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.

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…As proof of concept, DNA hydrogels (Dgel) is exploited for RNA synthesis and in particular, to prevail the naked siRNA instability. Accordingly, design of Dgel composed of a plasmid‐encoded gene is reported which transcribed siRNA that further targeted the corresponding mRNA with significantly superb RNAi performance (i.e., cellular uptake turnover), transcription (i.e., GFP expression), and stability (i.e., against nucleases) (Song et al, 2018). Also, nano‐scale hydrogels derived from Dgel, rather than proteins of therapeutic functions, have distinguished applications in plasmonics, protein production system, diagnostics, and drug delivery (Mao et al, 2018; Song et al, 2017, 2018).…”

“…Accordingly, design of Dgel composed of a plasmid‐encoded gene is reported which transcribed siRNA that further targeted the corresponding mRNA with significantly superb RNAi performance (i.e., cellular uptake turnover), transcription (i.e., GFP expression), and stability (i.e., against nucleases) (Song et al, 2018). Also, nano‐scale hydrogels derived from Dgel, rather than proteins of therapeutic functions, have distinguished applications in plasmonics, protein production system, diagnostics, and drug delivery (Mao et al, 2018; Song et al, 2017, 2018). Over and above being practical for CFPS, high yield production, selection of expressed bio‐products, and protein display could be actualized using DNA microgels (Ruiz, Kiatwuthinon, Kahn, Roh, & Luo, 2012).…”

“…Expression and display Production of extremely high local gene quantities and protein display Kahn et al (2016) RNAi synthesis DNA hydrogel produces RNA to suppress protein expression inside cells Song et al (2018) Protein synthesis Building minimal cells capable of in vitro genetic oscillations to maintain regulated long-lived protein synthesis (>11days) Zhou et al (2018) Protein synthesis High efficiency (300 times more than current, solution-based systems). High yield (5 mg/ml of functional proteins).…”

DNA-GEL, Novel Nanomaterial for Biomedical Applications and Delivery of Bioactive Molecules

“…As previously discussed, DNA-hydrogels are biodegradable and, in principle, the versatile nature of DNA sequences and their ability to respond to external stimuli allow their utilization for efficient encapsulation and release of drugs and other molecules. Several systems based on DNA-hydrogels made of NSs have been designed for loading and release of drugs (Um et al, 2006;Nishikawa et al, 2011;Nishikawa et al, 2014;Song et al, 2015b;Ishii-Mizuno et al, 2017;Umeki et al, 2017;Wei et al, 2019;Yue et al, 2019), nucleic acids, (Li et al, 2015;Nishida et al, 2016;Ma et al, 2018;Song et al, 2018;Komura et al, 2020), and proteins (Liu et al, 2018). The DNA-hydrogel can easily trap molecules by varying the stiffness and pore size of the DNA structure, as shown by Yue et al (2019).…”

A Simple Blocking PCR‐Based Method for the Synthesis of High‐Copy dsDNA Tandem Repeats

“…[3][4][5][6] For example, DNA repeats have been used to produce DNA hydrogels with unusual mechanical properties. [7][8][9][10] In general, DNA hydrogels are tunable, programmable, responsive to various stimuli, biocompatible, and biodegradable. Because of these unique features, DNA hydrogels have shown great advantages in a broad…”