa546 and cognate hybrids labeled with Alexa488. (c) Intracellular FRET experiments: cells were cotransfected with hybrid nanorings and cognate hybrids labeled with Alexa546 and Alexa488, respectively. Images were taken the next day. (d) GFP knockdown assays. Three days after transfection of MDA-MB-231/GFP cells with hybrid nanorings and cognate hybrids programmed to release DS RNAs, eGFP expression was statistically analyzed with flow cytometry experiments. As the control, DS RNA duplexes against eGFP were used. Please note that individually neither hybid nanorings nor hybrids cause decrease in eGFP production. Image numbers in (c) correspond to differential interference contrast (DIC) images (1), Alexa488 emission (2), Alexa546 emission (3), bleed-through corrected FRET image (4), and 3D chart representation of zoomed fragment indicated by a yellow box of bleed-through corrected FRET image with the yellow dot indicating the correspondence (5).

Figure 5

In vivo studies of nanorings functionalized with six DS RNAs in a tumor xenograft mouse model. Fluorescent imaging of tumors and corresponding quantification after 5 days postinjections in vivo demonstrate significant levels of eGFP silencing caused by nanorings functionalized with six DS RNAs compared to free siRNAs. Free DS RNAs were used at six times higher concentrations. Error bars denote ±SEM; N = 2.

Figure 6

HIV-1 expression and production is inhibited by functional nanorings. (a) HIV-1 expression inside the cell was measured at 48 h post-transfection. HeLa cells were lysed and probed by Western blotting for HIV-1 proteins. Positions of Pr55Gag (Pr55), matrix-capsid (p41), and capsid/capsid-SP1 (p24/p25) are indicated. Quantification of total cell-associated Gag: Pr55 + p41 + p25 + p24. Total Gag in virus control (HIV-1) without nanorings or Dicer substrate (DS) RNAs set at 100. Error bars denote ±SEM; N = 4. (b) HeLa cells were transfected with pNL4-3 (full-length HIV-1 molecular clone), with and without nanorings or DS RNAs. Virus supernatant was harvested 48 h post-transfection, and the reverse transcriptase (RT) production was measured (this assay quantifies the amounts of virus produced by the cells); data are shown normalized to virus controls (HIV-1) without functional nanorings or DS RNAs. Mock represents untrasfected HeLa cells. Corresponding mixtures of six different anti-HIV DS RNAs (A and B) were used as positive controls. Nanoring control without any anti-HIV DS RNAs was used as a negative control. Error bars denote ±SEM; N = 4.