Bubbled RNA‐Based Cargo for Boosting RNA Interference

As ribonucleic acid (RNA) nanotechnology has advanced, it has been applied widely in RNA‐based therapeutics. Among the range of approaches, enzymatically synthesized RNA structures for inducing RNA interference in cancer cells have potential for silencing genes in a target‐specific manner. On the other hand, the efficiency of gene silencing needs to be improved to utilize the RNA‐based system for RNAi therapeutics. This paper introduces a new approach for efficient generation of siRNA from bubbled RNA‐based cargo (BRC). The presence of bubbles in between to avoid nonfunctional short dsRNAs allows the RNA‐based cargoes to contain multiple Dicer‐cleavage sites to release the functional siRNAs when introduced to cells. BRCs can be synthesized easily in a one‐pot process and be purified by simple centrifugation. Furthermore, efficient target gene silencing by the bubbled structure is confirmed both in vitro and in vivo. Therefore, this bubbled RNA cargo system can be utilized for target‐specific RNAi therapeutics with high efficiency in the generation of functional siRNAs in the target cells.


Sequence design for BRCs
Template linear DNAs are designed to have four different regions that are complementary to the following RNA sequence: 1) T7 promoter, 2) sense or anti-sense strand of siRNAs, 3) bubble (non-complementary) and 4) sense or anti-sense strand of siRNAs. All DNA sequences are listed in Table S1. In result, RNA strands synthesized by T7 RNA polymerase have the following sequence: [siRNA-bubble-siRNA-bubble]n.

Synthesis of BRCs
For the synthesis of BRCs, linear DNA1 and linear DNA2 are circularized as previously reported [1]. Then, circular DNA1 and circular DNA2 at the final concentrations of 0.5 μM were mixed with 8 mM of ribonucleotide solution mix (New England BioLabs), reaction buffer (80 mM Tris, 40 mM NaCl, 12 mM MgCl2, 4 mM spermidine, 20 mM dithiothreitol, pH 7.8) and 80 units μl -1 of T7 RNA polymerase (Ambion). For the RCT reaction, the reaction solution was incubated for 20 h at 37°C. The final reaction solution was briefly sonicated, then the BRCs were washed three times with nuclease-free water before further analysis. For the synthesis of cy5-labeled BRCs, cy5-UTP (final concentration of 20 μM) was added to the RCT reaction mixture at the beginning of the incubation process. To remove unincorporated cy5-UTP, the BRCs were washed three times with nuclease-free water after RCT reaction.

Characterization
FE-SEM (Hitachi, S-5000H) and AFM (Park Systems, Park NX10) were used to obtain high resolution digital images of the BRCs. The BRCs for SEM imaging were deposited onto silicon wafer, and coated with Pt after being dried. The nanoparticles for AFM imaging were deposited S3 onto freshly cleaved mica (Ted Pella), and air-dried. The samples were scanned in non-contact mode with NC-NCH tips (Park Systems). Nanoparticle tracking analysis (NTA) was carried out with NanoSight NS300 (Malvern). TEM (JEOL, JEM-2100F) was employed to characterize the BRCs operating at an accelerated voltage of 200 kV. For the preparation of samples, the BRCs were deposited onto a Lacey Formvar/carbon-coated copper grids (Ted Pella, 01883-F), then air-dried at room temperature.

Dicer-mediated in vitro generation of siRNAs
For assessment of siRNA generation from BRCs, the nanoparticles were incubated with 0.1 U μl -1 of recombinant human Dicer enzyme and reaction buffer (1 mM ATP, 2.5 mM MgCl2, 40% Dicer reaction buffer) at 37ºC for 24 h. The cleaved RNAs were examined by 10% nondenaturing PAGE in 0.5X Tris-borate-EDTA (TBE) buffer carried out at 120 V for 60 min.
Then, the gels were stained with 1X GelRed in 0.5X TBE and analyzed under UV with GelDoc EZ Imager (Bio-Rad). The lane profiles were analyzed by GelDoc software (Bio-Rad).

Calculation of amount of siRNA in BRCs
RNA contents in BRCs were measured with UV-Vis spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific) by measuring absorbance at 260 nm, and BRC particle concentration was measured by NTA. From the measured RNA contents and BRC particle concentration, RNA contents in a single BRC can be calculated as follows: RNA contents in a single BRC ( RNA BRC particle ⁄ ) = RNA contents ( RNA ⁄ ) × 1 BRC particle concentration ( BRC particles ⁄ )

S4
By design, a half of total RNA contents can potentially be siRNAs. The amount of siRNAs in one BRC can be calculated as follows: The amount of siRNAs in one BRC = RNA contents in a single BRC ( Therefore, about 49000 of siRNA can be maximally generated from one BRC in theory. Experimentally, the amount of cleaved siRNA from one BRC was determined by PAGE ( Figure 2A). According to the results following Dicer treatment, about 7600 siRNA strands were generated from single BRC under optimal conditions. As previously reported, some portion of the RNA is not as readily accessed by Dicer in a more close-packed self-assembled RNA structure, and multimers of repeated RNA unit as incomplete dicing products could be produced [2].

In vitro gene knockdown analysis
HeLa-GFP cells were grown in DMEM supplemented with 10% FBS, 100 U ml -1 of penicillin, 100 μg ml -1 of streptomycin and 1% Antibiotic-Antimycotic at 37ºC in a humidified atmosphere supplemented with 5% CO2. The cells were passaged routinely to maintain exponential growth. One day prior to transfection (~90% confluence), the cells were trypsinized, diluted with fresh medium and transferred to 96-well plates (7000 cells per well).
The BRCs were covered with the transfection reagent, Stemfect TM RNA Transfection Kit  Table   Table S1. DNA sequences for synthesizing BRCs and shRNA-NPs. Complementary DNA sequence for promoter region for T7 RNA polymerase is shown in red, and primer for T7 RNA polymerase binds to the red region to form promoter region for T7 RNA polymerase.
Complementary DNA sequence for sense and anti-sense strands for siRNAs are presented as purple and orange, respectively.

DNA strands Length (nt) Sequence
Linear