RNA Interference Sponge Particle

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To perform RCT (ref. 35), circular DNA needs to be synthesized first (see Supplementary Information). Linear single-stranded DNA that includes antisense and sense sequences of anti-luciferase siRNA is hybridized with an equal molar amount of short DNA strands containing the T7 promoter sequence. The nick in the circular DNA was chemically closed by T4 DNA ligase. By RCT of the closed circular DNA, multiple tandem repeats of hairpin RNA structures from both antisense and sense sequences are generated to be able to form spherical sponge-like structures.a, SEM image of RNAi-microsponges. b, Fluorescence microscope image of RNAi-microsponges after staining with SYBR II, an RNA-specific dye. c,d, SEM images of RNAi-microsponges after sonication. Low-magnification image of an RNAi-microsponges (c). High-magnification image of an RNAi-microsponge (d).a–e, SEM images of RNA products of time-dependent RCT at 37 °C for 1 h (a), 4 h (b), 8 h (c), 12 h (d) and 16 h (e). Inset: scale bar, 500 nm. f, Image of mature RNAi-microsponges after 20 h RCT. g, Schematic of the formation of RNAi-microsponges. The spherical sponge-like structure is formed through a series of preliminary structures. A tandem copy of RNA strands from the RCT reaction are entangled and twisted into a fibre-like structure 1. As the RNA strands grow, they begin to organize into lamellar sheets that gradually become thicker 2; as the internal structure of the sheets begins to get very dense, some of the RNA sheets begin to grow in the z direction, possibly owing to the limited packing area for the RNA polymer as it is produced by the reaction. This process could generate a wrinkled semi-spherical structure on the sheet 3. Finally, the entire structure begins to pinch off to form individual particles consisting of gathered RNA sheets 4. h, Polarized optical microscopy of RNAi-microsponges. i, X-ray diffraction pattern of RNAi-microsponges. j, TEM images of RNAi-microsponges and schematic representation of the proposed crystal-like ordered structure of an RNA sheet in the microsponge. Inset: scale bar, 500 nm.a, Schematic of the generation of siRNA from RNAi-microsponges by Dicer in the RNAi pathway. b, Gel-electrophoresis result after Dicer reaction. On the left, lanes 1 and 2 indicate double-stranded RNA ladder and RNAi-microsponges (MS) after treatment with Dicer (1 unit) for 36 h, respectively. On the right, lanes 1 and 2 indicate double-stranded RNA ladder and RNAi-microsponges without Dicer treatment. Lanes 3–8 correspond to 12 h, 24 h, 36 h and 48 h reaction with 1 unit of Dicer, and 36 h reaction with 1.25 and 1.5 units of Dicer, respectively. Increasing the amount of Dicer did not help to generate more siRNA (lane 7 and 8 of b). The amount of generated siRNA from RNAi-microsponges was quantified relative to double-stranded RNA standards. 21% of the cleavable double-stranded RNA was actually diced to siRNA because Dicer also produced the two or three repeat RNA units. The results suggest the possibility that in a more close-packed self-assembled structure, some portion of the RNA is not as readily accessed by Dicer. c, Particle size and zeta potential before and after condensing RNAi-microsponges with PEI. d, SEM image of further condensed RNAi-microsponges with PEI. The size of the RNAi-microsponges was significantly reduced by linear PEI because the RNAi-microsponges with high charge density would be more readily complexed with oppositely charged polycations. The porous structure of the RNAi-microsponges disappeared following condensation.a, Intracellular uptake of red fluorescent dye-labelled RNAi-microsponges without PEI (top) and RNAi-microsponge/PEI (bottom). To confirm the cellular transfection of RNA particles, both types of particles, labelled for red fluorescence, were incubated with T22 cells. Fluorescence labelled RNAi-microsponges without a PEI outer layer showed relatively less cellular uptake by the cancer cell line (T22 cells) suggesting that the larger size and strong net negative surface charge of RNAi-microsponges probably prevents cellular internalization. Inset: scale bar, 5 μm. b, Suppression of luciferase expression by siRNA, Lipofectamine complexed with siRNA (siRNA/Lipo), siRNA complex with PEI (siRNA/PEI), RNAi-microsponge (RNAi-MS), and RNAi-microsponge condensed by PEI (RNAi-MS/PEI). The same amount of siRNA is theoretically produced from RNAi-microsponges at the concentration in parentheses. c, In vivo knockdown of firefly luciferase by RNAi-MS/PEI. Optical images of tumours after intratumoral injection of RNAi-MS/PEI into the left tumour of a mouse and PEI solution only as a control into the right tumour of the same mouse.
Professor Paula Hammond
Department of Chemical Engineering, MIT
External Link (hammondlab.mit.edu)
Jong Bum Lee
Department of Chemical Engineering, MIT
External Link (bna.uos.ac.kr)
Young Hoon Roh
Department of Chemical Engineering, MIT
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Nucleic Acid Particles, Methods and Use Thereof

US Patent Pending 2016-0151404
Self-assembled RNA interference microsponges for efficient siRNA delivery
Nature Materials, Feb 26, 2012, p. 316-322


RNAi sponge particle (RNAi-SP) technology can be used in a variety of therapeutic settings where siRNA mediated-gene silencing is a viable treatment strategy.


In recent years, RNA interference (RNAi) has gained interest as a powerful tool for suppressing gene expression. RNAi involves the intracellular delivery of double stranded RNAs (dsRNAs) which have sequences that target a gene of interest. Once inside the cell, these dsRNAs activate the RNA interference pathway to silence their target genes. The ability to safely deliver stable RNA into the cell continues to be a key challenge in the nascent field, and a new method such as RNAi-SP has the potential to greatly advance RNAi therapeutics. 


This technology takes advantage of rolling circle transcription (RCT), an artificial nucleotide replication method, to synthesize concatemerized strands of cleavable RNA. The concatemerized RNA strands self-assemble into therapeutically deliverable sponge-like particles with a diameter of approximately 2 µm. The RNA is protected from degradation because it forms double-stranded structures within these particles. These nanoparticles can be reduced to a 200 nm diameter size through treatment with cationic polyethylenimine (PEI). Each particle contains approximately a half a million of cleavable RNA strands, which makes their delivery efficiency far superior to currently existing transfection methods. When these cleavable strands are introduced into the cell, they activate the RISC (RNA-induced silencing complex) pathway to knock down intracellular production of any proteins of interest. Test trials on T22 cells have shown that RNAi-SPs can achieve the same knockdown efficiency as siRNA packaged by traditional liposome-based delivery methods at concentrations that are roughly three orders of magnitude less. Furthermore, the cells showed approximately 100% viability, indicating that the RNAi-SP delivery method exhibits low cytotoxicity. In vitro injection of RNAi-SP in mice have shown similar results. 


  • New route for effective, highly efficient delivery of siRNA
  • Ability to generate large amounts of siRNA in a form that assembles directly into a drug carrier that can be sued for direct transfection
  • siRNA is protected within sponge particle in the form of double-stranded polymeric RNAi
  • Broad clinical applications with RNA interference