A RNA producing DNA hydrogel as a platform for a high performance RNA interference system

Abstract

RNA interference (RNAi) is a mechanism in which small interfering RNA (siRNA) silences a target gene. Herein, we describe a DNA hydrogel capable of producing siRNA and interfering with protein expression. This RNAi-exhibiting gel (termed I-gel for interfering gel) consists of a plasmid carrying the gene transcribing siRNA against the target mRNA as part of the gel scaffold. The RNAi efficiency of the I-gel has been confirmed by green fluorescent protein (GFP) expression assay and RNA production quantification. The plasmid stability in the I-gel results in an 8-times higher transcription efficiency than that of the free plasmid. We further applied the I-gel to live cells and confirmed its effect in interfering with the GFP expression. The I-gel shows higher RNAi effect than plasmids in free form or complexed with Lipofectamine. This nanoscale hydrogel, which is able to produce RNA in a cell, provides a platform technology for efficient RNAi system.

Fig. 1

Illustration of RNAi mechanism of I-gel. a A schematic diagram illustrating the I-gel mechanism in a living cell. b A schematic diagram illustrating the I-gel mechanism in a cell lysate assay

Fig. 2

Optimization and evaluation of I-gel. a The FL spectra of GFP in each condition (no plasmid, only GFP expression, addition of scrambled shRNA 1 eq, scrambled shRNA 100 eq, scrambled plasmid mixture, scrambled plasmid gel, I-plasmid, and I-gel). b A comparison of the fluorescence intensities at the peak points of the graph (a) and the inset is fluorescence images of each sample. The relative FL intensity (c) with the addition of increasing amount of I-plasmid and I-gel (*P < 0.05, **P < 0.005 and ***P < 0.001, analyzed by t test. Asterisks indicate statistically significant differences between I-plasmid and I-gel), (d) with the variation of X-DNA and I-plasmid ratio in I-gel. For all experiments, 57 ng of I-plasmid has been used except (c). This data was obtained by in vitro transcription/translation with HeLa cell lysate system. Error bars refer to standard deviations from three replicates

Fig. 3

Comparison of RNA expressions. a, d The RT-PCR products in agarose gel electrophoresis for (a) shRNA and (d) GFP mRNA detection in extracted RNA samples from the cell lysates. a Samples on gel are lane M: molecular weight standard marker; cDNAs reverse transcribed respectively from Lane 1: standard shRNA, Lane 2: shRNA expressed from I-plasmid, Lane 3: shRNA expressed from I-gel; (d) cDNAs reverse transcribed respectively from the GFP mRNAs produced in the cell lysates transcribed/translated in the presence of Lane 1: only GFP plasmid, Lane 2, 3, 4, 5, 6: the addition of scrambled shRNA, scrambled plasmid mixture, scrambled plasmid gel, I-plasmid, and I-gel. b, e Copy number of RNA expressed from a template plasmid for (b) shRNA and (e) GFP mRNA expression in each condition (†concentration of scrambled shRNA was 102-fold increased in consideration of the template to RNA transcription rate of the I-gel) (b) (***P < 0.001, analyzed by t test. Asterisks indicate statistically significant differences between I-plasmid and I-gel) (e) (***P < 0.001, analyzed by t test. Asterisks indicate statistically significant differences between each condition marked by line and I-gel). c, f The relative RNA expression value for (c) shRNA and (f) GFP mRNA in 5.7 ng, 57 ng and 114 ng of I-plasmid amount condition. The insets of (c) and (f) represent zoomed up images of dashed box (***P < 0.001, analyzed by t test. Asterisks indicate statistically significant differences between I-plasmid and I-gel). Error bars refer to standard deviations from three replicates

Fig. 4

Comparison of intracellular uptake of plasmids in different formats. Second from left: I-plasmid labeled with Cy5, third: I-plasmid labeled with Cy5 complexed with lipofectamine, fourth: shRNA labeled with Cy5, fifth: shRNA labeled with Cy5 complexed with lipofectamine and the last: I-gel labeled with Cy5. Scale bar: 20 μm

Fig. 5

Gene-silencing effect by I-gel i n a cellular level. a Fluorescence images of MDCK-GFP-expressing (MDCK-GFP) cells coincubated with I-plasmid, I-plasmid complexed with lipofectamine, and I-gel. Scale bar: 20 μm. b Fluorescence-activated cell sorter analysis of GFP-expressing cell line, MDCK-GFP cells after treatment of sample/polymerase complexes in serum-deficient medium. The number means the percentage of GFP-overexpressing cells sorted within a prefixed gate region as indicated by a bar. c, d The relative amounts of (c) GFP mRNA and (d) shRNA from GFP-expressing MDCK cells after incubation in various conditions. (^†concentration of these conditions were 102-fold increased in consideration of the template to RNA transcription rate of the I-gel) (c: *P < 0.05, analyzed by one-away ANOVA, followed by Holm–Bonferroni multiple comparisons post test. Asterisks indicate statistically significant differences between between each condition and Cell-only) (d: *P < 0.05 and **P < 0.005, analyzed by t test. Asterisks indicate statistically significant differences between each condition and I-gel). Error bars refer to standard deviations from three (c) or six (d) replicates