Cellular siRNA Delivery Mediated by a Cell-Permeant RNA-Binding

RNA interference (RNAi), discovered in Caenorhabditis elegans, is a phenomenon in which gene expression is down-regulated by a double-stranded RNA ...
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Bioconjugate Chem. 2008, 19, 1017–1024

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Cellular siRNA Delivery Mediated by a Cell-Permeant RNA-Binding Protein and Photoinduced RNA Interference Tamaki Endoh, Masahiko Sisido, and Takashi Ohtsuki* Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan. Received January 14, 2008; Revised Manuscript Received March 21, 2008

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5′-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

INTRODUCTION RNA interference (RNAi), discovered in Caenorhabditis elegans, is a phenomenon in which gene expression is downregulated by a double-stranded RNA containing a sequence homologous to a specific gene (1). In mammals, the introduction of short double-stranded RNAs, termed small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), into the cytoplasm can silence genes (2–4). RNAi-mediated gene silencing is now an essential method for analyzing protein function and an attractive therapeutic tool for interfering with the expression of undesirable genes such as oncogenes and viral genes. Experimental techniques to introduce siRNAs or shRNAs into mammalian cells usually involve either direct delivery of RNA or introduction of a gene encoding an shRNA (5). In therapeutic use, the latter strategy could cause undesirable gene recombination. Thus, an efficient and safe method for direct RNA delivery is essential for RNAi therapy. Recently, cell-penetrating peptides (CPPs), such as partial sequences of the HIV-1 TAT protein and the Drosophila Antennapedia homeodomain, have been used to deliver fused or conjugated macromolecules into cells (6–9). CPPs enable the delivery of biologically active molecules into mammalian cells with high efficiency and low toxicity (10, 11). To deliver siRNAs, several groups have covalently linked CPPs to siRNAs by introducing an amino or thiol group at one terminus of the siRNA (12–14). However, siRNAs can be introduced into mammalian cells by noncovalent, electrostatic conjugation to CPPs (15–17), a method which is much easier than covalent linking. One possible drawback of the noncovalent conjugation strategy is that the carrier molecule has no sequence specificity for the RNA. A nonspecific carrier could internalize naturally occurring extracellular RNAs (18, 19) into cells, which may cause unexpected results. Thus, a cell-penetrating peptide or protein that noncovalently binds a specific RNA sequence is needed. The U1 small nuclear ribonucleoprotein A (U1A) has a relatively small RNA binding domain (RBD) of 98 amino acids at its N-terminus. The U1A RBD recognizes a short RNA * To whom correspondence should be addressed. Tel: 81-86-2518220; Fax: 81-86-251-8219; E-mail: [email protected].

sequence with high specificity and affinity (20, 21). In this study, we genetically fused the U1A RBD to the HIV-1 TAT peptide (-YGRKKRRQRRR-), a well-characterized CPP. As a cargo partner, we prepared siRNAs bearing a short sequence that binds to the U1A RBD (U1A RNA sequence) and examined the specific binding of the fused protein (TatU1A) to the cargo RNA and internalization of the complex. We also examined the photoinduced release of RNA from endosomes, since internalized TatU1A/RNA complexes were entrapped in the endosomes. We refer to this delivery system as CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photo-induced RNAi).

EXPERIMENTAL SECTION Plasmid Construction. Coding sequences for the TAT peptide and U1A RBD (amino acids 2 to 98) (22) (TatU1A) fusion construct were prepared by PCR ligation using two-step PCR. For the first PCR, the U1A RBD was amplified from human cDNA (Ambion Inc.) using the following primers: 5′-GGCAGTTCCCGAGACCC-3′ and 5′-GCGCTCGAGTTATTTCATCTTGGCAATGATA-3′ (3XhoU1A). Doublestranded DNA encoding the Tat peptide was prepared by primer extension using the following primers: 5′-CATCATCACAGCAGCGGCTACGGCCGCAAGAAACGCCGCCAGCGCCGTCGCGGCTACCCA-3′ and 5′-CGGGTCTCGGGAACTGCGCCGCTAGCGTAATCCGGAACATCGTATGGGTAGCCGCGACGGC-3′. For the second PCR, the first PCR product and primer extension product were ligated by PCR using 5′GGCGCCATGGGCAGCCATCATCATCATCATCACAGCAGCGGCTACGGC-3′ and 3XhoU1A as primers. The coding sequence of TatU1A was cloned into the Nco I-Xho I site of pET-28b to construct the expression vector pET-TatU1A. Expression vectors for U1A RBD only [U1A(-Tat)] and TatU1A bearing a C-terminal Cys (TatU1A-C) were constructed from pET-TatU1A using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Mutagenesis was performed using two sets of primers: 5′-CACAGCAGCGGCTACGGCTACCCATACGATGTTCC-3′ and 5′-GGAACATCGTATGGGTAGCCGTAGCCGCTGCTGTG-3′ for preparation of the U1A(-Tat) expression vector, and 5′-CAGATATCATTGCCAAGATGAAAGGATCCTGCTAACTCGAGCACCACC-3′ and 5′-GGTGGTGCTCGAGTTAGCAGGATCCTTTCATCTTGG-

10.1021/bc800020n CCC: $40.75  2008 American Chemical Society Published on Web 04/29/2008

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CAATGATATCTG-3′ for preparation of the TatU1A-C expression vector. Underlined regions are inserted sequences encoding Gly-Ser-Cys residues just upstream of the stop codon. The coding sequence for destabilized EGFP (dEGFP) (23) was prepared by PCR ligation. For the first PCR, EGFP was amplified from pEGFP-N3 (Clontech) using the following primers: 5′CGCGCTAGCACCATGGTGAGCAAGGGCGAG-3′ (5pcDNAeGFP) and 5′-CTTGTACAGCTCGTCCATG-3′. Double-stranded DNA encoding the mutated degradation domain of mouse ornithine decarboxylase was prepared by primer extension using the following primers: 5′-CATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTC-3′ and 5′-CACATTGATCCTAGCAGAAGCACAGGCTGCAGGGTGACGGTCCATCCCGCTCTCCTGGGCACAAGACATGGGCAGCGTGCC-3′. For the second PCR, the first PCR product and primer extension product were ligated by PCR using the 5pcDNAeGFP and 5′-CGCCTCGAGTTACACATTGATCCTAGCAGAAG-3′ primers. The coding sequence of dEGFP was cloned into the Nhe I-Xho I site of pcDNA5/FRT (Invitrogen) to construct the expression vector pcDNA/FRTdEGFP. All coding sequences were confirmed by sequencing analysis using an ABI PRISM 310 genetic analyzer. Preparation and Fluorescent Labeling of Proteins. E. coli strain BL21 (DE3) was transformed with pET-28b-derived expression vectors harboring TatU1A, U1A(-Tat), and TatU1A-C with N-terminal His-tags. The transformed cells were cultured, harvested by centrifugation, and then lysed by sonication and fractionated as described (24) with slight modifications. Buffer A [50 mM HEPES-KOH (pH 7.5), 150 mM (NH4)2SO4, 7 mM MgCl2, 20% glycerol, 7 mM β-mercaptoethanol, and 100 µM phenylmethylsulfonyl fluoride] was used to resuspend the cells, Buffer B [50 mM HEPES-KOH (pH 7.5), 1 M NH4Cl, 40 mM imidazole, 20% glycerol, and 5 mM β-mercaptoethanol] was used to wash the Ni-NTA column, and Buffer C [50 mM HEPES-KOH (pH 7.5), 100 mM (NH4)2SO4, 150 mM imidazole, 20% glycerol, and 5 mM β-mercaptoethanol] was used to elute the His-tagged protein. Purified proteins were then dialyzed against T buffer [20 mM HEPES-KOH (pH 7.4), 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 13.8 mM glucose] using Slide-A-Lyzer (Pierce). To label TatU1A-C with Alexa Fluor 546, we mixed eluted TatU1A-C with 100 µM Alexa Fluor 546 C5 maleimide (Molecular Probes) in Buffer C and incubated the mixture at room temperature for 2 h. The Alexa-modified TatU1A-C (TatU1A-Alexa) was purified in a Centri-Sep spin column (Princeton Separations) equilibrated with T buffer. Concentrations of all recombinant proteins were quantified using a Protein Assay Kit (Bio-Rad). Preparation and Fluorescent Labeling of RNAs. All RNAs used in this study were purchased from Japan Bio Services Co., Ltd. In order to construct an siRNA-type cargo RNA, antisense and U1A-fused sense strands were dissolved in distilled water at final concentrations of 20 µM, and were then annealed by incubation at 90 °C for 1 min followed by a 60 min incubation at 37 °C. Hybridization was confirmed by polyacrylamide gel mobility shift assays. shRNA-type cargo RNA was annealed by incubation at 90 °C for 1 min followed by slow cooling (1 °C/min) to 30 °C to enhance intramolecular annealing. RNAs that targeted dEGFP mRNA (the coding region 274-292 relative to the first nucleotide of the start codon), luciferase mRNA (1188-1206), and epidermal growth factor receptor (EGFR) mRNA (2121-2139) are referred to as siGFP (or shGFP), siLuc (or shLuc), and shEGFR, respectively. RNA was labeled with Cy3 or fluorescein using a LabelIT siRNA Tracker Intracellular Localization Kit (Mirus). To adjust

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Figure 1. Construction of cell-permeable TatU1A/U1AsiRNA complexes. (A) Sequences of the 5′-extended U1AsiRNA and standard siRNA. The U1A-binding sequence and following UCU linker sequence were added to the 5′ end of the sense strand (upper). (B) TatU1A specifically bound to the 5′-extended U1A binding sequence, forming the cell permeable TatU1A/U1AsiRNA complex.

Figure 2. Specific interaction between TatU1A and U1AsiRNA detected by gel mobility shift assays. Complex formation was initiated by mixing 500 nM siRNA with 1 µM of the indicated proteins, and 20 µL of the mixtures were loaded on the gel.

the RNA/fluorophore molar ratio of labeled siRNA and U1Afused siRNA, only the antisense strand was labeled and the labeled antisense strand was mixed with the sense strand or the U1A-fused sense strand before the annealing procedure. Annealing using the labeled strand was performed by incubation of 20 µM each of the sense and antisense RNAs at 65 °C for 3 min followed by a 60 min incubation at 37 °C. Gel Mobility Shift Assay. Purified proteins (1 µM) were mixed with 500 nM U1AsiRNA or standard siRNA in T buffer. Streptavidin was purchased from Amresco (US) and used as a negative control protein. After a 10 min incubation at 37 °C, protein-RNA mixtures were analyzed in 6% native polyacrylamide gels with Gel-Shift buffer (50 mM Tris-HCl [pH 6.8], 10 mM Mg(OAc)2, 65 mM NH4OAc, 1 mM EDTA) at 4 °C. The gel was stained with ethidium bromide to detect RNAs. Cell Culture and Construction of a Cell Line Expressing Destabilized EGFP. Chinese hamster ovary (CHO) cells (Invitrogen Flp-In-CHO cell line) were cultured at 37 °C under 5% CO2 in Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS). The human epidermoid carcinoma cell line A431 was kindly provided by Professor Seno (Okayama University) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Both media contained 100 units/mL penicillin and 100 µg/mL streptomycin. pcDNA/FRT-dEGFP was cotransfected with pOG-44 (Invitrogen) into Flp-In-CHO cells using the Effectene Transfection Reagent (Qiagen) and cultured in medium containing 500 µg/ mL hygromycin B in order to construct a cell line stably expressing dEGFP (dEGFP-CHO). RNA Delivery and Photoaccelerated Endosomal Escape. For RNA delivery experiments, a carrier protein (TatU1A or TatU1A-Alexa) and cargo RNA were mixed in 50 µL of

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Figure 3. Cy3-U1AsiRNA internalization into CHO cells mediated by TatU1A. Cellular images were obtained after incubation in the presence of TatU1A only (A), TatU1A and Cy3-U1AsiRNA (B), TatU1A and Cy3-siRNA (C), Cy3-U1AsiRNA only (D), U1A(-Tat) and Cy3-U1AsiRNA (E), or T-buffer (F). Concentrations of RNA and protein were 200 nM and 1 µM, respectively. The cells were treated with RNase A to remove extracellularly bound RNAs before imaging. Scale bars ) 50 µm. Fluorescence images were obtained with an excitation wavelength of 530-550 nm.

Figure 4. Photoinduced release of TatU1A-Alexa/Flc-U1AsiRNA from endocytic compartments. TatU1A-Alexa only (A), TatU1A-Alexa and FlcU1AsiRNA (B), or TatU1A-Alexa and Flc-siRNA (C) were mixed to induce complex formation, added to CHO cells, and then incubated as described in the Materials and Methods section. Images were obtained before a 10 s light stimulation at 540 ( 10 nm and at 2 min after photostimulation. The excitation wavelengths were 530-550 nm for Alexa Fluor 546 and 460-495 nm for fluorescein.

T buffer at the indicated concentrations, and incubated at 37 °C for 10 min. Cells grown to 70% confluence in 96-well culture plates were then overlaid with the mixture. After a 3 h incubation at 37 °C in T buffer, cells were treated twice with culture medium containing 34 µg/mL RNase A for a total of 1 h at 37 °C to remove RNAs adhering to the extracellular surface of the cells. Cells were then washed with

T buffer, and fluorescence images were obtained using a fluorescence microscope (IX51/IX2-FL-1/MP5Mc/OL-2, Olympus, Japan). In order to induce photoaccelerated endosomal escape of the internalized complex, cells were irradiated at 540 ( 10 nm for the indicated lengths of time with a 100 W halogen lamp (Olympus USH-1030L) passed through the 40× or 4× objective lens.

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at 37 °C. At 72 h after light stimulation, cells were incubated with the Alexa488/EGF complex (Molecular Probes) (5 µg/mL) in medium for 30 min at 37 °C. The cells were then washed with T buffer, and fluorescence images were obtained. Cell Viability Assay. In order to determine the effect of RNA delivery and light stimulation on cell viability, cell proliferation and cytotoxicity were evaluated using the Cell Counting Kit-8 (Dojindo, Japan) and a Cytotoxicity Detection Kit (LDH) (Roche, Switzerland), respectively. At 22 h after light stimulation, as described above, the culture medium was replaced with a medium containing 10 µL/well of cell counting kit substrate. The absorbance at 450 nm of each well was measured after a 2 h incubation at 37 °C. Simultaneously, an aliquot of medium was removed and mixed with the cytotoxicity detection substrate according to the manufacturer’s protocol, and the absorbance at 490 nm was measured. The absorbances at 450 and 490 nm were measured using a FLUOstar OPTIMA instrument, and the values were normalized by subtracting the values measured in wells containing no cells. In the case of the cell proliferation assay, the values for cells incubated with T buffer without carrier protein and cargo RNA were considered to represent 100% proliferation. In the case of the cytotoxicity assay, the values for cells that were incubated with medium containing 0.2% NP40 were considered to represent 100% cytotoxicity.

RESULTS AND DISCUSSION

Figure 5. Region-specific photostimulation of cells. TatU1A-Alexa and Flc-U1AsiRNA were mixed for complex formation, added to CHO cells, and incubated, as described in the Materials and Methods section. Images were obtained at 2 min after a 10 s light stimulation at 540 ( 10 nm. The excitation wavelengths were 530-550 nm for Alexa Fluor 546 and 460-495 nm for fluorescein. The irradiated area is to the right of the dotted line.

Induction of RNAi-Mediated EGFP Silencing. One day prior to RNA delivery, dEGFP-CHO cells were plated in a 96well culture plate at a density of 2 × 104 cells per well. U1Afused siRNA or shRNA was delivered into the cells using TatU1A-Alexa, and photoaccelerated endosomal escape was induced as described above. The cells were cultured at 37 °C. At 24 h after light stimulation, the culture medium was replaced with T buffer followed by two washes. The fluorescence intensity of adhering dEGFP-CHO cells was directly quantified by FLUOstar OPTIMA (BMG Labtech, Germany) using a 480 nm excitation and 540 nm emission filter set. The fluorescence intensity values were normalized by subtracting the values of the wells containing no cells. Cellular fluorescence images were subsequently obtained using a fluorescence microscope. Induction of RNAi-Mediated Epidermal Growth Factor Receptor (EGFR) Silencing. One day prior to RNA delivery, A431 cells were plated in a 96-well culture plate at a density of 1 × 104 cells per well. U1A-fused shRNA was delivered to the cells using TatU1A-Alexa and photoaccelerated endosomal escape was induced as described above. The cells were cultured

Specific Interaction Between TatU1A and U1AsiGFP. We hypothesized that TatU1A binding to a U1A-fused cargo RNA (Figure 1) would generate a cell-permeable complex that delivers the RNA to cells. We confirmed the specific interaction between TatU1A and U1AsiGFP (Figure 1A) using a gel mobility shift assay (Figure 2). Shifted bands were present in the U1AsiGFP and TatU1A or U1A(-Tat) preparations, but not in the control mixture of U1AsiGFP and streptavidin. An siGFP lacking the U1A sequence did not exhibit a mobility shift in the presence of the U1A protein (Figure 2). These results demonstrate a specific interaction between the U1A RBD and U1A RNA and the formation of a TatU1A/U1AsiGFP complex. When an excess of TatU1A was incubated with the U1AsiGFP (2.5 µM TatU1A and 500 nM U1AsiGFP), the U1A(-Tat)/U1AsiGFP complex was observed as a very broad, faint band and no free U1AsiRNA was detected (Supporting Information Figure 1). Furthermore, the siGFP band was also faint in the presence of excess TatU1A, but not in the presence of excess U1A(-Tat) (Supporting Information Figure 1). These results indicate that TatU1A selectively bound to U1AsiGFP, but that nonspecific RNA binding also occurred, most likely due to electrostatic interactions between the polycationic Tat-peptide and polyanionic RNA. siRNA Delivery into Mammalian Cells Using TatU1A as Carrier. Cy3-labeled U1AsiGFP (Cy3-U1AsiGFP) was prepared to examine whether siRNAs can be delivered to cells after forming a specific complex with TatU1A. Using a gel mobility shift assay, we confirmed that the Cy3 label does not affect U1AsiGFP binding to TatU1A (data not shown). No Cy3 fluorescence was observed in CHO cells treated with U1AsiGFP/ TatU1A obtained by incubating 200 nM Cy3-U1AsiGFP with 200 nM or 400 nM TatU1A (Supporting Information Figure 2). A 5× molar excess of TatU1A (1 µM) in complex formation was required to generate detectable fluorescence in cells. As shown in Figure 3, strong fluorescence was observed in cells treated with TatU1A/Cy3-U1AsiGFP (Figure 3B), but no fluorescence was observed in cells treated with U1A(-Tat)/Cy3U1AsiGFP or Cy3-U1AsiGFP alone (Figure 3D,E). Since both TatU1A and U1A(-Tat) form a protein-U1AsiGFP complex, as shown in Figure 2, the results of Figure 3B,E indicate that cellular internalization of U1AsiGFP was mediated by the

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Figure 6. dEGFP silencing after siRNA redistribution mediated by light stimulation. U1AsiGFP (200 nM) was mixed with TatU1A-Alexa for complex formation, added to dEGFP-CHO cells, and incubated as described in the Materials and Methods section. The relative fluorescence intensity (A) and fluorescent images (B) were evaluated after a 24 h incubation following a 60 s light stimulation or no stimulation. Irradiation was performed at 540 ( 10 nm for 60 s through a 4x objective lens. In (A), the fluorescence intensity was normalized by cell proliferation, estimated using a Cell Counting Kit-8. The dEGFP intensity in (A) is the mean ( standard deviation of three independent measurements.

Tat-peptide. Unexpectedly, weak fluorescent signals were also observed in cells treated with TatU1A/Cy3-siGFP (Figure 3C), indicating that a small amount of Cy3-siGFP entered the cells, potentially as a result of nonspecific interactions between Cy3siGFP and TatU1A, as shown in Supporting Information Figure 1. Thus, U1AsiGFP was selectively delivered to cells by TatU1A, probably because of the synergistic effect of two types of RNA-protein interactions: specific interactions between the U1A sequence and the U1A RBD and nonspecific interactions between the Tat-peptide and RNAs. Release of siRNA from Endocytic Compartments by Photostimulation of TatU1A-Alexa. The internalized Cy3U1AsiGFP exhibited a punctuate cytoplasmic distribution (Figure 3B). This distribution indicated that Cy3-U1AsiGFP was localized in endocytic compartments, as demonstrated by colocalization with LysoTracker Green (Molecular Probe), which labels lysosomes and other types of acidic compartments (Supporting Information Figure 3). Several groups have previously reported that Tat-peptide-mediated cellular internalization occurs through an endocytotic pathway by macropinocytosis (25). The transfer of Tat-peptide-conjugated macromolecular cargos from endocytic compartments to the cytosol is thus a critical step in activating the cargo’s function in cells. Chloroquine and influenza-virus-derived hemagglutinin peptide (HA2) have been used to destabilize the endosomal membrane and enhance delivery of entrapped Tat-peptide-conjugated macromolecules to the cytosol (25–27). Recently, as an alternative strategy, two groups reported the efficient release of fluorescently labeled CPP or CPP-fused proteins from the endosome using photostimulation of the fluorophores (28, 29). This is a very attractive strategy because controlled light stimulation would enable region-specific delivery of cargo molecules. We prepared TatU1A-Alexa bearing an Alexa Fluor 546 fluorophore to confirm whether the release of TatU1A-Alexa from endosomes could be induced by light stimulation. Since TatU1A-C contains a Cys residue only at the C-terminus, Alexa Fluor 546 C5 maleimide reacts specifically with the C terminus. We confirmed that TatU1A-Alexa binds to U1A-fused cargo RNA using a gel mobility shift assay (data not shown). We also prepared fluorescein-modified siRNAs (Flc-U1AsiGFP and Flc-siGFP) to examine whether the entrapped siRNA could be released into the cytoplasm following TatU1A-Alexa-mediated internalization. Fluorescent signals were monitored after incubation of CHO cells with TatU1A-Alexa/Flc-U1AsiGFP complexes, TatU1A-Alexa/Flc-siGFP complexes, or TatU1A-Alexa alone (Figure 4). Prior to light stimulation, TatU1A-Alexa (red images) exhibited a punctuate distribution (Figures 4A-C)

similar to that of Cy3-U1AsiGFP (Figure 3B). The FlcU1AsiGFP signal (green) was very weak compared to the Cy3U1AsiGFP signal. The low pH in the endosomes diminishes the fluorescence, because fluorescein is pH-sensitive (30). Next, we irradiated the cells at 540 ( 10 nm for 10 s using a 40× objective lens. In this procedure, the light from a 100 W halogen lamp was reduced to 12% of the total output using a neutral-density filter (Olympus 32-ND12) to avoid damaging the fluorophores and cells. Following this photostimulation, TatU1A-Alexa was widespread throughout the cytosol (Figures 4A-C). A widespread fluorescein signal was also observed in cells which were incubated with TatU1A-Alexa/Flc-U1AsiGFP complexes (Figure 4B). The RNA released from endosomes by light stimulation was localized to the cytosol and nucleus as shown in Supporting Information Figure 4. The green fluorescence signal indicates that Flc-U1AsiGFP but not TatU1A-Alexa is released from the endosome, because green fluorescence was not observed in cells treated only with TatU1A-Alexa (Figure 4A). The fluorescein signal from the Flc-U1AsiGFP intensified after photostimulation (Figure 4B), reflecting the higher pH of the cytoplasm relative to the endosomes. In cells treated with TatU1A-Alexa/Flc-siGFP, the dispersed fluorescein signal was much weaker than in those treated with TatU1A-Alexa/FlcU1AsiGFP (Figure 4C), indicating that complex formation and uptake was enhanced in the latter. Figure 5 shows the regionspecific redistribution of Flc-U1AsiGFP. Green fluorescence in the cytoplasm was observed only in the irradiated area. These results indicate that U1AsiGFP was delivered to CHO cells by the TatU1A protein and released into the cytoplasm by photostimulation of the Alexa 546 fluorophore. Although the mechanism of the cytosolic redistribution of TatU1A-Alexa is not well understood, reactive oxygen species generated by photostimulation may damage the membranes of endosomal compartments (28, 29). Several groups have used photostimulation of photosensitizers to produce reactive singlet oxygen and to induce the cytosolic redistribution of macro-biomolecules entrapped in the endocytic compartment (31, 32). dEGFP Silencing Mediated by U1A-Fused Interfering RNA. In order to examine whether the redistributed cargo RNA can induce RNAi-mediated gene silencing, dEGFP-CHO cells were treated with complexes generated using 200 nM U1AsiGFP and various concentrations of TatU1A-Alexa (Figure 6). Cells in the wells of a 96-well plate were then irradiated at 540 ( 10 nm for 60 s through a 4× objective lens. For irradiation through the 4× objective lens, we removed the neutral-density filter and extended the irradiation time because the light intensity is considerably weaker than that transmitted through the 40×

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Figure 8. EGFR silencing in A431 cells. (A) Sequence of shRNAtype cargo RNA targeted EGFR. (B) A431 cells were incubated with a mixture of 2 µM TatU1A-Alexa and 200 nM cargo RNA. Cells were stained with an Alexa488/EGF complex (Molecular Probes) after 72 h of culture after a 60 s light stimulation through a 4× objective lens. Fluorescence images were obtained at an excitation wavelength of 460 to 495 nm.

Figure 7. dEGFP silencing using U1A-fused RNAs. (A) Sequences of U1A-fused cargo RNA variants. RNA delivery to dEGFP-CHO cells was performed using complexes formed from incubating 2 µM TatU1AAlexa and 200 nM cargo RNA. Cargo RNAs internalized with TatU1AAlexa were redistributed into the cytosol using an 80 s photostimulation at 540 ( 10 nm through a 4× objective lens. Fluorescence intensity (B) and cytotoxicity (C) were evaluated at 24 h post-photostimulation. In (B), fluorescence intensity was normalized by cell proliferation, estimated using a Cell Counting Kit-8. The values in B and C are the mean ( standard deviation of three independent measurements.

objective lens. The dEGFP intensity was unchanged in cells treated with the TatU1A-Alexa/U1AsiGFP complex, compared to cells treated with T buffer only, but decreased following irradiation of TatU1A-Alexa/U1AsiGFP treated cells (Figure 6). We also confirmed the reduction in the mRNA level by RTPCR (data not shown). These results indicate that the photostimulation-induced redistribution of U1AsiGFP resulted in RNAi-mediated dEGFP silencing, and that the U1A-fused siGFP retains silencing activity. These results suggest that the internalized TatU1A-Alexa/U1AsiGFP complex could not mediate gene silencing without light stimulation due to endosome trapping. In the subsequent RNAi experiments, we used a TatU1A-Alexa concentration of 2 µM, to increase the level of gene silencing (Figure 6). We prepared various cargo RNA constructs (Figure 7A) in order to improve the RNAi efficiency. For siRNA type variants, the U1A sequence was linked to the 3′ terminus of the sense strand directly [siGFPU1A(no-linker)] or through a 9-mer linker [siGFPU1A(9-linker)]. For the shRNA-type variant, a minimum

U1A sequence was inserted in the loop region, which connects the sense and antisense strands (shGFPU1A). All the variants and standard siRNA (siGFP) were incubated with 2 µM TatU1A-Alexa for complex formation. Cells were treated with the complexes and siRNAs were redistributed using an 80 s light stimulation through a 4× objective lens. All the variants containing the U1A sequence induced dEGFP silencing, with the highest RNAi efficiency being induced by shGFPU1A (Figure 7B). The efficiency of dEGFP silencing by the standard siGFP was considerably weaker than the other variants, indicating that TatU1A-Alexa selectively delivers the U1A containing cargo RNAs. The shLucU1A, an shRNA-type cargo RNA targeted to the luciferase gene, did not change the dEGFP intensity in cells, suggesting that there were no nonspecific effects resulting from treatment with the RNA (Figure 7B). In order to compare the RNAi efficiency of this method with common transfection, cells were transfected with siGFP and siLuc using the TransIT-TKO transfection reagent (Mirus) according to the manufacturer’s protocol (Supporting Information Figure 5). Our method, using 200 nM U1AsiGFP, induced dEGFP silencing as effectively as TransIT-TKO mediated transfection with 10 nM siGFP (a concentration of approximately 10 nM is recommended in the manufacturer’s protocol) (Figure 7B and Supporting Information Figure 5A), and the cytotoxicity associated with our method was considerably lower than that observed with transfection (Figure 7C and Supporting Information Figure 5B). We estimated the effects of RNA concentration and photoirradiation time on dEGFP silencing (Supporting Information Figure 6). First, 2 µM TatU1A-Alexa was used to complex with various concentrations of shGFPU1A, and the cells were then incubated with the complexes and a 60 s light stimulation was then used to redistribute the RNA to the cytoplasm. On the basis of the results of this experiment, we used 200 nM RNA in the subsequent RNAi experiments. Cells were treated with complexes formed from 200 nM shGFPU1A and 2 µM TatU1AAlexa and the length of photostimulation was varied. Longer irradiation times induced more efficient dEGFP silencing, and the effect was saturated at irradiation times longer than 60 s (Supporting Information Figure 6B). When cells were irradiated

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Figure 9. Region-specific silencing of the dEGFP gene. CHO cells expressing dEGFP were incubated with TatU1A-Alexa only (A), TatU1A-Alexa and shGFPU1A (B), TatU1A-Alexa and shLucU1A (C), or TatU1A-Alexa and siGFP (D), and stimulated with light at 540 ( 10 nm as described in the Materials and Methods section. dEGFP fluorescence images of the photoirradiated area were obtained 20 h after photostimulation. In panel (B), a phase contrast image is shown below the fluorescence image. The irradiated area is to the left side of the dotted line.

for 120 s, cell proliferation was reduced to less than 80% (Supporting Information Figure 6C). Although prolonged irradiation damaged the cells, cellular damage was minimal with irradiation times of 60 s or less (Supporting Information Figure 6C), indicating that the silencing activity could be efficiently and safely regulated by photostimulation. EGFR Silencing Mediated by shEGFRU1A. We prepared an shRNA-type U1A-fused cargo RNA, shEGFRU1A (Figure 8A), to reduce endogenous EGFR expression. EGFR is overexpressed in many tumors, and is a convenient target for cancer therapy. We selected the A431 cell line as a model of EGFRoverexpressing carcinoma cells. A431 cells were treated with complexes formed from 2 µM TatU1A-Alexa and 200 nM shEGFRU1A, and then irradiated with light at 540 ( 10 nm for 60 s through a 4× objective lens. Photostimulation of the cells reduced the Alexa488 fluorescence, which is proportional to the amount of EGF bound to EGFR (Figure 8B). Neither the cells without light stimulation nor the cells incubated with TatU1A-Alexa and shLucU1A (nonspecific cargo RNA) reduced the fluorescent signal compared to control cells incubated with T buffer only. These results indicate that TatU1A-mediated RNA delivery and photoaccelerated endosomal escape was correlated with silencing of the endogeneous EGFR gene. Region-Specific Gene Silencing Mediated by CLIP-RNAi. shGFPU1A and control RNAs (shLucU1A and standard siGFP) were delivered to dEGFP-CHO cells using 2 µM TatU1A-Alexa for complex formation. TatU1A-Alexa only was used as a negative control. After a 4 h incubation, the cells were photostimulated for 5 s through the 40× objective lens, using a neutral-density filter to reduced the light intensity to 12%. Figure 9 shows the dEGFP fluorescence in the irradiated area at 20 h after photostimulation. The cells treated with TatU1A-Alexa and shGFPU1A exhibited a decrease in dEGFP only within the irradiated area (Figure 9B). In contrast, control cells treated with TatU1A-Alexa alone (Figure 9A), TatU1A-Alexa and shLucU1A (Figure 9C), or TatU1A-Alexa and siGFP (Figure 9D) did not induce a decrease in dEGFP irrespective of photostimulation. These results indicate that RNA is only released from endosomes in areas of photostimulation and that RNAi is thus restricted to the irradiated areas.

CONCLUSIONS We have demonstrated the efficient and selective delivery of an siRNA into mammalian cells mediated by TatU1A. Although

several studies have induced RNAi in mammalian cells using CPPs, the release of siRNAs from the endosome is a ratelimiting step in activating RNAi. We achieved the cytosolic redistribution of cargo RNA using TatU1A-Alexa and controlled light stimulation. The method demonstrated here is a simple strategy to deliver any shRNA or siRNA bearing a short extension with a specific recognition sequence. In addition, the light stimulation strategy (CLIP-RNAi) enables the implementation of regionally and temporally controlled RNAi.

ACKNOWLEDGMENT We thank H. Sakakoshi (Okayama Univ.) for his help with RNA purification, K. Kishimoto (Okayama Univ.) for his help with plasmid construction, Prof. M. Seno (Okayama Univ.) for the material, and Dr. J. Futami for Hoechst33342. This work was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO) (05A02707a). Supporting Information Available: Supplementary figures. This material is available free of charge via the Internet at http:// pubs.acs.org.

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