Enzymatic Synthesis of Self-assembled Dicer Substrate RNA

process to produce geometry defined nanostructures for tailored uses. 2, 4, 5 Previously, various DNA and RNA nanostructures were reported as novel pl...
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Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing Bora Jang, Boyoung Kim, Hyunsook Kim, Hyokyoung Kwon, Minjeong Kim, Yunmi Seo, Marion Colas, Hansaem Jeong, Eun Hye Jeong, Kyuri Lee, and Hyukjin Lee Nano Lett., Just Accepted Manuscript • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing Bora Jang1, Boyoung Kim1, Hyunsook Kim1, Hyokyoung Kwon1, Minjeong Kim1, Yunmi Seo1, Marion Colas 1,2, Hansaem Jeong1, Eun Hye Jeong1, Kyuri Lee1, and Hyukjin Lee1* 1

College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of Korea; 2 Faculté de Pharmacie de Paris, Université Paris Descartes, Paris 75006, France

ABSTRACT: Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes. KEYWORDS: RNA nanostructures, Dicer substrate RNA, Gene silencing, Enzymatic synthesis, Programmable gene regulation Self-assembled nucleic acid nanostructures are widely utilized for applications in drug delivery and molecular imaging.1, 2 They offer unique advantages over the conventional nanoparticles made of polymers, inorganic, and carbon-based materials. These include the high-yield synthesis of molecularly identical nanostructures with precise control of shape and size.3 In addition, selfassembly of oligonucleotides is an easy and care-free process to produce geometry defined nanostructures for tailored uses. 2, 4, 5 Previously, various DNA and RNA nanostructures were reported as novel platforms for the intracellular delivery of macromolecules such as siRNA, transcriptional factors, and gene editing proteins.4, 6, 7 These studies received much attention due to their early attempts on expanding the use of nucleic acid nanostructures for target specific gene regulation in biological systems. Later, other studies also demonstrated the wide use of threedimensional nucleic acid nanostructures for the intracellular delivery of therapeutics in mammalian cells and animals.8-10 Previously, large-scale production of DNA nanostructures was reported by rolling circle amplification (RCA) carrying multiple synthetic short interfering RNAs (siRNAs) into target cells.11 Small amount of circular DNA templates can be easily amplified to produce large quantities of self-assembled DNA junctions. Various synthetic siRNAs can be incorporated within the DNA junctions by simple overhang hybridization. Although

DNA nanostructures are particularly useful for delivering synthetic siRNAs into the cells, their major role is quite limited to provide physical and structural support for the region-specific hybridization of siRNAs within the systems.4, 7, 8 Besides the DNA nanostructures, several RNA nanostructures were also reported for gene silencing applications and they utilized natural or artificially selected RNA motifs and modules for the pre-defined selfassembly.5, 6 These studies showed other possibilities, since RNA structures can provide bi-functional roles as a physical frame for intracellular delivery as well as a biological trigger to induce RNAi by itself. However, they required multiple long synthetic RNA strands to generate functional RNA nanostructures and it was very costly to prepare them in large quantities. Herein, we report the large-scale preparation of RNA nanostructures through an enzymatic synthesis. As shown in Figure 1a, three arm junction RNA nanostructure (YRNA) was synthesized in large quantities by the two-step reactions including isothermal rolling circle transcription (RCT) of DNA templates and site-specific cleavage of amplified RNA strands by RNase H treatment. Selfassembled DNA template was used for in vitro transcription of RNA strands having three distinct regions such as i) core, ii) RNAi, and iii) loop. Different regions of Y-RNA provided a specific function such as i) stabilizing the nanostructure, ii) inducing sequence specific RNAi, and iii) protecting the RNA structure against nuclease

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Figure 1. Enzymatic synthesis of Y-RNA and its characterization. (a) Schematic illustration of enzymatic preparation of Y-RNA through rolling circle transcription (RCT) and site-specific cleavage. (b) PAGE analysis of sequential self-assembly and RCT process (Lane 1, 2, and 3: hybridization of linear template strand 1, 2, and 3, lane 4: ligated circular template, lane 5: RCT product, and lane 6: self-assembled Y-RNA). (c) AFM images of amplified RCT products (left) and self-assembled Y-RNA (inset: phase image of Y-RNA) (right).

degradation. For the RCT process, T7 RNA polymerase was used to recognize the single stranded (ss) circular loop of DNA template as an open transcription complex to initiate in vitro transcription without the addition of complementary DNA primers.12-14 This was particularly advantageous for the synthetic process, since very long single stranded (ss)-RNA with a tandem repeat of circular DNA template could be produced. In order to cleave long ss-RNA products into smaller fragments with a defined size, 2’-O-methyl modified DNA helper strands were used. These modified DNA helper strands have been widely used for the site-specific cleavage of ss-RNA products by RNase H.15-17 Since RNase H does not recognize 2’-O-ME DNA/RNA hybrid, modified region of helper strand prevents non-specific cleavage. As shown in Figure 1b, each step of enzymatic synthesis was confirmed by polyacrylamide gel electrophoresis (PAGE) analysis: i) self-assembly of DNA templates (Lane: 1~3), ii) ligation and RCT of DNA templates (Lane 4, 5), iii) site-specific cleavage of amplified RNA products and self-assembly of Y-RNA (Lane 6) were achieved. Large amount of amplified RNA products was visualized in the gel loading area due to their high molecular weight. After RNase H treatment, cleaved short RNA fragments could self-assemble to form a well-

defined Y-RNA. Residual strands were removed by centrifugal filtration and the production yield of Y-RNA was more than 92 % by gel densitometry analysis (Figure S1, supporting information). In addition, atomic force microscopy (AFM) analysis further validated the RCT process and self-assembly of Y-RNA. Each sample was deposited on the freshly cleaved mica surface and its morphology was imaged in non-contact mode (Figure 1c and Figure S2a, supporting information). The amplified ss-RNA products showed very long thread like morphology with the lateral size of several hundred nanometers. This confirmed the successful in vitro transcription of complementary RNA strands from the DNA templates. Physical entanglement of ss-RNA products was also visualized as reported elsewhere.1, 18 After the site-specific cleavage by RNase H, self-assembled Y-RNA (20 nm in size) can be obtained. There were no large aggregates formed and only mono-dispersed Y-RNA was observed. The hydrodynamic size of Y-RNA was also measured by dynamic light scattering (DLS) confirming the narrow distribution of Y-RNA (Figure S2b, supporting information). Through the enzymatic synthesis, we could produce more than 2 nmol of Y-RNA within 12 hrs by using 1 pmol of circular DNA templates.

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Figure 2. Evaluation of in vitro Dicer cleavage and gene silencing activity. (a) in vitro Dicer cleavage of substrate ds-RNA (L-RNA and Y-RNA) and PAGE analysis of Dicer processing to confirm the length dependent cleavage of substrate ds-RNA (left) and the effect of 2nt overhang on Dicer cleavage of Y-RNA (right). (-: before Dicer treatment, +: after Dicer treatment) (b) Dose dependent gene silencing test in GFP-KB cells using different RNA samples (siRNA, L-RNA, and Y-RNA). (**: p-value ൑ 0.01, ****: p-value ൑ 0.0001). (c) Effects of structural variations of Y-RNA on gene silencing (different arm lengths and overhang structures (19+2, 23+2, 25+2 Y-RNA and 19+0, 23+0 Y-RNA)

It is well known that long double stranded (ds)-RNA undergoes endogenous Dicer processing to generate short ds-RNA (20-21 bp).19-21 The cleaved ds-RNA is subsequently loaded with Ago2 to form a catalytic RISC having antisense RNA as a guide strand. Previous studies on Dicer substrate RNA highlighted that more catalytic RISC can be generated by facilitating the Ago2 loading of short ds-RNA through the Dicer cleavage process.22 Therefore, it is important to figure out the structural criteria of Y-RNA for proper interaction with Dicer. To investigate this, we examined the in vitro cleavage of Y-RNA with different arm lengths using recombinant human Dicer (Figure 2a). Linear ds-RNA (L-RNA) was also tested as a control. After 6 hr incubation, the cleavage of various RNA samples was evaluated. The canonical 19+2 ds-RNA showed no cleavage as expected (Lane 1), while longer dsRNA (23+2 and 25+2) generated short cleaved ds-RNA products (Lane 2 and 3). Length of cleaved products were

about 20 bp length (Figure S3, supporting information). YRNA also showed different Dicer processability depending on its arm length. For example, 19+2 Y-RNA resulted minimal cleavage (Lane 4) due to its short arm length.23 There seems to be suitable arm length of Y-RNA for proper Dicer cleavage and the core of junction may not be accessible by Dicer when the arm length is too short. On the other hand, longer arm Y-RNA (23+2 and 25+2) was cleaved by Dicer and short ds-RNA products were generated similar to that of the linear ds-RNA (lane 5 and 6). Next, we evaluated the effect of 3’-2nt overhang on YRNA for Dicer processing. As shown in Figure 2a, both of blunt Y-RNA(19 and 23) failed to generate any cleaved dsRNA products nonetheless of their arm length. This confirmed the structural importance of 3’-2 nt overhang on Y-RNA for Dicer recognition.24 As a result, blunt Y-RNA maintained its intact structure up to 16 hrs under in vitro Dicer processing condition.

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Figure 3. Simultaneous gene regulation of GFP, RFP, and BFP in cells by Y-RNA. Co-transfection of multiple siRNA strands was directly compared with that of Y-RNA transfection. 3D flow cytometry plot of fluorescence expressing HeLa cells was generated after (a) co-transfection of 1 nM of each siRNA strand (siR+G+B). (b) transfection of 1 nM Y-RNA (Y-RNA). In addition to 3D plot, 2D cytometry plot clearly showed the broad distribution of FL level in co-transfection of multiple siRNA as compared to that of Y-RNA. (gray plot: white fluorescence cells (NC), blue plot: down regulated fluorescence expression in cells)

Figure 4. Programmable gene regulation by Y-RNA. (a) Preparation of ON/OFF Y-RNA using RNase H with DNA helper (OFF) and RNA helper (ON), (b) PAGE analysis of the preparation of ON/OFF-Y RNA (Lane 1: circular template, 2: RCT product, 3: R-OFF, 4: RG-OFF, and lane 5: RGB-OFF), (c) Flow cytometry plot of GFP KB-cells after transfection of 1 nM of RGB-OFF, RG-OFF, and ROFF. (d) Evaluation of gene silencing effect (top) and confocal microscope images (bottom) of fluorescent HeLa-cells after the transfection of various ON/OFF Y-RNA (1 nM)

After in vitro evaluation of Dicer cleavage of Y-RNA, gene silencing activity of Y-RNA was investigated. The inhibition of GFP expression in cells was monitored after transfecting various RNA samples (19+2 siRNA, 25+2 L-

RNA, 25+2 Y-RNA) (Figure 2b). Dose-dependent gene silencing was achieved in all tested groups. Negative control samples (transfection reagent only and scramble siRNA) were also tested to verify the sequence specific

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Figure 5. Evaluation of in vivo gene silencing by Y-RNA. (a) characterization of LNP formulation with Y-RNA. in vivo delivery of YRNA using LNP formulation for liver specific gene regulation. (b) Live animal imaging using IVIS Lumina showed the liver specific uptake of LNP by exhibiting strong luminescent signal. (c) ex vivo organ images for liver specific delivery of Y-RNA. (d) Comparative evaluation of dose responsive FVII knockdown using siRNA and Y-RNA (n=3) (*: p-value ൑ 0.05, **: p-value ൑ 0.01).

gene silencing of Y-RNA (Figure S4, supporting information). No significant cellular cytotoxicity was observed in all tested doses (Figure S5, supporting information). Compared to the canonical siRNA (19+2), both Dicer substrate RNA samples (25+2 L-RNA and 25+2 Y-RNA) showed enhanced gene silencing at lower dose (50 and 200 pM). This result confirmed that endogenous Dicer processing of Y-RNA could facilitate the RISC formation for enhanced gene silencing. We also evaluated the effects of structural variations on Y-RNA such as having different arm lengths (19+2, 23+2, 25+2) and blunt ends (19+0, 23+0) (Figure 2c). Guo and co-workers prevsiouly reported the Dicer substrate pRNA 3WJ and confirmed successful survivin gene inhibition in cells.5 This study utilized phi29 RNA mimic to prepare multi-module RNA nanostructures with RNAi sequences. In our study, we aimed to demonstrate the importance of structural criteria for Dicer substrate RNA nanostructures for enhanced RNAi. Similar to the results of in vitro Dicer cleavage test, Y-RNA with longer arms showed effective gene silencing (EC50 of 150 pM for 23+2, EC50 of 125 pM for 25+2), while blunt ends (19+0, 23+0) failed to induce RNAi. It is clear that structural variations of RNA nanostrcutres can highly affect the binding affinity of substrate RNA with Dicer and other cofactors. It is interesting to observe that Y-RNA with short arms (19+2) also exhibited gene silencing in some extent (EC50 of 230 pM). Unlike the result of in vitro Dicer cleavage, it is possible that other RNA binding proteins in cells may participate on the Dicer cleavage of 19+2 Y-RNA to induce gene silencing.25 In addition to the enhanced gene silencing, simultaneous and programmable gene regulation can be achieved in cells using Y-RNA. Figure 3 shows the 3D

flow cytometry plot of fluorescence expressing HeLa cells. White fluorescence cells were prepared by the coexpression of three distinct fluorescent proteins such as DsRed Express2 RFP (R), eGFP (G), and mTagBFP2 (B) (Figure S6, supporting information). After successful expression of R, G, B fluorescence in cells, merged white fluorescence color was obtained as shown in the 3D cytometry plot and fluorescence microscope images. The expression level of R, G, and B fluorescence was carefully modulated to express equal amount of fluorescence intensity in cells (Supporting Movie S1). By employing these cells, we demonstrated the effects of delivering controlled ratio of RNAi sequences in each cell. When multiple siRNA strands were co-transfected, it remained difficult to deliver equal ratio of siRNA strands in cells. Each cell had different copies and ratio of siRNA strands, therefore the precise control of gene expression could not be achieved. As shown in 3D cytometry plot, there was a discrepancy among fluorescence levels of cells after codelivery of multiple siRNA strands (Figure 3a, Supporting Movie S2). This caused the broad FL distribution of cells appearing throughout the unintended regions of 3D plot. 2D plot also indicated that the distribution of FL for all three fluorescent proteins (GFP, RFP, and BFP) was much borader. On the other hand, when Y-RNA was treated to the cells, precise gene regulation was accomplished (Figure 3b, Supporting Movie S3). Since controlled ratio of RNAi strands was introduced by Y-RNA, each cell displayed in 3D plot showed much narrower distribution of FL. In addition, Dicer mediated gene silencing by Y-RNA resulted enhanced FL inhibition and induced the left shift of FL distribution in 2D plot with narrow distribution.

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During the enzymatic synthesis of Y-RNA, various YRNA structures can be generated by preserving their ssRNA loop structure. Depending on the hybridization of helper strands, Y-RNA with open or closed loop structure can be prepared (Figure 4a). Since RNase H can only recognize the DNA/RNA hybrids on the loop structure, addition of RNA helper strands allows the protection of specific loop structure within Y-RNA. As we mentioned earlier, Dicer only recognizes the 3’ end of ds-RNA. Therefore, arms of Y-RNA with the closed loop structure cannot be accessed by Dicer. As shown in Figure 4b, the selective synthesis of YRNA with open and closed-loop structure was confirmed by PAGE analysis. Gene silencing of Y-RNA with different open-loop structure was evaluated in cells. As given in the cytometry plot, selective gene silencing was achieved with Y-RNA regulating RG and RGB expression (Figure 4c). Y-RNA with the closed G-loop (R-OFF) did not inhibit GFP expression in cells. To further elaborate this programmable gene regulation, 7 different Y-RNA samples were prepared having various combination of open and closed-loop structures (Figure S7, supporting information). It is important to note that all of Y-RNA samples were synthesized from a single DNA template, but the different choices of helper strands allowed the generation of selective Y-RNA for specific uses. By applying these Y-RNA, full control of fluorescence color expression was achieved in cells. As shown in Figure 4d, various color spectrum could be obtained including C, M, Y (one-open), R, G, B (two-open), and faint black (allopen). In addition to the cellular study, in vivo gene silencing of Y-RNA was investigated after the systemic injection of Y-RNA using a lipid nanoparticle (LNP).26, 27 C12-200 lipidoid was utilized for liver specific delivery of Y-RNA. Formulation of LNP was validated by confirming its hydrodynamic size as well as encapsulation efficiency (Figure 5a). Both of formulations having either siRNA or Y-RNA showed similar mean particle size of 100 ~ 120 nm (Figure S8, supporting information). Encapsulation efficiency (EE) of Y-RNA (87.8 %) within LNP was higher than that of siRNA (57.3 %). It is likely that Y-RNA with higher charge density could complex more easily with ionizable lipid by stronger charge interaction.28 This result confirmed that various conventional lipid formulations can be employed for the delivery of Y-RNA without altering their standard protocol. In our animal study, FVII inhibition in liver hepatocytes was attempted and dose responsive gene silencing of Y-RNA was directly compared with that of canonical siRNA. Liver specific delivery of Y-RNA was confirmed by the both of live animal and ex vivo organ imaging (Figure 5b and c) by co-delivering luciferase mRNA. Strong luminescence signal was observed only in liver, while other organs showed minimal expression. Different dose of RNA samples was treated by tail vein injection and the level of FVII in mouse blood was

evaluated after 3 days. Figure 5d shows the dose responsive FVII gene inhibition of RNA samples at three different concentrations. In all tested doses, Y-RNA resulted enhanced FVII knockdown as compared to that of siRNA. EC50 of Y-RNA was estimated and its value was 0.05 mg/kg that was substantial lower than that of canonical siRNA (0.09 mg/kg). Our data clearly confirmed that the Y-RNA prepared by enzymatic synthesis could effectively induce in vivo gene silencing as an alternative to the conventional siRNA. In conclusion, we have demonstrated the large scale enzymatic synthesis of Dicer substrate Y-RNA for programmable gene silencing. Synthesis of Y-RNA was consisted of two-step enzymatic reactions of rolling circle transcription (RCT) and site-specific cleavage by RNase H. The resultant Y-RNA showed different gene silencing activity depending on i) its arm length and ii) existence of 2nt overhang. The Y-RNA offered the simultaneous regulation of multiple target genes by controlling the ratio of delivered RNAi sequences in cells. In addition, our enzymatic synthesis method allowed the preparation of various Y-RNA derivatives for programmable gene regulation in cells. Collectively, our results showed the versatile potential of Dicer substrate RNA nanostructures and this appraoch may offer new strategies to develop future RNAi therapeutics.

ASSOCIATED CONTENT Supporting Information Additional details on materials and methods are provided in supporting information file. This includes figures showing co-expresion of GFP, RFP, and BFP in HeLa cells; confocal microscope images of programable gene regulation after transfection of ON/OFF Y-RNAs; particel size distribution of LNPs formulated with siFVII and YsiFVII. Supporting movies showing 3D flow cytometry plot of HeLa cells expressing GFP, RFP, and BFP; 3D flow cytometry plot after transfection of each siRNA (siRFP, siGFP, and siBFP); 3D flow cytometry plot after transfection of Y-RNA.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported by National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT & Future

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Planning Pioneer Research Center Program (2014M3C1A3054153), GiRC Program (2012K1A1A2A01056092), Basic Science Research Program (2015R1A1A1A05027352). Authors express special thanks to Prof. Daniel G. Anderson (MIT) and Dr. Kevin Kauffman for providing lipidoid materials and helping animal study.

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