(E-188) Rolling circle transcription (RCT)-based s ize-controllable RNAi modules

Introduction::

RNA nanotechnology has advanced extensively for its potential biomedical applications by leveraging a wide range of biological functions of RNA. Especially, RNA nanostructures fabricated by rolling circle transcription (RCT)-based enzymatic RNA self-assembly has gained much attention as one of the promising approaches to enhancing therapeutic RNA's loading capacity and delivery. Here, we synthesized size-controllable RNAi modules to fully potentiate the functionality of its cargo siRNA. 

Materials and Methods:: Template DNA, rNTP, T7 RNA polymerase were mixed with different amount of glycerol to regulate the size of synthesized RNAi modules. After reaction, the RNAi modules were wash three times with nuclease-free water.
Hela luciferase cells were seeded in 96 well before RNAi module transfection. After 24 h, the RNAi modules complexed with RNAiMAX transfection reagent was treated for 48 h. Then, the viability and luciferase activity was measured and normalized to untreated groups.

Results, Conclusions, and Discussions::

RNAi modules are fabricated by complementary Rolling Circle Transcription methods. Two circular DNA templates contain complementary sequences to form double-strand siRNA and non-complementary sequences to form bubble regions. T7 RNA polymerase recognizes the T7 promoter region in template DNA and transcribes RNA strands continuously. Finally, sub-micrometer-sized RNAi modules are generated which structures have flower-like morphologies.

The RNA transcription condition impacted the sizes of fabricated RNAi modules. Here, the reaction solution viscosity was increased by adding glycerol. When the reaction solution viscosity was increased from 10% glycerol to 40%, the sizes of the RNAi modules were reduced from about 500 nm to 250 nm.

Increasing viscosity in the RCT reaction solution resulted in a reduced RNA polymerization rate. Furthermore, the inorganic crystallization of magnesium pyrophosphate, which has an important role as a structural basis of the RNAi module, decreased as the solution viscosity increased. As a result, the size of the RNAi module was decreased as the solution viscosity increased.

To study the impacts of the sizes of RNAi modules, the cytotoxicity and RNAi efficiencies of each RNAi module were tested. The largest RNAi module exhibited high cellular toxicity and negligible RNAi effects. The middle RNAi module showed alleviated cytotoxicity and the highest knockdown efficiencies. The smallest RNAi module showed more reduced toxicity than the middle RNAi module, but the RNA efficiencies were also decreased. Therefore, it is concluded that middle RNAi modules have the highest potency as RNAi therapeutics.

Here, we fabricated the size-controllable RNAi modules and studied the cytotoxicity and RNAi efficiencies of each RNAi module with different sizes. These results indicate the importance of precisely controlling the size of nanostructures to develop RNAi therapeutics.

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