Amino-Modified and Lipid-Conjugated Dicer-Substrate siRNA

Article pubs.acs.org/bc

Amino-Modified and Lipid-Conjugated Dicer-Substrate siRNA Enhances RNAi Efficacy Takanori Kubo,*,† Yoshifumi Takei,‡ Keichiro Mihara,§ Kazuyoshi Yanagihara,† and Toshio Seyama† †

Laboratory of Molecular Cell Biology, Department of Life Science, Yasuda Women’s University Faculty of Pharmacy, Hiroshima, Japan ‡ Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan § Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan S Supporting Information *

ABSTRACT: The development of Dicer-substrate small interfering RNAs (DsiRNAs) has been pursued in recent years because these molecules exhibit a much more potent gene-silencing effect than 21-nucleotide (nt) siRNAs. In the present study, we designed eight different types of aminomodified DsiRNAs and a palmitic acid-conjugated DsiRNA expected to result in improved biological properties of siRNAs, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. The DsiRNAs were modified with an amine at the 5′- and/or 3′-end of the sense and/or antisense strand. Dicer enzyme cleaved most of the amino-modified DsiRNAs to lead to the release of 21-nt siRNA; some of them, however, were not or partly cleaved. All amino-modified DsiRNAs exhibited strong resistance against nuclease degradations. Among the amino-modified DsiRNAs, the DsiRNA modified with an amine restricted at the 3′-end of the sense strand showed the most enhanced gene-silencing effect and maintained its potent gene suppression after one week of cell transfection against Renilla luciferase activity. For further improvement, palmitic acid was conjugated to DsiRNA at the 3′-end of the sense strand (C16-DsiRNA) to facilitate the membrane permeability and potent gene-silencing activity. The C16-DsiRNA showed enhanced membrane permeability to HeLa cells. The C16-DsiRNA exhibited extremely high inhibition of Renilla luciferase activity.

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against nuclease degradation.11−20 Modifications at 2′-OH of the nucleotide of 21-nt siRNAs, such as 2′-O-methyl, 2′-fluoro, and locked nucleic acid (LNA), demonstrated a high nuclease stability.11−13 In addition, in order to increase the nuclease resistance of the siRNA, the phosphate backbone in siRNA was replaced with a phosphorothioate or boranophosphate backbone.14−16 A series of nucleobase-modified siRNA duplexes were reported to exhibit enhanced thermodynamic stability and gene-silencing activity.17−20 Direct conjugation of bioactive molecules, including polymers,21,22 peptides,23−25 and lipids,26−29 to siRNAs has been reported to improve the biological properties of siRNAs in vitro and in vivo. Among these conjugations, the covalent conjugation of cholesterol, bile acids, and long-chain fatty acids to siRNAs at the 3′-end of the sense strand were shown to mediate siRNA uptake in cells.26,27 These lipid-conjugated siRNAs interacted with lipoprotein particles, lipoprotein receptors, and transmembrane proteins, and they influenced the uptake behaviors of the siRNA.27 Although the

INTRODUCTION RNA interference (RNAi) technology, discovered by Fire et al. using long double-stranded RNA (dsRNA),1 has potent gene silencing in a sequence specific manner, and has attracted much attention for applications to biosciences and medicines.2−5 In the cytoplasm of mammalian cells, long dsRNAs are cleaved to small dsRNAs [called small interfering RNAs (siRNAs) consisting of 21-nucleotides (nt), having a phosphate at the 5′-end and a 2-nt overhang at the 3′-end] by Dicer.6,7 The 21-nt siRNA is bound to a protein complex called an RNA-induced silencing complex (RISC).8 The RISC cleaves the target mRNA at a sequence-specific position, guided by the antisense strand of the siRNA. The 21-nt siRNA has advantages over the long dsRNA in that the 21-nt siRNA can be easily prepared by chemical synthesis, easily handled during biological assays, and avoids interferon activation of the RNAi. Although chemically synthesized 21-nt siRNAs showed potent RNAi effects, they still have the problems of poor membrane permeability and nuclease resistance which limit their applicability to therapeutic use.9,10 A variety of chemically modified siRNAs have been developed, mostly with the goal of improving their resistance © 2012 American Chemical Society

Received: June 24, 2011 Revised: December 6, 2011 Published: January 12, 2012 164

dx.doi.org/10.1021/bc200333w | Bioconjugate Chem. 2012, 23, 164−173

Bioconjugate Chemistry

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chemical modifications and direct conjugations to the siRNAs, as described above, could solve some of the problems of RNAi, such as nuclease stability or cell permeability, most of them weaken the gene-silencing efficacy. The structurally accurate designed siRNAs showed enhanced gene-silencing efficacy and nuclease resistance compared with a normally designed 21-nt siRNA to the same target gene. Bluntended 27-nt dsRNAs, which are cleaved by Dicer to lead to the release of 21-nt siRNAs, exhibited a several-fold higher genesilencing effect than 21-nt siRNAs.30,31 Dicer-substrate siRNA (DsiRNA), which is an asymmetric duplex RNA composed of a 25-nt sense strand with a blunt-end at the 3′-end and a 27-nt antisense stand with a 2-nt overhang at the 3′-end (25D/27-nt), could be directly cleaved by Dicer, leading to the release of a single primary 21-nt siRNA product, and exhibited a potent gene-silencing effect.32−34 The asymmetric terminal structures of siRNA, which were composed of a 19-nt sense strand and 21-nt antisense strand with unilateral 2-nt 3′-overhangs on the antisense strand, and strand asymmetry siRNAs and shRNAs having a mismatch near the 5′-end of the antisense strand, exhibited enhanced RNAi activity with a reduced off-target effect.35−38 Although these structurally accurate designed siRNAs exhibited enhanced RNAi potency, they still have problems of poor membrane permeability and low resistance against nuclease degradation. In the present study, we designed eight different types of amino-modified DsiRNAs and palmitic acid conjugated DsiRNA (C16-DsiRNA) to improve the biological properties of DsiRNA, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. All DsiRNA modifications and conjugations were with respect to Dicer cleavage, stability in serum, cellular uptake, and RNAi efficacies. We showed that the C16-DsiRNA revealed the best potential for development into a new generation of RNAi molecules.

Figure 1. Design and structure of amino-modified DsiRNAs, and Dicer cleavage. (A) Sequence of 21-nt siRNA and DsiRNA molecule targeted to Renilla luciferase gene. Ribonucleotides are upper-case, and deoxyribonucleotides are bold lower-case. The letter ‘p’ represents 5′phosphate. Arrows are the expected Dicer cleavage sites. (B) Structures of unmodified (DsiRNA A) and amino-modified DsiRNAs (DsiRNA B−I). Eight different types of amino-modified DsiRNAs were designed. (C) Dicer-substrate of amino-modified DsiRNAs. The amino-modified DsiRNAs were reacted with Dicer for 12 h at 37 °C. The reaction products were electrophoresed on 20% PAGE and visualized by silver staining.

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EXPERIMENTAL PROCEDURES Design and Synthesis of 21-nt siRNA, DsiRNA, and Amino-Modified DsiRNAs. 21-nt siRNA and 25D/27-nt DsiRNA were designed to target the Renilla luciferase gene (Figure 1A). The 21-nt and 27-nt single-stranded RNAs (ssRNAs; antisense and sense strand), and 23-nt ssRNA plus 2nt DNA at 3′-end (sense strand), including amino-modifications at the 5′-end or 3′-end, were purchased from Integrated DNA Technologies Inc. (IDT, Coralville, IA). The molecular weights of all ssRNAs were confirmed by MALDI-TOF mass spectrometry (Ultraflex; Bruker Daltonics, Bremen, Germany) using saturated solutions of 2,4,6-trihydroxyacetophenone (Sigma-Aldrich, St. Louis, MO) in 50 mg/mL diammonium hydrogen citrate in 50% acetonitrile as a matrix.39 All MALDITOF mass spectrometry measurements were carried out in linear-negative mode. The concentrations of all ssRNAs were calculated using their absorbance at 260 nm as detected spectrophotometrically (V-670 spectrophotometer; Jasco, Tokyo, Japan). The sense and antisense strands of RNAs were annealed in buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl] to prepare dsRNAs following the manufacturer’s instructions. Briefly, each ssRNA was diluted using sterile RNase-free water to a final concentration of 50 μM, and then 20 μL of each ssRNA (sense and antisense) solution was combined with 10 μL of annealing buffer (5×). The solutions were incubated for 1 min at 90 °C and cooled slowly afterward to room temperature (the final concentration of the siRNA was 20 μM (20 pmol/μL)). The dsRNA formation was confirmed

by 20% polyacrylamide gel electrophoresis (PAGE; 30 mA, 70 min) and visualized by silver staining (DNA Silver Stain Kit; GE Healthcare, Piscataway, NJ). Synthesis of Palmitic Acid-Conjugated DsiRNA. Amino-modified ssRNA at the 3′-end (4 nmol in 20 μL water) was reacted with 40 nmol palmitic acid Nhydroxysuccinimide ester (Sigma-Aldrich) dissolved in 10 μL N,N-dimethylformamide (DMF; Sigma-Aldrich) containing 0.7 μL N,N-diisopropylethylamine (DIEA; Sigma-Aldrich) in a 100 μL isopropanol/water (1:1) mixture, for 12 h at room temperature. The ssRNA with palmitic acid conjugation at the 3′-end (C16-ssRNA) was purified by reversed-phase HPLC (RP-HPLC) using an ODS column (4.6 × 150 mm, 5 μm) under a linear gradient from 7% to 70% acetonitrile over 40 min in 20 mM triethylammonium acetate (TEAA) (pH 7.0). The molecular weight of the conjugate was confirmed by MALDI-TOF mass spectrometry (Ultraflex; Bruker Daltonics) as predicted under the same conditions above (calculated mass: 8320.3, found mass: 8319.2). The yield of the conjugate was spectrophotometrically calculated on the basis of absorbance at 260 nm wavelength. 165



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DsiRNAs at different concentrations (200, 100, 50, and 25 nM) without any transfection reagents were added to the cells and incubated for 48 h. To evaluate the long-term RNAi activity of DsiRNAs, HeLa cells were seeded at 1 × 104 cells/mL in 100 μL medium in each well of a 96-well multiplate and cultured under the same conditions as described above. Twelve hours later, 5 pmol of the DsiRNAs preincubated with 0.2 μL LF2000 were added to each well of a 96-well multiplate. Eight hours after the DsiRNAs were transfected, the culture medium was replaced with 100 μL fresh medium and the cells were cultured for 48, 96, and 168 h to assess RNAi. Forty-eight hours before RNAi analysis, the psiCHECK-2 vector was transfected using LF2000 by the same procedure as described above, and then the cells were washed three times with culture medium after 4 h of transfection. Gene Silencing of Renilla Luciferase. The efficacy of RNAi against Renilla luciferase was evaluated by the Dual-Glo Luciferase Assay System (Promega), which is designed to analyze both Renilla and Firef ly luciferase activities. To detect Firef ly luciferase activity as an intraplasmid control, 50 μL of Dual-Glo luciferase reagent-1 (beetle luciferin) was added to each well of a 96-well multiplate containing 100 μL culture medium including DsiRNA samples. The plates were incubated in the dark for 10 min at room temperature. Luminescence emitted from the Firef ly luciferase catalytic reaction was measured for 1 s for each well on a microplate reader (Wallac 1420 ARVO MX; Perkin-Elmer, Waltham, MA). To measure the Renilla luciferase activity and to quench the luminescence from the Firef ly luciferase catalytic reaction, 50 μL of Dual-Glo Stop and Glo reagent-2 (containing coelenterazine) were added to each well. The luminescence arising from the Renilla luciferase catalytic reaction was measured in the same way as described above for Firef ly luciferase activity, and normalized by the luminescence of Firefly luciferase activity in each well of the 96-well multiplates. The RNAi of DsiRNA, including aminomodifications and C16-conjugation, toward the Renilla luciferase was assessed as a percentage of the control (DsiRNA nontreated) sample. Intracellular Delivery of DsiRNAs. To prepare fluorescence (FAM)-labeled DsiRNA, antisense ssRNA was labeled with 5′-fluorescein phosphoramidite (6-FAM; Glen Research, Sterling, VA) at the 5′-end. The FAM-labeled antisense 27-nt ssRNA, which was prepared for Renilla luciferase, was annealed with palmitic acid-conjugated (3′-end) and unconjugated sense 23-nt ssRNA plus 2-nt DNA at the 3′-end, respectively, in an annealing buffer as in the protocols described above. To deliver the prepared DsiRNAs intracellularly, 200 pmol of DsiRNAs, including C16-conjugation labeled with FAM, were incubated with 2 μL LF2000 in 100 μL Opti-MEM diluted twice for 30 min at room temperature. Then, 100 μL of the mixture was added to 900 μL of culture medium supplemented with 10% heat-inactivated FBS (Invitrogen), 100 U/mL penicillin (Wako), and 100 μg/mL streptomycin (Wako) of HeLa cells (5 × 104 cells) and incubated for 6 h in the dark under a humidified atmosphere (5% CO2, 37 °C). The cells were washed several times with fresh medium, and an intracellularly incorporated amount of DsiRNAs labeled with FAM in cells were examined under a fluorescent confocal microscope (IX70; Olympus, Tokyo, Japan). Another approach was used to investigate the intracellular delivery of DsiRNA, including C16-conjugation labeled with FAM, by flow cytometry. The forward- and side-scatter parameters were adjusted to accommodate the inclusions of

To prepare the C16-DsiRNA, antisense 27-nt ssRNA, which was complementary to the mRNA strands of the Renilla luciferase gene and was synthesized independently, was annealed with the C16-ssRNAs described above in an annealing buffer according to the manufacturer’s protocol. The quality of the C16-DsiRNA was confirmed by PAGE with a 20% gel. in Vitro Cleavage of DsiRNAs by the Recombinant Dicer. DsiRNAs, including amino-modifications and C16conjugation (20 pmol), were mixed with 1 U of recombinant Dicer (Gene Therapy Systems Inc., San Diego, CA) in 10 μL of 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 2.5 mM MgCl2. The mixtures were incubated at 37 °C for 12 h and the reaction was stopped by adding 2 μL of the stop-solution (Gene Therapy Systems Inc.). The reaction products were detected and analyzed by PAGE (20% gel), silver staining, and LAS4000 imaging system (Fujifilm, Tokyo, Japan). Stability against Nuclease Degradation in CellCulture Medium. Ten microliters of DsiRNAs, including amino-modifications and C16-conjugation (200 pmol), were added to 90 μL of Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka, Japan) containing 10% heat-inactivated FBS (Invitrogen, La Jolla, CA). The samples were incubated for different time intervals (0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h) at 37 °C. Aliquots (10 μL) were taken from each sample. The samples were frozen in liquid nitrogen to stop the nuclease reaction and were kept at −20 °C. The RNA products of nuclease degradation were detected and analyzed as described the above section. Cell Cultures and Transfections. HeLa cells were cultured in DMEM (Wako) supplemented with 10% heatinactivated FBS (Invitrogen), 100 U/mL penicillin (Wako), and 100 μg/mL streptomycin (Wako). To evaluate the RNAi potency of DsiRNAs, including amino-modifications and C16conjugation, after short-term (48 h) and long-term (96 and 168 h) targeting the Renilla luciferase gene in either the presence or absence of Lipofectamine 2000 (LF2000; Invitrogen), we used psiCHECK-2 vector (Promega, Madison, WI) as a reporter gene, which contains both the Firef ly and Renilla luciferase genes, in HeLa cells. Renilla luciferase activity was used to estimate the RNAi efficacy. The second reporter, Firef ly luciferase, was used as a control. To evaluate the short-term RNAi activity of DsiRNAs, HeLa cells were seeded at 5 × 104 cells/mL in 100 μL medium in each well of a 96-well multiplate and cultured in a 100% humidified atmosphere (5% CO2, 37 °C). Twelve hours later, 0.02 μg of psiCHECK-2 vector was first incubated with 0.2 μL of LF2000 in 10 μL of Opti-MEM (Invitrogen) for 30 min according to the manufacturer’s protocol, and then 10 μL of the mixture was added to each well of a 96-well multiplate containing the cells in 90 μL fresh culture medium without antibiotics. To investigate RNAi in the presence of LF2000, the DsiRNAs at different concentrations (10, 5, 2, 1, 0.5, and 0.2 nM) were preincubated with LF2000 as described for the psiCHECK-2 vector. Four hours after the vectors were transfected, 10 μL of the preincubated mixtures of DsiRNAs with LF2000 were added to each well containing 90 μL of fresh culture medium. After another 8 h of incubation, the culture medium was replaced with 100 μL fresh medium and the cells were cultured for 48 h to assess RNAi. To investigate the RNAi effect in the absence of LF2000, first the psiCHECK-2 vector was transfected using LF2000 by the same procedure as described above, and then the cells were washed three times with culture medium after 4 h of vector transfection. The 166



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Figure 2. Stability of amino-modified DsiRNAs against nuclease degradation. Samples (21-nt siRNA and DsiRNA A-I) were incubated in culture medium containing 10% FBS at 37 °C for 0, 0.5, 1, 2, 4, 6, 8, 12, 24, or 48 h, and aliquots were analyzed on 20% PAGE. (A) Representative PAGE patterns of 21-nt siRNA and DsiRNAs, including amino-modifications, after treated with culture medium. (B) Survivals of 21-nt siRNA and DsiRNAs, including amino modifications, in culture medium. The mean and SD values from three independent experiments. ***, P < 0.001 at 48 h of incubation time.

approaching to the DsiRNA from 3′-overhang (2-nt) of the antisense strand might play a role in attaching the modifications at the 3′-blunt-end of the sense strand in the DsiRNA. Other reports have also described that the 2-nt 3′-overhang structure is important for recognition of the PAZ domain, which is one of the essential domain of Dicer, and consequently Dicer measures from the 3′-end of the dsRNA to be cut by its RNaseIII domain leading to release of 21-nt siRNA.40−43 Stability of Amino-Modified DsiRNAs in Serum. All amino-modified DsiRNAs showed strong stability in the cellculture medium containing 10% FBS, whereas the 21-nt siRNA was immediately degraded (Figure 2). Among them, the DsiRNAs with the 3′-end amino-modifications (DsiRNA B, C, and D) exhibited appreciably strong resistance against nuclease degradation in comparison with not only DsiRNA A, but also DsiRNAs modified with amine at 5′-end. The aminomodifications at the 3′-end might protect the DsiRNA against 3′-exonuclease degradation. The degradation of siRNAs in cellculture medium supplemented with FBS could be explained by the primary 3′-exonuclease and RNase A activities in serum. It is widely accepted that the hydrolysis of single-stranded oligonucleotides in plasma occurs exclusively by 3′- to 5′exonuclease,44 while the hydrolysis of dsRNA in serum occurs by RNase A.39 We previously described the hypothetical mechanism of the 21-nt siRNA degradation in serum confirmed by MALDI-TOF mass and PAGE analysis.45 The 2-nt overhangs of the 21-nt siRNA rapidly degraded to release a 19-nt dsRNA catalyzed by 3′-exonuclease, and then the 19-nt dsRNA was gradually degraded in a reaction catalyzed by RNase A. Thus, the acquisition of the exonuclease resistance is linked to the protection of the 3′-end in the siRNA. The other factors of the stability of dsRNAs are length and sequence. We previously demonstrated that the blunt-ended dsRNAs manifested a higher stability in comparison with the 21-nt siRNA.46−48 Therefore, our amino-modified DsiRNAs, especially those with 3′-end modifications, appeared to have strong stability against nuclease degradation. RNAi Efficacy of Amino-Modified DsiRNAs. The RNAi efficacies of 21-nt siRNA, DsiRNA A, and amino-modified DsiRNAs were evaluated using a luciferase gene reporter assay in the presence of LF2000 (Figure 3). Although the Renilla luciferase activity was reduced to two-thirds of the control value (nontreated cells) by the RNAi of 21-nt siRNA, the effect of gene-silencing using the DsiRNA A was intensified to a much

each of the dissociated cell lines with the aid of FAM as a marker. Five thousand cells were analyzed and no cell was excluded from the analysis. Data were collected and analyzed using Epics XL and XL System II software (Beckman Coulter, Fullerton, CA). Effects of Unaltered Serum on the Function of C16DsiRNA. The stability, RNAi efficacy, and membrane permeability of DsiRNA, including C16-conjugation, were investigated in the presence of unaltered serum (serum without heat-inactivation). The stabilities of these siRNAs in unaltered FBS were studied as described above. Finally, the RNAi efficacies and membrane permeabilities of the siRNAs in the presence of unaltered FBS were investigated using HeLa cells as described above.

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RESULTS AND DISCUSSION The Dicer Substrate of Amino-Modified DsiRNAs. We synthesized eight different types of DsiRNAs modified with amines at the terminal of the sense and/or antisense strand targeting the Renilla luciferase gene (Figure 1B: DsiRNA B−I). We investigated whether the amino-modified DsiRNAs were cleaved by Dicer leading to release of the 21-nt siRNAs (Figure 1C). Nonmodified DsiRNA (DsiRNA A) was cleaved to the 21-nt siRNA by Dicer. The DsiRNAs modified with amines at the 3′- or 5′-end of the sense and/or 5′-end of the antisense strand (DsiRNA B, E, F, G, and I) were substrates of Dicer leading to release of the 21-nt siRNA. In contrast, the DsiRNAs modified with amines at least at the 3′-end of the antisense strand (DsiRNA C and H) were partly cleaved. A DsiRNA modified with amines at the 3′-end of both the sense and antisense strands (DsiRNA D) was not cleaved. These results can help to elucidate how Dicer approaches the DsiRNA. It was realized from the results for DsiRNA D and I that the 3′-end site of the DsiRNA was essential for approaching to Dicer, because the DsiRNA I was cleaved to the 21-nt siRNA, whereas the DsiRNA D was not converted to any product after reaction with Dicer. Kim et al. also reported that the presence of the 3′end modification in long dsRNA blocked the Dicer processing.30 In addition, despite the finding that DsiRNA B was almost completely cleaved, the DsiRNA C was partly cleaved to produce the 21-nt siRNA under the same reaction conditions. Thus, the entrance of Dicer to the DsiRNA was, in part, obstructed by the amino-group at the 3′-end of the antisense strand on the DsiRNA. In other words, the Dicer 167



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that the RNAi potency was appreciably dependent on the behavior of Dicer-cleavage, even though the amino-modified DsiRNAs had a high level of stability in culture medium containing 10% FBS. The DsiRNA C and DsiRNA H also showed a weak gene-silencing efficacy. We performed an analysis of the long-term RNAi activity against Renilla luciferase gene using the 21-nt siRNA, DsiRNA A, and DsiRNA B, which were analyzed after 48, 96, and 168 h of cell transfection at a concentration of 50 nM (Figure 3B). All samples exhibited a high-level gene-silencing effect, inhibiting Renilla luciferase activity by about 80−90%, after 48 h of cell transfection. However, a significant difference in the genesilencing efficacy between 21-nt siRNA and DsiRNAs after 96 h cell transfection was verified. The 21-nt siRNA decreased the gene-silencing effect, which suppressed the Renilla luciferase activity of cells by about 60%; in contrast, the DsiRNA A and DsiDNA B still exhibited a high level gene-silencing effect that was almost the same as the RNAi activity after 48 h. After 168 h of transfection, although both DsiRNA A and DsiRNA B showed a slight decrease in inhibitory effect, they still maintained potent RNAi activity. In particular, the Renilla luciferase activity was reduced to one-fifth of the control value by the RNAi of the DsiRNA B that was almost the same as the RNAi activity after 48 h. In contrast, the 21-nt siRNA showed a remarkable loss of gene suppression after one week of cell transfection at the same concentration. Another report showed that the chemical modification pattern of DsiRNAs using 2′-Omethyl or 2′-fluoro had high stability in serum and a long-lived gene-silencing effect, but it showed a reduction in RNAi activity compared with nonmodified DsiRNA.46 Our results suggested that the amino-modified DsiRNAs at the 3′-end of sense strand had several advantages over the chemically modified DsiRNAs: (i) strong gene-silencing was exhibited; (ii) many kinds of functional molecules are acceptable for conjugation; (iii) synthesis can be performed at low cost. Therefore, we proposed that the modifications and conjugations to the DsiRNA ought to be confined to the 3′-end of the sense strand. Synthesis and Biological Properties of Lipid-Conjugated DsiRNA. High RNAi potency of lipid-conjugated siRNA molecules such as cholesterol and fatty acid has been reported both in vitro and in vivo.26−29 However, in the literature,26−29 the direct conjugation of lipids to the siRNAs has been limited by the conjugating position to the siRNA, mostly to the 3′-end using lipophile-bearing solid supports27 or to the 5′-end using phosphoramidite derivatives,28 which involves complicated synthesis. We previously reported a simple and efficient synthesis, and demonstrated the potent RNAi efficacy of lipid-conjugated 21-nt siRNAs.50 However, lipid-conjugated 21-nt siRNAs may, in part, obstruct certain functions of RISC. We developed the C16-DsiRNAs to overcome the problems of siRNAs. Because we previously demonstrated that C16 was most influenced by cellular uptake and the inhibitory effect of direct conjugation of 21-nt siRNA to functional molecules,50 conjugation of C16 to DsiRNA was restricted to the 3′-end of the sense strand. The C16-DsiRNA was expected to have a high membrane permeability and to be cleaved by Dicer to the 21-nt siRNA, leading to release of the C16. To prepare the ssRNA (sense strand) conjugated with C16 at the 3′-end (C16-ssRNA), amino-modified ssRNA at the 3′end was condensed with palmitic acid N-hydroxysuccinimide ester in solution (Figure 4A). The crude C16-ssRNA was purified by RP-HPLC, and the molecular weight of C16-ssRNA

Figure 3. Short-term (A) and long-term (B) RNAi activity of aminomodified DsiRNAs. (A) RNAi against Renilla luciferase activity in HeLa cells after 48 h of incubation with amino-modified DsiRNAs. psiCHECKTM-2 vector was transfected into HeLa cells by LF2000. Four hours after the vector transfection, amino-modified DsiRNA (0.2 nM) was transfected into the same cells by LF2000. The Renilla luciferase activity was measured 48 h after the vector transfection. The mean and SD values are from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (t test). (B) RNAi against Renilla luciferase activity in HeLa cells after one-week incubation with amino-modified DsiRNA. Amino-modified DsiRNA (50 nM) was transfected into HeLa cells by LF2000. After 8 h, the medium was changed and the cells were cultured within 2, 4, and 7 days in a humidified atmosphere (5% CO2, 37 °C). psiCHECKTM-2 vector was transfected into the same cell by LF2000, and the Renilla luciferase activity was detected 48 h after the vector transfection. The mean and SD values are from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (t test).

greater extent (by less than 10%) than the 21-nt siRNA value at a concentration of 0.2 nM after incubation for 48 h (Figure 3A). Almost all amino-modified DsiRNAs manifested strong RNAi efficacy at the same concentrations, but exhibited different RNAi potencies. The strongest gene-silencing effect was exhibited in DsiRNA B, which suppressed the Renilla luciferase activity of cells by about 80%. A slightly increased inhibitory effect was found in the DsiRNA E and DsiRNA G, but the other amino-modified DsiRNAs showed a decrease in RNAi potency compared with DsiRNA A. This suggested that the potent gene-silencing activity of DsiRNA B was attributable to its ability to serve as a substrate of Dicer (Figure 1B) and its strong resistance against nuclease degradation (Figure 2). On the other hand, DsiRNA D exhibited the weakest gene-silencing efficacy of all amino-modified DsiRNAs. The results suggest 168



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Figure 4. Synthesis and characterization of C16-DsiRNA. (A) Simple synthesis of C16-ssRNA for C16-DsiRNA. (B) HPLC profiles of ssRNA and C16-ssRNA. HPLC was performed using an ODS column (4.6 × 150 mm, 5 μm) under a linear gradient condition of acetonitrile while shifting the concentrations from 7% to 70% for 40 min in 20 mM TEAA (pH 7.0). (C) Molecular-weight confirmation by MALDI-TOF mass spectrometry. MALDI-TOF mass was carried out in linear-negative mode using a saturated solution of 2,4,6-trihydroxyacetophenone in 50 mg/mL diammonium hydrogen citrate in 50% acetonitrile as a matrix. (D) Sequence and summarized character data of C16-ssRNA.

LF2000, Renilla luciferase gene expression was dose-dependently suppressed in all samples (21-nt siRNA, DsiRNA A, and C16-DsiRNA) with high potency (Figure 6A). It is noteworthy that the RNAi efficacy of C16-DsiRNA was superior to that of 21-nt siRNA and DsiRNA A at below 1 nM concentrations. For development of siRNA-based gene-silencing therapy for clinical applications, establishment of sufficient delivery of the molecules to the target cells is one of the important challenges. Various types of delivering regents for siRNAs, such as lipid-like delivery materials,51 peptide-mediated carriers,52,53 proteinmediated carriers,54 and polyethylenimine (PEI)-based carriers,55 have been developed, but most of them were cytotoxic to the cells and were limited in terms of their in vivo application. A naked siRNA is most safe and convenient, but it cannot penetrate the cell membrane, and is rapidly degraded by nuclease. In fact, many papers have reported that naked siRNA exhibited no gene-silencing effect at a high concentration.22,23,56,57 Our data showed that the naked siRNA did not inhibit Renilla luciferase activity at any concentration even at concentrations as high as 200 nM in the absence of transfection reagent (Figure 6B). On the other hand, the cells treated with C16-DsiRNA showed a reduction in Renilla luciferase activity in a dose-dependent manner. Thus, the data suggested that C16DsiRNA was able to efficiently interact with the cellular membrane and penetrate cells affected by C16. The C16DsiRNA developed herein can be used for clinical application without cytotoxic transfection reagents. We investigated the RNAi efficacy of C16-DsiRNA and its effects on unaltered FBS using a Renilla luciferase reporter assay in HeLa cells. All of the samples (21-nt siRNA, DsiRNA A, and C16-DsiRNA) in the presence of unaltered serum exhibited decreased RNAi efficacy, compared with the values in heatinactivated serum (see Figure S2 in the Supporting Information). However, even in the unaltered serum, C16DsiRNA exhibited strong and remarkably high RNAi potency, compared with those of 21-nt siRNA and DsiRNA A, irrespective of the presence of LF2000. These results could

was confirmed by MALDI-TOF mass spectrometry. The RPHPLC retention time of C16-ssRNA was found to be slower than that of nonconjugated ssRNA under the purified condition (Figure 4B). The precise molecular weight of C16-ssRNA was determined by MALDI-TOF mass analysis (Figure 4C). The C16-ssRNA was annealed with the antisense 27-nt ssRNA, which was complementary to mRNA strands of the target Renilla luciferase, and was synthesized independently in order to make C16-DsiRNA (Figure 5A). The C16-DsiRNA was the substrate of Dicer cleaved to 21-nt siRNA, leading to release of the C16 (Figure 5B). Thus, we demonstrated that Dicer recognized and cleaved the C16-DsiRNA even though the C16 was covalently bound to the 3′-end of the sense strand. It is predicted that the C16-DsiRNA does not obstruct certain functions of RISC linked to potent RNAi activity. The nuclease stability of C16-DsiRNA in culture medium containing 10% heat-inactivated FBS was evaluated using PAGE analysis (Figure 5C1). The C16-DsiRNA exhibited higher resistance compared with not only 21-nt siRNA but also DsiRNA A (Figure 5C2). It was suggested that the pronounced nuclease resistance of C16-DsiRNA was conferred by properties of lipid-molecules. Another report also described that the lipid-siRNAs showed much greater survival compared to nonmodified siRNAs interacting with lipoprotein particles.27 The stability of C16-DsiRNA in culture medium containing 10% unaltered FBS was also investigated. All of the samples (21-nt siRNA, DsiRNA A, and C16-DsiRNA) in unaltered FBS were degraded faster than those in heat-inactivated FBS (see Figure S1 in the Supporting Information). The 21-nt siRNA showed an especially fast disappearance. In contrast, C16DsiRNA survived after 48 h of incubation. On the other hand, DsiRNA A was almost degraded after 48 h. RNAi Efficacy of Lipid-Conjugated DsiRNA. The RNAi efficacy of C16-DsiRNA was compared with that of 21-nt siRNA and DsiRNA A targeted to the Renilla luciferase in HeLa cells cultured in DMEM containing heat-inactivated FBS in either the presence or absence of LF2000. In the presence of 169



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Figure 5. Structure, Dicer cleavage, and serum stability of C16DsiRNA. (A) Structure of C16-DsiRNA. The C16 was covalently attached to DsiRNA at the 3′-sense end. (B) Dicer-substrate C16DsiRNA. The C16-DsiRNA was reacted with Dicer for 12 h at 37 °C. The reaction products were electrophoresed on 20% PAGE and visualized by silver staining. (C) Stability of C16-DsiRNA against nuclease degradation. Samples (21-nt siRNA, DsiRNA A, and C16DsiRNA) were incubated in culture medium containing 10% FBS at 37 °C for 0, 0.5, 1, 2, 4, 6, 8, 12, 24, or 48 h, and aliquots were analyzed on 20% PAGE. Representative PAGE patterns (C1) and survivals (C2) of 21-nt siRNA, DsiRNA A, and C16-DsiRNA after incubation with culture medium were shown. The mean and SD values are from three independent experiments. *, P < 0.05; and ***, P < 0.001 (t test) at 48 h of incubation time.

Figure 6. Gene-silencing efficacy of C16-DsiRNA targeted to Renilla luciferase in HeLa cells in either the presence (A) or absence (B) of LF2000. The controls were given only PBS (-). The luminescence of Renilla luciferase activity was normalized by the luminescence of Firefly luciferase activity. The mean and SD values are from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (t test).

open a new avenue for the application of C16-DsiRNA in in vivo treatments. Intracellular Delivery of C16-DsiRNA. The membrane permeability of DsiRNA A and C16-DsiRNA were investigated in HeLa cells cultured in DMEM containing heat-inactivated FBS in the presence of LF2000 using confocal microscopy and FACS analysis (Figure 7). The confocal microscopy images of the cells treated with the DsiRNAs are shown in Figure 7A. No fluorescence signals were detected in control cells (nontreated), whereas the cells treated with DsiRNAs labeled with FAM exhibited bright fluorescence. Among them, the cell treated with the C16-DsiRNA labeled with FAM showed an extremely high fluorescence intensity. The results of FACS analysis showed similar results as the microscope observations. In Figure 7B, the logarithm of the fluorescence intensity is shown on the horizontal axis, and the number of cells is shown on the longitudinal axis. The area of the histogram is separated into four parts (a−d) along with the fluorescence intensity. The histogram of the cells treated with C16-DsiRNA labeled with FAM exhibited a higher fluorescence intensity region (c and d) than the histogram of the cells treated with DsiRNA A. In contrast, the low fluorescence intensity area (a and b) was

Figure 7. Confocal microscopic images (A) and FACS analysis (B) of HeLa cells treated with DsiRNA A and C16-DsiRNA labeled with FAM, in the presence of LF2000. FL, fluorescent image; Merge, merged image of FL and Trans. In the FACS analysis, the logarithm of the fluorescence intensity is shown on the horizontal axis, and the number of cells is shown on the longitudinal axis. The area of the histogram is separated into four parts (a−d) along with the fluorescence intensity. 170



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decreased in the cells treated with C16-DsiRNA in comparison with the cells treated with DsiRNA A. Accordingly, the C16DsiRNA showed enhanced cell membrane permeability in culture cells, as predicted by the properties of palmitic acid. The membrane permeabilities of DsiRNA A and C16DsiRNA in unaltered FBS were also evaluated. The fluorescence intensity of the cells treated with the DsiRNA A and C16-DsiRNA in the presence of unaltered FBS was decreased, compared with that in heat-inactivated FBS via confocal microscopy and FACS analysis (see Figure S3 in the Supporting Information). It seemed from the results of nuclease stability (Figure 5C2 and Figure S1) that the numbers of DsiRNA molecules, including C16-conjugates, in the cells in the presence of unaltered FBS were less than those in the cells in the presence of heat-inactivated FBS. Therefore, in the confocal microscopy images, the surviving DsiRNAs, including C16-conjugates, in unaltered FBS appeared to be almost all in the cytoplasm, whereas the surviving DsiRNAs, including C16conjugates, in heat-inactivated FBS in both the nucleus and cytoplasm. Despite this, the cells treated with C16-DsiRNA revealed brighter fluorescence, compared with the cells treated with DsiRNA A. The FACS analysis also showed similar results. These results suggested the potential feasibility of C16-DsiRNA for therapeutic use.

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Article

ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-82-878-9473; Fax: +81-82-878-9540; E-mail: [email protected].

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CONCLUSION

We designed eight different types of amino-modified DsiRNAs to improve nuclease stability, membrane permeability, and RNAi potency. Almost all of the amino-modified DsiRNAs were cleaved by Dicer leading to release of the 21-nt siRNA, but some of them (DsiRNA C, D, and H) were partly or hardly cleaved by Dicer. We confirmed that the 5′-end of the antisense strand including the 2-nt overhang of DsiRNA is essential for the first contact of Dicer to the DsiRNA. Here, we focused on the properties of DsiRNA B, which was completely cleaved by Dicer, leading to release of 21-nt siRNA, and exhibited excellent RNAi potency with longevity of gene suppression. Thus, the DsiRNA B was superior to the other amino-modified DsiRNA as an RNAi molecule. In addition, the DsiRNA B can be directly conjugated with many kinds of functional molecules through the amine at the 3′-end of the sense strand. For further improvement, we developed C16-DsiRNA. The C16 was conjugated to the 3′-end of the sense strand of DsiRNA. The C16-DsiRNA is cleaved by Dicer cleaved to 21nt siRNA, leading to release of lipid, and can avoid the reduction of RNAi potency induced by lipids. The C16DsiRNA exhibited efficient gene-silencing activity in HeLa cells cultured in DMEM containing either heat-inactivated FBS or unaltered FBS in the presence or absence of LF2000. In particular, finding a remarkable gene-silencing effect by C16DsiRNA in the absence of any transfection reagent was notable, since it suggested that C16-DsiRNA might be applicable to various biotechnologies, including in vivo applications. Moreover, the C16-DsiRNA had excellent cell membrane permeability. Our newly developed C16-DsiRNA was found to be an appropriate set of next-generation RNAi molecules to facilitate the intracellular delivery of siRNA, to enhance RNAi potency, and potentially to achieve gene silencing in vivo. 171



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Amino-Modified and Lipid-Conjugated Dicer-Substrate siRNA

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