Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription

Abstract

Since the discovery of RNA interference (RNAi), small interfering RNA (siRNA) has been powerful tools for gene downregulation in biomedical applications. Despite the outstanding efficacy of siRNA, the development of a therapeutic delivery system remains a challenge owing to the instability of RNA. In this study, we describe a new method for the design of siRNA-generating nanosponges by using complementary rolling circle transcription (cRCT), a technique that requires two complementary circular DNA. The sequences of one of the circular DNA are designed to have complete complementarity to the target mRNA resulting in double stranded RNA (dsRNA) that can be digested to siRNA by cellular Dicer activity. This siRNA design, called 'library siRNA', could be universally applied to fabricate RNA nanosponges targeting any known mRNA sequence.

Figure 1

Schematic illustration of GFP lib-NSs synthesis. (a) Preparation of sense and antisense circular DNA templates from a target mRNA sequence. (b) The process of complementary rolling circle transcription (cRCT). Two RNA strands hybridize and become entangled, producing lib-NSs. (c) Lib-NSs enter tumour cells by endocytosis and are cleaved by Dicer (green ellipses), producing library siRNA, which complexes with RISC (orange ellipses) and target mRNA, resulting in mRNA degradation.

Figure 2

Characterization of GFP lib-NSs. (a) Size distribution by DLS. (b) SEM images. Inset: Magnified images of each GFP lib-NSs. Scale bars: 500 nm. (c) TEM images. Scale bars: 200 nm. (d) Confirmation of GFP lib-NSs components by EDS mapping.

Figure 3

Generation of siRNA from GFP lib-NSs. Lanes 1–4 indicate the dsRNA ladder, and GFP lib-NSs 0, 24, and 48 h after Dicer treatment for 0.3 μM/RP40 (a), 2.5 μM/RP40 (b), and 0.5 μM/RP80 (c), respectively. dsRNA ladder and the GFP lib-NSs were loaded at the amount of 1 $\mathrm{\mu }\mathrm{g}$ in all agarose gels. The exposure was set automatically. The red boxes indicate the product generated at 21-bp region.

Figure 4

GFP lib-NSs-mediated viability and gene knockdown assays. (a) Fluorescence microscopy of HeLa GFP cells 24 h after treatment with 0.5 and 2.5 pM GFP lib-NSs. Control cells were left untreated for 24 h. Scale bar: 10 μm. (b) Viability was assayed 24 and 48 h after treatment with 0.5 and 2.5 pM GFP lib-NSs. (c) GFP knockdown was assayed 24 and 48 h after treatment with 0.5 and 2.5 pM of GFP lib-NSs. GFP intensities were normalized to the intensity of untreated cells (n = 4). The P-value was calculated using one-way ANOVA with a Tukey’s procedure (*p < 0.05 compared with the untreated: GFP lib-NSs).