Self-assembled DNA-Guided RNA Nanovector via Step-wise Dual

Subscriber access provided by The University of Melbourne Libraries

Article

Self-assembled DNA-guided RNA Nanovector via Step-wise Dual Enzyme Polymerization (SDEP) for Carrier-free siRNA Delivery Yongkuk Park, Hyejin Kim, and Jong Bum Lee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.5b00554 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Self-assembled DNA-guided RNA Nanovector via Step-wise Dual Enzyme Polymerization (SDEP) for Carrier-free siRNA Delivery Yongkuk Park†, Hyejin Kim† and Jong Bum Lee†,* †

Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero,

Dongdaemungu, Seoul 130-743, Korea *Address correspondence to [email protected] KEYWORDS: Biotechnology, Materials science, Nanotechnology, Bioengineering, siRNA

ABSTRACT Nucleic acid-based therapeutics are being used increasingly for biomedical applications. Despite this, the development of nontoxic and cell-targetable delivery systems for practical use is still a challenge. This paper reports a novel enzymatic synthetic approach to produce cell-targetable RNA nanovectors. The RNA nanovectors were generated by the hybridization of DNA and RNA strands produced by temperature-dependent dual polymerization, which is also called step-wise dual enzyme polymerization. The RNA strands are designed to contain siRNA precursors and the DNA strands include aptamers that bind specifically to the target molecules for cell-targeting. The RNA nanovector can also enhance the resistance to nuclease degradation and help overcome the limitations associated with the nature of RNA via DNA1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 27

RNA hybrids. Therefore, the proposed strategy, step-wise dual enzyme polymerization, is an innovative solution for successful carrier-free siRNA delivery.

1. INTRODUCTION RNA interference (RNAi) is considered a powerful tool for manipulating target gene expression and opening a promising therapeutic strategy.1-4 The current studies for achieving efficient RNAi have focused on the development of delivery vehicles.5-9 On the other hand, the delivery of small interference RNA (siRNA) has limitations associated with the nature of RNA. Instability is one of the obstacles to overcome because naked siRNA is quite sensitive to nuclease degradation.10 In addition, the negative charges of naked siRNA are unfavorable for cellular uptake.6 To address these issues, cationic polymers and liposomes have been used to form a complex with siRNA or encapsulate siRNA. This complexation or encapsulation can enhance the cellular uptake significantly and increase the retention time of siRNA under physiological condition by offering protection from nuclease degradation.11-15 Despite these developments, several issues, such as the potential toxicity of complexation agents, decomplexation of the siRNA16, non-specific delivery to healthy cells, and low loading efficiency, need to be addressed. DNA and RNA have attracted considerable attention in recent years because of their potential applications in the delivery of RNA molecules.17-20 DNA nanostructures, such as DNA tetrahedra or branched DNA, have been applied to the delivery of siRNA.21-24 In addition, the incorporation of DNA aptamer into the DNA nanostructures can result in high affinity to the specific targets for target-specific cellular uptake without a transfecting agent.25-27 On the other hand, the amount of siRNA loaded onto the DNA nanostructures is still limited. RNA technologies including rolling circle transcription (RCT) have increased

2 ACS Paragon Plus Environment

Page 3 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the loading efficiency of siRNA.28 Previous studies have reported that long RNA transcripts containing repeated siRNA precursors by RCT can self-assemble into RNA structures.29-31 By taking the advantages of both DNA and RNA, this paper suggests a new enzymatic synthetic method with DNA and RNA polymerase, which is termed step-wise dual enzyme polymerization (SDEP) (Figure 1a). The resulting structures were generated by the hybridization of DNA and RNA strands produced through temperature-dependent dual polymerization. From this rational design, the RNA strands contain tandem copies of siRNA precursors and the DNA strands include repeated copies of aptamers that specifically bind to the target molecules (Figure 1b). The multi-functions from RNA and DNA are amenable to target-specific siRNA delivery by binding to a cell-surface receptor overexpressed in the target cancer cells. With these nanostructures, siRNA can be delivered without the need for complexation with cationic polymers or transfection agents. In addition, siRNA in the nanostructures is more stable in the blood serum than naked siRNA because of the resistance of RNA-DNA hybrid to nuclease digestion.10,32

2. MATERIALS & METHODS Circular DNA preparation. Long linear DNA and Primer DNA were purchased from Integrated DNA Technologies, Inc. A phosphorylated long linear DNA (1 μM) and a Primer DNA (1 μM) were mixed in nuclease free water and annealed by heating at 95 °C for 2 min, followed by gradual cooling to 25 °C for more than 1 h. The annealed product was mixed in T4 DNA ligase (0.06 U μl-1, Promega) and ligation buffer (300 mM Tris-HCl [pH 7.8], 100 mM MgCl2, 100 mM DTT and 10 mM ATP, Promega) at room temperature over an 8 h period. Circularization of the DNA strands was analyzed by gel electrophoresis (Figure S1). 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 27

Self-assembly of the RNA nanovector by SDEP. For rolling circle DNA replication, the first circularized template (NCL-aptamer linear ssDNA and primer for DNA) was incubated with Phi29 DNA polymerase (10 U μl-1, Epicentre), dNTP mix (8 mM, Epicentre) and reaction buffer (80 mM Tris-HCl, 100 mM KCl, 20 mM MgCl2, 10 mM (NH4)2SO4, 8 mM DTT) for 10 min at 30 °C. To perform RCT, the second circularized template (Antiluciferase linear ssDNA and primer for RNA) was mixed in T7 RNA polymerase (10 U μl-1, NEB), rNTP mix (8 mM, NEB) and reaction buffer (80 mM Tris-HCl, 12 mM MgCl2, 2 mM DTT, 4 mM spermidine, NEB) for 10 min at 37 °C. After 10 min, the two separated reactants (DNA polymerase reaction and RNA polymerase reaction) were mixed together. The reaction temperature was then changed periodically between the optimal activity temperatures of DNA or RNA polymerase for 20 h. To break up the potential aggregated portions, the final reactant was sonicated for 3 min. This process was repeated three times. For labeling, Cy5dCTP (0.2 mM, GE Healthcare) and Cy3-UTP (0.2 mM, Enzo Lifescience) were added to the reaction mixture. Characterization of the RNA nanovector. An XL30-FEG (FEI) environmental scanning electron microscopy, a CM 30 (Phillips) transmission electron microscopy and Park NX10 (Park Systems) atomic force microscopy were used to obtain high resolution digital images of the RNA particles. The sample was coated with Pt for SEM, and the TEM sample was stained with 2 % uranyl acetate for 20 seconds. The AFM sample was dropped and dried on mica. All AFM images were recorded with Non-Contact Cantilever (PPP-NCHR 5M, Nanosensors) in non-contact mode at room temperature. The images were analyzed using XEI software (Park Systems). Dynamic Light Scattering (DLS). The size of the RNA nanovectors were measured using a Zetasizer (Nano-ZS90, Malvern) and the results were analyzed using Zetasizer 4 ACS Paragon Plus Environment

Page 5 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


software. The RNA nanovectors were diluted in nuclease-free water and all measurements were carried out at 25 °C. Image cytometry analysis. The RNA nanovectors were stained with DAPI (Sigma Aldrich). A solution of the RNA nanovectors was deposited on NC-Slide A2 (Chemometec). To confirm the fluorescence of the fluorescently-labelled RNA nanovectors, two-color analysis was performed using a Nucleocounter (NC-3000, Chemometec) and the results were analyzed using NucleoViews NC-3000 software (Chemometec) Evaluation of RNA nanovectors after RNase H treatments. The RNA nanovector was treated with RNase H (2 U μl-1, Epicentre) at 37 °C. Then, the reactants were heated at 95 °C for 2 min by using Thermal cycler (Bio-Rad). The resulting products were examined by gel electrophoresis with 1.0 % agarose gel in the Tris-acetate-EDTA (TAE) buffer (40 mM Tris-acetate, 1 mM EDTA [pH 8.0], Biosesang) at 95 V for 100 min. The agarose gel was stained with gel red (Biotium) and examined under UV. The results were analyzed using Image Lab software (Bio-Rad). Generation of siRNA by Dicer enzyme. The recombinant human Dicer enzyme kit (Genlantis) was used to generate siRNA from the RNA nanovectors. The RNA nanovectors were incubated with ATP (1 mM), MgCl2 (2.5 mM), Dicer reaction buffer (2.5X) and recombinant Dicer enzyme (0.1 U μl-1) at 37 °C. The resulting products were examined by gel electrophoresis with 3.0 % agarose gel at 95 V for 120 min. Because 21 bp ~ 50 bp of RNA strands cannot be divided into each single bands in 1 % agarose gel, 3 % agarose gel was selected for Dicer enzyme experiment. Cell culture. MDA-MB-231 (kindly provided by Korea Institute of Science and Technology), HeLa and HeLa-Luc cells were cultured, as recommended by the American 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 27

Type Culture Collection (ATCC) in RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM). The cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 and routinely passaged to maintain exponential growth. Both cell were used to evaluate for targeting capability of RNA nanovectors in different cell types. Cell-specific uptake study. The MDA-MB-231 cells were seeded at 50,000 cells per well in an 8 well slide chamber (SPL). The cells were incubated in Opti-MEM I (Gibco) with the Cy3-UTP labeled RNA nanovector (150 μg per well) at 37 °C for 4 h. After 4 h, the RNA nanovectors were removed, and the cells were fixed with 3.7 % formaldehyde in PBS. The cells were stained with DAPI (Sigma Aldrich), Alexa Fluor 488 phalloidin (Invitrogen) and washed twice with DPBS. Then, the cells were then examined by confocal microscopy (Leica TCS SP5, Leica Microsystems GmbH) and the results were analyzed using Leica LAS AF Lite software. Cell viability assay. The HeLa cells were seeded at 7,000 cells per well in a 96-well flat-bottomed plate. The cells were incubated in Opti-MEM I with the RNA nanovector for 4 h. Subsequently, the media was removed and replaced with 10 % fetal bovine serum (FBS)containing growth media. After 48 h incubation, the MTT stock solution (5 mg mL-1 in DPBS), which was assayed to equal one-tenth of original medium volume, was added to each well and incubated for 4 h. The medium was then removed and formazan crystals were solubilized in DMSO:isopropanol (1:1). After 20 min, the absorbance is measured at a wavelength of 570 nm. Error bars in Figure 5e are standard deviation. siRNA knock-down experiments. The HeLa-Luc cells were seeded at 7,000 cells per well in a 96-well plate. The cells were incubated in Opti-MEM I with the RNA nanovector for 12 h. After incubation, the media was replaced with 5 % FBS containing growth media. The luciferase activities were measured using Dual-Glo luciferase assay system (Promega) at 6 ACS Paragon Plus Environment

Page 7 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


48 h after media change. Renilla luciferase was used as a house keeping gene in order for cell densities to be taken into account. By obtaining normalized firefly luminescence/Renilla luminescence ratio for each sample well, knockdown percentage of firefly luciferase was calculated. The luminescence was measured using microplate reader (Synergy HT, BioTek) and the results were analyzed using Gene5 2.01 software (BioTek). For statistical analysis, pvalue was calculated using One-way ANOVA with post hoc Fisher’s test. Error bars in Figure 5g are standard deviation.

3. RESULTS AND DISCUSSION 3.1. Self-assembly of the DNA-Guided RNA nanovector Figure 1 presents a schematic diagram of the overall process to generate nanostructures by step-wise

dual

enzyme

polymerization.

To

perform

temperature-dependent

dual

polymerization, two polymerases with different optimal reaction temperatures, T7 RNA polymerase and Phi29 DNA polymerase, were chosen. The entire synthesis process was then performed by manipulating the reaction temperature and incubating time for T7 RNA polymerase and Phi29 DNA polymerase with two different optimal activity temperatures, 37 °C and 30 °C, respectively. For SDEP, two different circular DNAs, which are partially complementary to each other with specific functional sequences complementary to NCLaptamer and anti-luciferase siRNA were first prepared using a previously reported method (sequences in Figure S2 and Table S1).29,33 Anti-luciferase circular DNA was used as the template DNA for T7 RNA polymerase to produce the RNA, and the NCL-aptamer (AS1411) circular DNA was used for Phi29 DNA polymerase to produce the DNA. Theoretically, Phi29 DNA polymerase could produce DNA from both circular DNAs, whereas T7 RNA polymerase can synthesize the RNA strands only from the anti-luciferase circular DNA that 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 27

is hybridized with a promoter. To avoid the production of unnecessary DNA strands from anti-luciferase circular DNA by Phi29 DNA polymerase, the two circular DNAs were incubated with the desired polymerases separately at the pre-incubation stage of SDEP for 10 min. Using this process, Phi29 DNA polymerase and T7 RNA polymerase could link efficiently with the desired circular DNA ([1] of Figure 1a and Figure S3). After the initial stage, the two reaction solutions were mixed ([2] of Figure 1a). The reaction solution was then heated with repeated temperature changes. One heating cycle involved heating the mixture to 37 °C, followed by cooling down to 30 °C, with each being held at those temperatures for 10 min to 1 h ([3] of Figure 1a). This heating cycle reaction was repeated for 20 h. As a result, the newly synthesized DNA and RNA strands were hybridized and selfassembled to form nano-size nucleic acid particles, called DNA-guided RNA nanovectors. To examine the formation of the DNA-guided RNA nanovector, the resulting products were visualized using three microscopic methods with a 10 min reaction step (RS). Atomic force microscopy (AFM) of the resulting particles showed a mono-dispersed spherical nanostructure, approximately 150 nm in diameter, suggesting that the resulting DNA and RNA strands had self-assembled readily into the nanostructures (Figure 1c). High magnification transmission electron microscopy (TEM) of the RNA nanovector revealed densely assembled spherical nanostructures (Figure 1d). The size and shape of the nanostructures determined by scanning electron microscopy (SEM) were consistent with the aforementioned results (Figure 1e).

3.2. Formation process of the DNA-guided RNA nanovectors The formation of the RNA-nanovector was examined by time-dependent experiments during SDEP. The formation process was confirmed by AFM after SDEP for 4 h, 12 h and 20 h. In the early stages of SDEP, highly entangled DNA-RNA strands with tiny globular structures 8 ACS Paragon Plus Environment

Page 9 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were observed (Figure 2a). After 12 h, clusters of the DNA-RNA granules were generated from the entangled structures (Figure 2b). At the final stage of SDEP, aggregated RNA nanovectors began to appear (Figure 2c). Because the products at the final stage were perfectly spherical and loosely connected to each other, they were separated easily into individual nanoparticles, i.e., RNA nanovectors, by short-time sonication. Based on the AFM results, Figure 2d presents a schematic diagram of the formation process of RNA nanovectors. Similar to traditional polymer crosslinking and the assembly of spatially localized nanogels or clusters at certain concentrations of polymer34, a comparable process could occur during SDEP by reaching a critical concentration. In addition, hydrogen-bonding between the RNA and DNA could promote the assembly process.

3.3. Control of the morphology of DNA-guided RNA nanovectors by SDEP with different reaction step This study also examined whether the morphology of the RNA nanovectors could be controlled by changing the reaction conditions. Interestingly, a morphological change in the resulting products was observed by manipulating the RS and concentrating the circular DNAs. Changing the RS produced a variety of structures, such as ring structures, sponge-like structures, microscopic spheres, nano-spheres and net-like structures (Figure 3). The products from SDEP without a heating cycle exhibited sponge-like structures (Figure 3a). In particular, the sponge-like spherical structures, which were similar to the previously published RNA microsponges29, were observed with an isothermal reaction at only 37 °C, the optimal activity temperature of T7 RNA polymerase. The connected meshed structures were observed after the isothermal reaction at only 30 °C (Figure 3b). This suggests that performing an isothermal reaction without a heating cycle could mostly induce a single polymerase reaction. Therefore, the products from SDEP performed at only 30 °C or 37 °C could be composed largely of 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 27

DNA or RNA, respectively. The resulting products from rolling circle DNA replication tended to be more connected than by RNA replication. To generate the optimal RNA vectors, SDEP was performed for various reaction times in each step, 5 min, 10 min, 20 min or 60 min. As mentioned above, ring structure was fabricated by SDEP with 5 min RS (Figure 3c). Spherical nanovectors were observed after SDEP with 10 min RS (Figure 3d). The resulting particles from SDEP with the 20 min RS appeared to be linked to each other like the short length of a chain (Figure 3e). With 60 min RS, large net-like structures were observed (Figure 3f). Hence, the degree of the hybridization of RNA and DNA appeared to be controlled by increasing the RS from 5 min to 60 min. Because each DNA or RNA strand generated in a longer single RS could become relatively longer, there could be a greater chance of the entanglement of DNA or RNA themselves instead of the hybridization between the elongated DNA and RNA strands, resulting in a net-like structure. Moreover, huge net-like structures were also observed by increasing the concentration of circular DNA with 0.3 µM of circular DNA (Figure S4). The DLS results revealed a mean size of RNA nanovectors and number percentage below 200 nm in size. The mean size increased with increasing time for the RS, whereas the number percentage below 200 nm in size decreased (Figure 3g and Figure S5). These results are consistent with the SEM and AFM results. For biomedical applications, the size and monodispersity of the RNA nanovectors are important for the enhanced cellular uptake and permeation and retention effect.7,35 The RNA nanovectors from SDEP with a 10 min RS have a monodispersed spherical structure with a mean size of 148 ± 37 nm, which is appropriate for cellular uptake.

3.4. Composition and nuclease resistance of DNA-guided RNA nanovectors


Page 11 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To confirm the composition of the RNA nanovectors, the reaction was performed with cyanine3-UTP (Cy3-UTP) and cyanine5-dCTP (Cy5-dCTP) for RNA polymerase and DNA polymerase, respectively. As expected, the image cytometry results reveal that fluorescently-labeled RNA nanovectors emitted a higher red and orange fluorescent intensity than non-labeled RNA nanovector as negative control, suggesting that the nanovectors were composed of both DNA and RNA (Figure 4a, b). To further examine the DNA-RNA hybridization, the RNA nanovectors were incubated with RNase H, which could specifically degrade the RNA strand hybridized to the DNA. The reactants were then examined by gel electrophoresis (Figure 4c). Because the RNA fragments degraded by RNase H can still be hybridized to DNA strands, the reactants were heated shortly to separate the RNA fragments from the DNA strands. As shown in Figure 4c, the band of RNA nanovectors with 10 min RS in the well was significantly dimmed after RNase H treatment (lane 3), indicating significant degradation of RNA nanovector by RNase H and high degree of RNA and DNA hybridization. However, any significant decrease from RNA products with 20 min RS (lane 5) and 60 min RS (lane 7) was not observed, indicating the relatively less hybridization of RNA and DNA. This result suggests that the RNA nanovector with 10 min RS is composed of well-hybridized DNA-RNA strands. The stability of the RNA nanovector was evaluated by the nuclease degradation with DNase I, RNase I and RNase III. After the nuclease treatments, the reactants were analyzed by gel electrophoresis (Figure S6). Owing to DNA-RNA hybridization, the RNA nanovectors were expected to show resistance to nuclease degradation. In addition, the RNA nanovectors with the NCL-aptamer could also enhance the stability because of the G-quartet structure.36,37 As shown in the gel electrophoresis results, the RNA nanovectors still remained after being treated with 0.05 U µl-1 nucleases, which is much higher than the nuclease concentration in human blood (lane 2 in Figure S6a).38,39 On the other hand, the products comprised of DNA only or RNA only were degraded easily by 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 27

nuclease degradation (lane 3 and lane 5 in Figure S6b), showing that the RNA nanovectors are more stable in the presence of nucleases than only RNA or DNA products. To confirm the enhanced cellular uptake by NCL-aptamer on RNA nanovectors, the MDA-MB-231 cells, which overexpress nucleolin receptors, were incubated with the red fluorescence-labeled RNA nanovectors for 4 h at 37 °C. As shown in Figure 5a and 5b, the RNA nanovectors with NCL-aptamer exhibited enhanced cellular uptake by the MDA-MB231 cells, compared to the RNA nanovectors without an aptamer (Figure S7). Highmagnification confocal microscopy further suggested that the RNA nanovectors could be internalized into the cells through NCL-mediated endocytosis without the need for a transfection agent (Figure 5c). Confocal microscopy image with xz- and yz-projections is clearly showing that the fluorescent particles were internalized into target cell (Figure 5d). These results highlight the potential ability of the RNA nanovectors for carrier-free siRNA delivery. In addition, the RNA nanovectors did not affect the cell viability (Figure 5e). Because the RNA nanovectors were designed to produce 21-25 bp siRNA fragments by an intracellular mechanism, they were incubated with the recombinant Dicer enzyme to confirm Dicer cleavage of the nanovectors. The resulting products were then analyzed by gel electrophoresis (Figure 5f and Figure S8). As shown in Figure 5f, the RNA nanovectors were converted successfully to siRNA by the Dicer. The yield of the siRNA fragments from the RNA nanovectors was improved by increasing the incubation time with the Dicer up to 48 h. With the ability to generate siRNA, the RNA nanovectors were evaluated for their ability to inhibit target gene expression. As shown in Figure 5g, gene expression of HeLa-luciferase (HeLa-Luc) cells which also overexpress nucleolin receptors, was reduced to about 20 % at a concentration of 37.5 nM, suggesting that the target gene activity was regulated efficiently by the RNA nanovectors with the NCL-aptamer. This result also indicates that our RNA 12 ACS Paragon Plus Environment

Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanovectors are able to be internalized into both MDA-MB-231 cells and HeLa cells which are different nucleolin-overexpressed cancer cell line. In addition, the luciferase activity of the HeLa-Luc cells transfected for 12 h was effectively inhibited suggesting that the RNA nanovectors were sustainedly internalized into target cells for 12 h of transfection time. Therefore, with the nuclease resistance of our RNA nanovectors, long-term delivery of siRNA could be achieved.

3.5. DNA-guided siRNA delivery of RNA nanovectors To confirm the targeting effect of NCL-aptamer, the RNA nanovectors without aptamer were also tested. As a result, the knock-down efficiency of the RNA nanovectors at 37.5 nM showed a significant difference compared to the without aptamer, suggesting that the aptamers within the nanovectors could improve the cellular uptake capability significantly. As another control, off-target effect of RNA nanovectors was confirmed by using anti-GFP siRNA as scrambled siRNA. As shown in purple and dark cyan columns in Figure 5g, there were no statistical significance compare the knock-down of RNA nanovectors with scrambled siRNA and untreated cells. Furthermore, the knock-down efficiency of RNA nanovector was compared with siRNA in combination with transfection agent as a positive control. According to the result, luciferase activity with positive control was reduced to 16 %, indicating slightly better knock-down efficiency than our RNA nanovectors. However, it is meaningful that RNA nanovector resulted in significant knock-down of the luciferase activity because the transfection agent was not needed for repression of target gene. These results suggest that the new carrier-free siRNA delivery system retains its unique properties, such as the target-specific cellular uptake without the need for conventional cationic transfection agents, effective target gene regulation and low cytotoxicity. Furthermore, the RNA nanovector could be applied to delivery of siRNA in different cell types. 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

4. CONCLUSIONS In conclusion, directed RNA or DNA assembly has been promoted as a means of creating structures that can form into nano- or micro-structures; however, these capabilities have rarely been translated to the development of carrier-free siRNA delivery systems.40-42 To take the advantages from DNA, RNA, and DNA-RNA hybridization, polymerization of RNA and DNA was designed and optimized for carrier-free siRNA delivery. Step-wise dual enzyme polymerization is a new strategy that can generate self-assembling material composed of DNA aptamer and siRNA precursor, called DNA-guided RNA nanovectors. From the timedependent experiment, we examined that the RNA nanovectors could be generated by the hybridization and entanglement of DNA-RNA strands. The experimental results from SEM, TEM and AFM suggested that RNA nanovectors have spherical nanostructure below 200 nm in size. Furthermore, the morphology of RNA products could be controlled by manipulating the reaction condition such as RS and concentration of circular DNA. We found that four different structures such as ring-like, spherical, short chain-like and large net-like structure were generated. More importantly, the DNA-guided RNA nanovectors could be designed with desirable properties, such as nuclease resistance and enhanced cellular uptake without a transfection agent. To achieve these unique properties, we designed the circular DNA templates to possess particular sequences so that the RNA nanovectors would be comprised of DNA-RNA hybridization and DNA aptamer as a key factor for targetable carrier-free siRNA delivery. As expected, the stability analysis indicated that the RNA nanovectors are more stable than the replicated DNA or RNA products. Moreover, the cell uptake experiment showed the RNA nanovectors were delivered specifically into target cells without any transfection agents, leading to significant repression of the target gene expression. These results suggest the 14 ACS Paragon Plus Environment

Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


possibility that SDEP may serve as platform for development of carrier-free nucleic acidbased therapeutics. In addition, the particles can be functionalized easily for a variety of purposes by designing DNA and RNA strands, and can be applied in target-specific simultaneous mRNA-siRNA delivery or plasmid-siRNA delivery.

SUPPORTING INFORMATION Additional experimental data microscopy images are availiable as Supporting Information for this article. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT This research was supported by the Global Innovative Research Center (GiRC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2012K1A1A2A01056093) and also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1A1A1A05001174).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

REFERENCES 1.

Afonin, K. A.; Grabow, W. W.; Walker, F. M.; Bindewald, E.; Dobrovolskaia, M.

A.; Shapiro, B. A.; Jaeger, L., Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine. Nat. Protoc. 2011, 6, 2022-2034. 2.

Pecot, C. V.; Calin, G. A.; Coleman, R. L.; Lopez-Berestein, G.; Sood, A. K., RNA

interference in the clinic: challenges and future directions. Nat. Rev. Cancer 2010, 11, 59-67. 3.

Li, H.; Lee, T; Dziubla, T.; Pi, F.; Guo, S.; Xu, J.; Li, C.; Haque, F.; Liang X.-J.; Guo,

P., RNA as a stable polymer to build Controllable and defined nanostructures for material and biomedical applications. Nano Today 2015, 10, 631-655 4.

Guo, P., The emerging field of RNA nanotechnology. Nat. Nanotechnol. 2010, 5,

833-842 5.

Douglas, S. M.; Bachelet, I.; Church, G. M., A logic-gated nanorobot for targeted

transport of molecular payloads. Science 2012, 335, 831-834. 6.

Jiang, S.; Eltoukhy, A. A.; Love, K. T.; Langer, R.; Anderson, D. G., Lipidoid-coated

iron oxide nanoparticles for efficient DNA and siRNA delivery. Nano Lett. 2013, 13, 105964. 7.

Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R.,

Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2, 751-760. 8.

Young, K. L.; Scott, A. W.; Hao, L.; Mirkin, S. E.; Liu, G.; Mirkin, C. A., Hollow

spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells. Nano Lett. 2012, 12 3867-3871. 9.

Khan, M.; Ong, Z. Y.; Wiradharma, N.; Attia, A. B. E.; Yang, Y. Y., Advanced

Materials for Co‐Delivery of Drugs and Genes in Cancer Therapy. Adv. Healthcare Mater. 2012, 1, 373-392. 16 ACS Paragon Plus Environment

Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10.

Hoerter, J. A.; Krishnan, V.; Lionberger, T. A.; Walter, N. G., siRNA-like double-

stranded RNAs are specifically protected against degradation in human cell extract. PLoS one 2011, 6, e20359. 11.

Patil, M. L.; Zhang, M.; Betigeri, S.; Taratula, O.; He, H.; Minko, T., Surface-

modified and internally cationic polyamidoamine dendrimers for efficient siRNA delivery. Bioconjugate Chem. 2008, 19, 1396-1403. 12.

Davis, M. E.; Zuckerman, J. E.; Choi, C. H. J.; Seligson, D.; Tolcher, A.; Alabi, C.

A.; Yen, Y.; Heidel, J. D.; Ribas, A., Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464, 1067-1070. 13.

Chien, P.-Y.; Wang, J.; Carbonaro, D.; Lei, S.; Miller, B.; Sheikh, S.; Ali, S. M.;

Ahmad, M. U.; Ahmad, I., Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo. Cancer Gene Ther. 2005, 12, 321-328. 14.

Cao, Z.; Tong, R.; Mishra, A.; Xu, W.; Wong, G. C.; Cheng, J.; Lu, Y., Reversible

Cell‐Specific Drug Delivery with Aptamer‐Functionalized Liposomes. Angew. Chem. Int. Ed. 2009, 48, 6494-6498. 15.

Liu, L.; Xu, K.; Wang, H.; Tan, P. J.; Fan, W.; Venkatraman, S. S.; Li, L.; Yang, Y.-

Y., Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent. Nat. Nanotechnol. 2009, 4, 457-463. 16.

Cavallaro, G.; Campisi, M.; Licciardi, M.; Ogris, M.; Giammona, G., Reversibly

stable thiopolyplexes for intracellular delivery of genes. J. Controlled Release 2006, 115, 322-334. 17.

Guo, P.; Coban, O.; Snead, N. M.; Trebley, J.; Hoeprich, S.; Guo, S.; Shu, Y.,

Engineering RNA for targeted siRNA delivery and medical application. Adv. Drug Delivery Rev. 2010, 62, 650-666. 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18.

Page 18 of 27

Afonin, K. A.; Kireeva, M.; Grabow, W. W.; Kashlev, M.; Jaeger, L.; Shapiro, B. A.,

Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs. Nano Lett. 2012, 12, 5192-5. 19.

Hao, C.; Li, X.; Tian, C.; Jiang, W.; Wang, G.; Mao, C., Construction of RNA

nanocages by re-engineering the packaging RNA of Phi29 bacteriophage. Nat. Commun. 2014, 5, 3890. 20.

Ohno, H.; Kobayashi, T.; Kabata, R.; Endo, K.; Iwasa, T.; Yoshimura, S. H.;

Takeyasu, K.; Inoue, T.; Saito, H., Synthetic RNA-protein complex shaped like an equilateral triangle. Nat. Nanotechnol. 2011, 6, 116-120. 21.

Saito, H.; Fujita, Y.; Kashida, S.; Hayashi, K.; Inoue, T., Synthetic human cell fate

regulation by protein-driven RNA switches. Nature Commun. 2011, 2, 160. 22.

Lee, J. B.; Peng, S.; Yang, D.; Roh, Y. H.; Funabashi, H.; Park, N.; Rice, E. J.; Chen,

L.; Long, R.; Wu, M., A mechanical metamaterial made from a DNA hydrogel. Nat. Nanotechnol. 2012, 7, 816-820; 23.

Roh, Y. H.; Lee, J. B.; Kiatwuthinon, P.; Hartman, M. R.; Cha, J. J.; Um, S. H.;

Muller, D. A.; Luo, D., DNAsomes: Multifunctional DNA‐Based Nanocarriers. Small 2011, 7, 74-78. 24.

Lee, H.; Lytton-Jean, A. K.; Chen, Y.; Love, K. T.; Park, A. I.; Karagiannis, E. D.;

Sehgal, A.; Querbes, W.; Zurenko, C. S.; Jayaraman, M., Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotechnol. 2012, 7, 389-393. 25.

McNamara, J. O.; Andrechek, E. R.; Wang, Y.; Viles, K. D.; Rempel, R. E.; Gilboa,

E.; Sullenger, B. A.; Giangrande, P. H., Cell type–specific delivery of siRNAs with aptamersiRNA chimeras. Nat. Biotechnol. 2006, 24, 1005-1015.


Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26.

Wu, C.; Han, D.; Chen, T.; Peng, L.; Zhu, G.; You, M.; Qiu, L.; Sefah, K.; Zhang,

X.; Tan, W., Building a multifunctional aptamer-based DNA nanoassembly for targeted cancer therapy. J. Am. Chem. Soc. 2013, 135, 18644-50. 27.

Chu, T. C.; Twu, K. Y.; Ellington, A. D.; Levy, M., Aptamer mediated siRNA

delivery. Nucleic Acids Res. 2006, 34, e73-e73. 28.

Seyhan, A. A.; Vlassov, A. V.; Johnston, B. H., RNA interference from multimeric

shRNAs generated by rolling circle transcription. Oligonucleotides 2006, 16, 353-363. 29.

Lee, J. B.; Hong, J.; Bonner, D. K.; Poon, Z.; Hammond, P. T., Self-assembled RNA

interference microsponges for efficient siRNA delivery. Nat. Mater. 2012, 11, 316-22. 30.

Han, D.; Park, Y.; Kim, H.; Lee, J. B., Self-assembly of free-standing RNA

membranes. Nature Commun. 2014, 5, 4367. 31.

Gallagher-Jones, M.; Bessho, Y.; Kim, S.; Park, J.; Kim, S.; Nam, D.; Kim, C.; Kim,

Y.; Noh do, Y.; Miyashita, O. et al., Macromolecular structures probed by combining singleshot free-electron laser diffraction with synchrotron coherent X-ray imaging. Nat. Commun. 2014, 5, 3798. 32.

Afonin, K. A.; Viard, M.; Martins, A. N.; Lockett, S. J.; Maciag, A. E.; Freed, E. O.;

Heldman, E.; Jaeger, L.; Blumenthal, R.; Shapiro, B. A., Activation of different split functionalities on re-association of RNA-DNA hybrids. Nat. Nanotechnol. 2013, 8, 296-304. 33.

Daubendiek, S. L.; Kool, E. T., Generation of catalytic RNAs by rolling transcription

of synthetic DNA nanocircles. Nat. Biotechnol. 1997, 15, 273-277. 34.

Seiffert, S.; Sprakel, J., Physical chemistry of supramolecular polymer networks.

Chem. Soc. Rev. 2012, 41, 909-30. 35.

Petros, R. A.; DeSimone, J. M., Strategies in the design of nanoparticles for

therapeutic applications. Nat. Rev. Drug Discovery 2010, 9, 615-627.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

36.

Page 20 of 27

Ireson, C. R.; Kelland, L. R., Discovery and development of anticancer aptamers.

Mol. Cancer Ther. 2006, 5, 2957-2962. 37.

Bates, P. J.; Laber, D. A.; Miller, D. M.; Thomas, S. D.; Trent, J. O., Discovery and

development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp. Mol. Pathol. 2009, 86, 151-164. 38.

Nadano, D.; Yasuda, T.; Kishi, K., Measurement of deoxyribonuclease I activity in

human tissues and body fluids by a single radial enzyme-diffusion method. Clin. Chem. 1993, 39, 448-452. 39.

Tamkovich, S. N.; Cherepanova, A. V.; Kolesnikova, E. V.; Rykova, E. Y.; Pyshnyi,

D. V.; Vlassov, V. V.; Laktionov, P. P., Circulating DNA and DNase activity in human blood. Ann. N. Y. Acad. Sci. 2006, 1075, 191-196. 40.

Khisamutdinov, E. F.; Jasinski, D. L.; Guo, P., RNA as a boiling-resistant anionic

polymer material to build robust structures with defined shape and stoichiometry. ACS Nano 2014, 8, 4771-4781 41.

Cui, D.; Zhang C.; Liu, B.; Shu, Y.; Du, T.; Shu, D.; Wang, K.; Dai, F.; Liu, Y.; Li,

C.; Pan, F.; Yang, Y.; Ni, J.; Li, H.; Brand-Saberi, B.; Guo, P., Regression of gastric cancer by systemic injection of RNA nanoparticles carrying both ligand and siRNA, Sci. Rep. 2015, 5 42.

Shu, D.; Li, H.; Shu, Y.; Xiong, G.; Carson III, W. E.; Haque, F.; Xu, R.; Guo, P.,

Systemic delivery of anti-miRNA for suppression of triple negative breast cancer utilizing RNA nanotechnology. ACS Nano 2015, 9, 9731-9740


Page 21 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Schematic diagram of the process and characterization of the synthesis DNAguided RNA nanovector. (a) Step-wise dual enzyme polymerization (SDEP). Heating cycle reaction consist of repeated reaction step (RS). x and y of RS are reaction time to maintain reaction temperature at 37 ºC and 30 ºC, respectively. (b) Self-assembly of the RNA nanovector. Characterization of the RNA nanovectors from SDEP with 10 min RS. (c) ChaNon-contact mode AFM image of the RNA nanovector. Inset scale bar: 200 nm. (d) High-magnification of the TEM image displaying the structure of the RNA nanovector. (e) SEM image of the RNA nanovector. Inset scale bar: 100 nm. 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 27

Figure 2. Formation of DNA-guided RNA nanovectors from SDEP. AFM images of the RNA nanovector of time-dependent SDEP for (a) 4 h, (b) 12 h and (c) 20 h, respectively. (d) Schematic diagram of the formation process of the RNA nanovectors.


Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Structural properties of the RNA nanovector with changes in the RS. SEM, TEM and AFM images showing the various structures of the RNA nanovector. SEM images of the RNA nanovector via SDEP w/o heating cycle. SDEP was performed at only (a) 37 °C or (b) 30 °C, respectively. Inset scale bar: 1 µm. (c) TEM and AFM images of the RNA nanovectors from SDEP with 5 min RS. Inset scale bar: 200 nm. SEM and AFM images of the RNA nanovector from SDEP with (d) 10 min, (e) 20 min and (f) 60 min RS. Inset scale bar: 200 nm. (g) Size distribution of the RNA nanovector showing the mean value and number percentage below 200 nm in size. 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

Figure 4. Confirmation of the DNA-RNA hybridization in the RNA nanovector. Image cytometry acquisition plots for two-color analysis of (a) non-labeled RNA nanovector and (b) Cy3 and Cy5 labeled RNA nanovector. Both of RNA nanovectors were synthesized via SDEP with 10 min RS. The green and red curves in image cytometry profiles of RNA nanovectors represent the histogram generated by fluorescently-labeled RNA nanovectors with Cy3-UTP and Cy5-dCTP. The black curves represent the histogram generated by nonlabeled RNA nanovectors as negative control. (c) 1% agarose gel image after RNase H treatment for 24 h. Lane 1 indicates 100 base pair DNA ladder. Lanes 2, 4 and 6 indicate the RNA nanovector which were made from SDEP with a 10 min, 20 min and 60 min RS, respectively. Lanes 3, 5 and 7 indicate the RNA nanovector which were made from SDEP with a 10 min, 20 min and 60 min RS, respectively, after treatment with RNase H (2,000 U mL-1) for 24 h.


Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Cellular uptake and gene repression of the RNA nanovector with 10 min RS. Confocal microscope images of MDA-MB-231 cells treated with the Cy-3 UTP labeled RNA nanovector (a) without and (b) with the aptamer, respectively. (c) Representative confocal microscope images showing the cellular distribution of the RNA nanovector with the NCLaptamer. (d) Confocal microscope image with xz- and yz-projections are shown on the left and at the top. (e) Cell viability assay of the RNA nanovector toward HeLa cells, which overexpress nucleolin receptors as well. Values represent the average ± SD of two independent experiments (n=3). (f) 3% agarose gel image after Dicer enzyme treatment. Lanes 1 and 2 indicate the double stranded RNA ladder and the RNA nanovector w/o Dicer, 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

respectively. Lanes 3 to 6 correspond to the RNA nanovector incubated with the Dicer enzyme for 4, 12, 24, and 48 h, respectively. (g) Suppression of firefly luciferase expression by the RNA nanovector toward the HeLa-Luc cells. HeLa-Luc cells were transfected for 12 h. Anti-GFP siRNA was used as non-targeted siRNA to confirm off-target effect of siRNA. Anti-luciferase siRNA (200 nM) in combination with transfection agent (TransIT-X2, Mirus) was used as positive control. Normalized luciferase activity indicates the ratio of firefly luciferase expression to Renilla luciferase expression, and Renilla luciferase is used as a house keeping gene. Values represent the average ± SD of three independent experiments (n=5). P-value was calculated using One-way ANOVA with post hoc Fisher’s test (* p < 0.05 compared with the RNA(scrambled):DNA(scrambled), ** p < 0.05).


Page 27 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only

Self-assembled DNA-guided RNA Nanovector via Step-wise Dual Enzyme Polymerization (SDEP) for Carrier-free siRNA Delivery Yongkuk Park, Hyejin Kim and Jong Bum Lee*


Self-assembled DNA-Guided RNA Nanovector via Step-wise Dual

Recommend Documents