Positional Effect of Chemical Modifications on Short Interference RNA

RNA interference (RNAi), the process of depleting RNA target by use of double-stranded RNA (dsRNA), has emerged as a powerful tool for gene regulation...
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J. Med. Chem. 2005, 48, 4247-4253

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Positional Effect of Chemical Modifications on Short Interference RNA Activity in Mammalian Cells Thazha P. Prakash,* Charles R. Allerson, Prasad Dande, Timothy A. Vickers, Namir Sioufi, Russell Jarres, Brenda F. Baker, Eric E. Swayze, Richard H. Griffey, and Balkrishen Bhat Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, California 92008 Received January 16, 2005

A systematic study on the effect of 2′-sugar modifications (2′-F (2′-F-2′-deoxy-nucleoside residues), 2′-O-Me (2′-O-methyl-nucleoside residues), and 2′-O-MOE [2′-O-(2-methoxyethyl)]nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5′-end of the antisense strand were less active relative to the 3′-modified ones. The 2′-F sugar was generally well-tolerated on the antisense strand, whereas the 2′-OMe showed significant shift in activity depending on the position of modification. The 2′-OMOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2′-OMe and 2′-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications. Introduction RNA interference (RNAi), the process of depleting RNA target by use of double-stranded RNA (dsRNA), has emerged as a powerful tool for gene regulation.1 The prospect of using this technology for therapeutic applications became a possibility since the demonstration that synthetic 19-21 nt RNA duplexes (short interference RNA or siRNA) exhibited RNAi activity in mammalian cells.2-4 Several groups have demonstrated the efficacy of siRNA mediated inhibition of clinically relevant genes in vitro.5-9 Recently, in vivo activity of nuclease-resistant siRNA has also been reported.10 The mechanistic understanding of RNAi mediated gene silencing is emerging in conjunction with the accumulation of biochemical and genetic evidence.11-18 It has been reported that mammalian RNAi is shortlived compared to RNAi in other eukaryotes.19,20 In mammalian cells, siRNA mediated gene silencing persists effectively only for an average of ∼66 h before the siRNA is likely to dilute out over the course of several cell divisions.21 Since persistence of RNAi occurs for only a short period of time in mammalian cells, finding methods for increasing the duration of the RNAi mechanism in human cells will be important for exploiting RNAi technology for novel therapeutic applications.22 Additionally, short unmodified oligoribonucleotides are not likely to be drug candidates because of their unfavorable metabolic stability and pharmacokinetic properties. Developing chemically modified siRNA duplexes with improved metabolic stability and pharmacokinetic properties is required for therapeutic applications. A number of chemical modifications are known to stabilize oligonucleotides against metabolic degradation, * To whom correspondence should be addressed. Phone: 760-6032590. Fax: 760-603-4654. E-mail: [email protected].

alter duplex stability, and improve pharmacokinetic properties.23,24 Modified oligonuleotides have been essential for the development of antisense oligonucleotides and their advancement in the clinic.25,26 Any modifications that improve plasma stability and alter pharmacokinetic properties should be valuable for developing pharmacologically active siRNA duplexes. Chemical modifications may also be used as tools for probing mechanistic details of the RNAi pathway and the role of various proteins that make up the RNA-induced silencing complex (RISC).27-30 A number of modifications developed for RNase H dependent antisense oligonucleotide therapeutics have been introduced into siRNA constructs and evaluated for their effect on activity in cell culture.22,27,31-32 The most critical issue in developing the chemically modified siRNA is the placement of the modification in an siRNA duplex because chemical modifications have been known to affect its activity.22,27,31-32 A systematic study on the positional effect of these modifications in the sense and antisense strands of siRNA duplexes on activity has yet to be reported. Such a study is necessary to understand the H-bond, electronic, and steric effects of chemical modifications with respect to position in the sequence and the effect of activating the RNAi pathway for target reduction. We envisaged that this study would be a guide in designing siRNA duplexes with multiple chemical modifications that could in part improve plasma stability, target affinity, and pharmacokinetic properties over siRNA duplexes without compromising the intrinsic potency. A systematic scanning of blocks of chemical modifications, such as 2′-F (2′-F-2′-deoxy-nucleoside residues), 2′-O-Me (2′-O-methyl-nucleoside residues), and 2′-O-MOE [2′-O-(2-methoxyethyl)]-nucleoside residues), was carried out (Figure 1), and their effect on

10.1021/jm050044o CCC: $30.25 © 2005 American Chemical Society Published on Web 05/27/2005

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Figure 1. 2′-Modifications evaluated for position-dependent effects on siRNA activity in cell culture.

Figure 2. PTEN siRNA duplex used for the study: P, phosphate; As, antisense strand; S, sense strand.

siRNA mediated target reduction in mammalian cells is presented in this report. Results and Discussion Gene silencing via the RNAi pathway consists of multiple biochemical processes. The synthetic siRNA duplexes, once delivered into the cytoplasm, get loaded into a protein complex known as RISC, which uses an ATPdependent RNA-helicase to unwind the duplex.15,18,33-34 Subsequently, the antisense strand of the duplex siRNA guides the RISC to the target mRNA, where the RISCassociated endonuclease cleaves the target mRNA at a single site in the center, which results in the degradation of the mRNA.3,35 We envisaged that chemical modifications to the siRNA duplex would influence RISC loading and subsequent processes differently as a function of position. Recently it has been shown that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release.36 It has also been noted that bases at the 5′-end of the siRNA disproportionately contribute to the target RNA binding, whereas base pairs formed by the central region and the 3′-region provide a helical geometry required for catalysis.36 This warranted a systematic study on the positional effect of chemical modifications to design modified siRNA duplexes with improved activity. We evaluated the positional effect of three chemical modifications, 2′-F, 2′-O-Me, and 2′-O-MOE (Figure 1) on siRNA mediated target reduction in mammalian cells. Target Gene. Active unmodified siRNAs targeting the human PTEN mRNA were identified by screening a series of siRNA constructs targeting the gene.37 The site utilized in this study was selected in part because of 100% homology between mouse and human. The siRNA duplex 1:2 (As-S, Figure 2) inhibited the expression of the message (Figure 3) when transfected into HeLa cells. siRNA Design. By use of a blockmer approach, three ribonucleotide residues of the sense and antisense strand of the PTEN siRNA duplex were substituted with modified residues (2′-F, 2′-O-Me, 2′-O-MOE, Figures 3-9). In some cases, modified antisense strand phosphorothioates (PS) were used for the study. The PS linkage was chosen to improve the nuclease stability of the siRNA duplex.3,27,38 The 5′- end of the modified

Figure 3. Antisense strand SAR, showing activity of 2′-OMe modified siRNA duplex (PO/PO) in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined, boldface italics. Modified antisense strands (4-10) were duplexed with sense strand RNA phosphodiesters 2: As, antisense strand; S, sense strand; P, phosphate; PO, phosphodiesters; PS, phosphorothioates; A, 2′-O-methyladenosine; G, 2′O-methylguanosine; C, 2′-O-methylcytidine; U, 2′-O-methyluridine; UTC, untreated control.

antisense strand was phosphorylated chemically. This was done to minimize the effect of chemical modification on cellular kinases, which is one of the steps involved in gene silencing by siRNA duplexes.3 Synthesis of Modified siRNA. Oligonucleotides 1-42 (Figures 3-9) were synthesized on a solid-phase DNA/RNA synthesizer using the 2′-O-TBDMS RNA phosphoramidites according to the reported protocols.39 2′-F, 2′-O-Me and 2′-O-MOE phosphoramidites with exocyclic amino groups protected with benzoyl (Bz for A and C) or isobutyryl (ibu for G) protecting groups were used for the synthesis of the RNA chimera. 0.12 M solution of the phosphoramidites in anhydrous acetonitrile was used for the synthesis. Oxidation of the internucleosidic phosphite to the phosphate was carried out using tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with a 10 min oxidation time. 3-H-1,2-Benzodithiol-3-one 1,1-dioxide40 (the Beaucage reagent, 0.2 M solution in anhydrous acetonitrile) was used as the sulfur-transfer agent for the synthesis of oligoribonucleotide phosphorothioates. The stepwise coupling efficiency of all modified phosphoramidites was more than 97%. Oilgoribonucleotide-bearing solid supports were heated with aqueous ammonia/ethanol (3:1) solution at 55 °C for 6 h to deprotect the base labile protecting groups. It has been reported that oligonucleotides with 2′-F pyrimidine residues are sensitive to basic conditions.41,42 Using aqueous ammonia-ethanol solution and heating at 55 °C for 6 h, we did not observe differences in the yield of the modified oligonucleotides or the formation of side products as evident from the MS analysis of the crude products. The 2′-O-TBDMS group

siRNA Activity

Figure 4. Antisense strand SAR, showing activity of 2′-OMe modified siRNA duplex (PS/PO) in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined, boldface italics. Modified antisense strands (11-17) were duplexed with sense strand RNA phosphodiesters 2: As, antisense strand; S, sense strand; P, phosphate; PO, phosphodiesters; PS, phosphorothioates; A, 2′-O-methyladenosine; G, 2′-O-methylguanosine; C, 2′-O-methylcytidine; U, 2′-Omethyluridine; UTC, untreated control.

was removed using a mixture of triethylamine trihydrofluoride/1-methyl-2-pyrrolidinone/triethylamine mixture at 65 °C for 1.5 h.39 After deprotection, all the modified oligoribonucleotides were isolated by HPLC on a strong anion exchange column. Oligoribonucleotides were desalted to yield RNA 1-42. The oligoribonucleotides 1-42 were characterized by electrospray MS and their purity was confirmed by HPLC and capillary gel electrophoresis. Inhibition of PTEN Expression by Modified (Blockmer) siRNA Duplex in HeLa Cells. Antisense Strand SAR. The modified antisense RNA 4-31 were paired with sense strand RNA phosphodiester 2. The duplexes were complexed with cationic lipid, and HeLa cells were treated with these complexes. The expression levels of PTEN mRNA were determined by real time quantitative RT-PCR (Figures 3-6). The potency of the modified siRNA duplexes was compared to the unmodified siRNA duplexes 1:2 and PS/PO siRNA 3:2 (Figure 3). To evaluate the positional effect of 2′-O-Me modification in siRNA activity, we introduced 2′-O-Me modified residues (three residues tandem) from the 5′-end to the 3′-end of the antisense strand (4-17, Figures 3 and 4) of PTEN siRNA and monitored the mRNA expression (Figures 3 and 4). The antisense strands 4-10 were with phosphodiester (PO, Figure 3) backbone, whereas 11-17 were phosphorothioates (PS, Figure 4). In general we observed less inhibition of PTEN mRNA expression with PS/PO siRNA relative to PO/PO siRNA (Figures 3 and 4). The 2′-O-Me modified siRNAs exhibited positional preference in activity (Figures 3 and 4). The siRNA duplexes with three 2′-O-Me residues at the 3′-end of the antisense strand (10:2 and 17:2) were more

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Figure 5. Antisense strand SAR, showing activity of 2′-F modified siRNA duplex in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined, boldface italics. Modified antisense strands (18-24) were duplexed with sense strand RNA phosphodiesters 2: As, antisense strand; S, sense strand; P, phosphate; PO, phosphodiesters; PS, phosphorothioates; A, 2′-deoxy-2′-fluoroadenosine; G, 2′-deoxy-2′-fluoroguanosine; C, 2′-deoxy-2′-fluorocytidine; U, 2′-deoxy-2′-fluorouridine; UTC, untreated control.

active than the 5′-modified siRNAs (4:2 and 11:2). The 2′-O-Me modification was relatively more tolerated at the center of the antisense strand (7:2 and 14:2, Figures 3 and 4). Even though the PS/PO siRNAs generally were less active than the PO/PO siRNA, the relative positional preferences of 2′-O-Me modification were similar. The 2′-F modified siRNAs were less potent (Figure 5) relative to unmodified siRNA 1:2 and PS/PO siRNA 3:2, depending on the position of the modification in the antisense strand of the duplexes. The 2′-F modified nucleotide residues were relatively well-tolerated throughout the antisense strand (Figure 5). The 3′-end of the antisense strand was less sensitive (Figure 5) for modification than the 5′-end (Figure 5). The siRNA duplex with three 2′-O-Me modified residues at the 3′-end of the antisense strand (17:2, Figure 4) and the corresponding 2′-F siRNA duplex (24: 2, Figure 5) showed comparable activity. However, the siRNA duplex 11:2 with three 2′-O-Me residues at the 5′-end of the antisense strand (11, Figure 4) was less active than the corresponding 2′-F modified siRNA duplex 18:2 (Figure 5). The 2′-O-Me group is larger in size than the 2′-F substitution; hence, 2′-O-Me substitutions may alter recognition of the 5′-terminus to a greater degree than the 2′-F substitutions or may be attributed to differences in the loading of the modified siRNA duplex into RISC or in the subsequent helicase activity. Interestingly enough, the 2′-O-Me and 2′-F modified oligonucleotides 14:2 and 21:2 (Figures 4 and 5), where modifications were placed 10 residues upstream from the 3′-end of the antisene strand, exhibited similar activity. It has been reported that the ribonuclease component (Argonaute2) of RISC cleaves the target mRNA at the center of the region that is complementary

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Figure 6. Antisense strand SAR, showing activity of 2′-OMOE modified siRNA duplex in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined, boldface italics. Modified antisense strands (25-31) were duplexed with sense strand RNA phosphodiesters 2: As, antisense strand; S, sense strand; P, phosphate; PO, phosphodiesters; PS, phosphorothioates; A, 2′-O-(2-methoxyethyl)adenosine; G, 2′O-(2-methoxyethyl)guanosine; C, 2′-O-(2-methoxyethyl)-5-methylcytidine; U, 2′-O-(2-methoxyethyl)-5-methyluridine; UTC: untreated control.

to the guide RNA.3,43 The results presented in this report suggest that 2′-O-Me and 2′-F modified nucleoside residues at the cleavage site of the guide sequence do not interfere with the nucleolytic activity. The 2′-O-Me modified siRNA provides enhanced plasma stability relative to the unmodified siRNA.27 The 2′-F modified RNA also forms a more stable duplex, compared to unmodified RNA.27 The duplex RNA was found to be relatively more stable in serum compared to single strand RNA.27 These data suggest that the siRNA duplexes with low thermal stability would cause the duplex to dissociate, increasing the probability of the degradation of RNA. Thus, modifications that provide additional thermal stability would be valuable in developing siRNA therapeutics. To design siRNA constructs with improved thermal stability and serum stability, one could use 2′-O-Me and 2′-F modifications in a synergistic design. The results from studies with sterically less demanding modifications, 2′-F and 2′-O-Me, prompted us to evaluate the positional effect of 2′-O-MOE substitution on siRNA activity. This modification is known to provide both thermal and serum stability to oligonucleotides and was developed for RNase-H based antisense technology.23 The 2′-O-MOE modification is bulkier than the 2′-O-Me or 2′-F substituents and would provide further insights into the effect of steric interference on the loading of siRNA into RISC as well as on the helicase activity.

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Figure 7. Sense strand SAR, showing activity of 2′-O-MOE modified siRNA duplex in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined boldface italics. Chemically modified sense strands (32-35) were duplexed with antisense strand RNA phosphodiesters 1 and antisense strand RNA phosphorothioates 3: P, phosphate; As, antisense strand; S, sense strand; PO, phosphodiesters; PS, phosphorothioates; A, 2′-O-(2-methoxyethyl)adenosine; G, 2′-O-(2methoxyethyl)guanosine; UTC, untreated control.

The antisense RNA strands 25-31 (Figure 6) with three 2′-O-MOE modified nucleoside residues placed at different positions from the 5′- to the 3′-end of the sequence were synthesized. These strands were then duplexed with sense strand 2 and were evaluated for their effect on inhibition of message in HeLa cells (Figure 6). The potency of the 2′-O-MOE modified siRNA duplexes was relatively limited compared to the unmodified siRNA 1:2 (Figure 6). This modification was not tolerated either at the 5′-end (25:2, Figure 6) or at the 3′-end (31:2, Figure 6) of the antisense strand. However, the 2′-O-MOE modifications were tolerated at the middle of the sequence (28:2, Figure 6) compared to 3′ and 5′ ends of the antisense strand. It is worth noting that, like the 2′-F and 2′-O-Me substitutions, siRNA duplexes with 2′-O-MOE nucleoside residues placed in proximity to the endonuclease cleavage site seemed to be better tolerated than other designs. A comparison of the potency of the modified siRNA (Figures 3-6) and the size of the subtituent shows that the sterically bulky groups are not well-tolerated at the 5′-end of the antisense strand of the duplex. In contrast, the bulkier substituents were tolerated at the 3′-end of the antisense strand. There was, however, a size threshold of tolerance at the 3′-end. The 2′-O-Me was well-tolerated, whereas 2′-O-MOE was not tolerated at the 3′-end of the antisense strand. One of the striking observations that emerged from our study was that at the cleavage site most of the substituents were allowed with a potency rank order of 2′-F > 2′-O-Me > 2′-OMOE. Sense Strand SAR. We then evaluated the 2′-OMOE modification on the sense strand. Three modified RNAs (32-35, Figure 7) were designed and synthesized as described before. These sense strand oligonucleotides

siRNA Activity

Figure 8. Sense strand SAR, showing activity of 2′-O-Me modified siRNA duplex in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined boldface italics. Chemically modified sense stands (36-42) were duplexed with antisense strand RNA phosphodiesters 1: As, antisese strand; S, sense strand; P, phosphate; PO, phosphodiesters; A, 2′-Omethyladenosine; G, 2′-O-methylguanosine; C, 2′-O-methylcytidine; U, 2′-O-methyluridine; UTC, untreated control.

were duplexed with anstisense RNA phosphodiester 1 and phosphorohioate 3, and their ability to reduce the PTEN mRNA was evaluated. A similar SAR was conducted with 2′-O-Me modification as well (Figures 8 and 9). Surprisingly, siRNA duplexes, with 2′-O-MOE substitutions at the 3′-end (1:32, 3:32, Figure 7) and 5′end (1:35, 3:35, Figure 7) of the sense strand, demonstrated activity comparable to that of their respective control siRNA 1:2 and 3:2. These observations were in contrast to the effect we observed with 2′-O-MOE substitution on the antisense strand. The activity of siRNA duplexes with 2′-O-MOE modified antisense strands (25-31, Figure 6) was less than the control 1:2 and 3:2, respectively. The observed differences in activity of siRNA with 2′-O-MOE modification on the antisense and sense strands may suggest that 2′-O-MOE modification on the sense strand drives loading of the antisense strand to the RISC. The siRNA duplexes with 2′-O-Me substitution in the sense strand exhibited activity similar to that from the 2′-O-MOE modifications (Figures 8 and 9). The sense strands 36-42, with 2′-O-Me substitutions when paired with the unmodified antisense strand 1 or the PS antisense strand 3, exhibited relatively similar activity compared to the corresponding controls (1:2 and 3:2), and activity was not dependent on the position of the substitutions. The remarkable tolerance of 2′-O-MOE and 2′-O-Me substitution at the 3′- and 5′-end of the sense strand of siRNA suggests that the recognition of sense strand by helicases may not be rate-limiting during the unwinding

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Figure 9. Sense strand SAR, showing activity of 2′-O-Me modified siRNA duplex in reducing PTEN mRNA in HeLa cells. The modified residues are in underlined boldface italics. Chemically modified sense stands (36-42) were duplexed with antisense strand RNA phosphorothioates 3: As, antisene strand; S, sense strand; P, phosphate; PO, phosphodiesters; PS, phosphorothioates; A, 2′-O-methyladenosine; G, 2′-Omethylguanosine; C, 2′-O-methylcytidine; U, 2′-O-methyluridine; UTC, untreated control.

process. Instead, the 5′-end of the antisense strand may be the recognition site for the helicases, and as a result, bulkier substituents like 2′-O-MOE and 2′-O-Me were less tolerated at the 5′-end of the antisense strand or binding to PAZ domain is critical and 2′-O-MOE at 5′terminus blocks binding. Conclusions In conclusion, we evaluated the effect of the steric hindrance of the 2′-substituents and the effect of the position of these modifications on siRNA activity. Three 2′-modifications, 2′-F, 2′-O-Me, and 2′-O-MOE, with different characteristics were incorporated into the siRNA duplex, and a systematic correlation of the position of the substituents on the sequence and the activity was studied. The mammalian cells were transfected with modified siRNA duplexes, and the level of expression of the PTEN mRNA was measured. A strong positional preference of the modifications on activity of the siRNA was observed. On the antisense strand the 2′-F modification was tolerated regardless of position in the sequence, while more sterically bulkier substituents were better tolerated at the 3′-end of the sequence over the 5′-end. In contrast, the sense strand was less sensitive to bigger substituents such as the 2′-O-MOE analogue. These data may provide guidance to designing siRNA duplexes with chemical modifications to achieve improved activity.

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Experimental Section General Procedures. The RNA phosphoramidites with the 2′-O-(tert-butyldimethylsilyl) protecting group and reagents were procured from Glen Research Inc., VA. All the reagents and anhydrous solvents were purchased from Aldrich and used without further purification. Thin-layer chromatography was performed on precoated plates (silica gel 60 F254, EM Science, NJ) and visualized with UV light and spraying with a solution of p-anisaldehyde (6 mL), H2SO4 (8.3 mL), and CH3COOH (2.5 mL) in C2H5OH (227 mL) followed by charring. 1H NMR spectra were referenced using internal standard (CH3)4Si, and 31 P NMR spectra were referenced using external standard 85% H3PO4. Mass spectra were recorded by Mass Consortium, San Diego, CA, and the College of Chemistry, University of California, Berkeley, CA. Synthesis of Modified Nucleoside Phosphoramidites. The 5′-O-(4,4′-dimethoxytrityl)]-2′-deoxy-2′-fluoro-nucleoside3′-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidites and 5′-O(4,4′-dimethoxytrityl)]-2′-O-(2-methoxethyl)-nucleoside-3′-[(2cyanoethyl)-N,N-diisopropyl]phosphoramidites (ABz, U, CBz, and Gibu) were synthesized as reported.41,44-45 5′-O-(4,4′Dimethoxytrityl)]-2′-O-(methyl)-nucleoside-3′-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidites were procured from Glen Research Inc., VA. The (2′-O-tert-butyldimethylsilyl)ribonucleoside phosphoramidites (U, N4-acetyl C, N6-Pac-A, N2-iPr-PacG), ribonucleoside CPG (U, N6-Pac-A), and 2′-O-methylribonucleoside CPG (U, N6-Bz-A) were purchased from Glen Research, VA. Synthesis of Modified siRNAs. The standard phosphoramidites and solid supports were used for incorporation of A, U, G, and C residues. A 0.12 M solution of the amidites in anhydrous acetonitrile was used for the synthesis. Chemical phosphorylation reagent procured from Glen Research Inc., VA, was used to phosphorylate the 5′-terminus of modified oligonucleotides. The modified oligonucleotides were synthesized on functionalized controlled pore glass (CPG) on an automated solid-phase DNA synthesizer. The universal solid support was used for the synthesis of oligonucleotides bearing a terminal 2′-deoxy-2′-fluoro modification.46 Twelve equivalents of phosphoramidite solutions were delivered in two portions, each followed by a 6 min coupling wait time. All other steps in the protocol supplied by the manufacturer were used without modification. A 0.15 M solution of Beaucage reagent in acetonitrile was used as a sulfurizing agent with a 1 min wait time. A solution of tert-butyl hydroperoxide/acetonitrile/ water (10:87:3) was used to oxidize internucleosidic phosphite to phosphate. The stepwise coupling efficiencies were more than 97%. After completion of the synthesis, solid support was suspended in aqueous ammonium hydroxide (30 wt %)/ethanol (3: 1) and heated at 55 °C for 6 h to complete the removal of all protecting groups except the TBDMS group at the 2′position. The solid support was filtered, and the filtrate was concentrated to dryness. The residue obtained was resuspended in anhydrous triethylamine trihydrofluoride/triethylamine/1-methyl-2-pyrrolidinone solution (0.75 mL of a solution of 1 mL of triethylamine trihydofluoride, 750 µL of triethylamine, and 1.5 mL of 1-methyl-2-pyrrolidine to provide a 1.4 M HF concentration) and heated at 65 °C for 1.5 h to remove the TBDMS groups at the 2′-position.37 The reaction was quenched with 1.5 M ammonium bicarbonate (0.75 mL), and the mixture was loaded onto a Sephadex G-25 column (NAP Columns, Amersham Biosciences Inc.). The oligonucleotides were eluted with water, and the fractions containing the oligonucleotides were pooled together and purified by highperformance liquid chromatography (HPLC) on a strong anion exchange column (Mono Q, Pharmacia Biotech, 16/10, 20 mL, 10 µm, ionic capacity 0.27-0.37 mmol/mL, A ) 100 mM ammonium acetate, 30% aqueous acetonitrile, B ) 1.5 M NaBr in A, from 0% to 60% B in 40 min, flow rate of 1.5 mL min-1, λ ) 260 nm). Fractions containing full-length oligonucleotides were pooled together (assessed by CGE analysis >90%) and evaporated. The residue was dissolved in sterile water (0.3 mL), and absolute ethanol (1 mL) was added and cooled in dry ice (-78 °C) for 1 h, The precipitate formed was pelleted

Prakash et al. out by centrifugation (NYCentrifuge 5415C; Eppendorf, Westbury, NY) at 3000 rpm. The supernatant was decanted, and the pellet was redissolved in 10 M ammonium acetate (0.3 mL) solution. Ethanol (1 mL) was added and cooled to -78 °C for 1 h to get a precipitate, and the precipitate pelleted out in a centrifuge (NYCentrifuge 5415C; Eppendorf, Westbury, NY) at 3000 rpm for 15 min. The pellet was collected by decanting the supernatant. The pelleted oligonucleotides were redissolved in sterile water (0.3 mL) and were precipitated by adding ethanol (1 mL) and cooling the mixture at -78 °C for 1 h. The precipitate formed was pelleted out and collected as described above. The oligonucleotides were characterized by ES-MS, and purity was assessed by capillary gel electrophoresis and HPLC (Waters, C-18, 3.9 mm × 300 mm, A ) 100 mM triethylammonium acetate, pH 7, B ) acetonitrile, from 5% to 60% B in 40 min, flow rate 1.5 mL min-1, λ ) 260 nm). siRNA Preparation. The equivalent molar concentrations of the sense and antisense strands were mixed together and annealed by heating the mixture at 90 °C for 1 min and subsequently incubating at 37 °C for 5-6 h. The successful duplex formation was confirmed by capillary gel electrophoresis (CGE, Beckman, MDQ CE). The running buffer (tris-borate, Beckman gel buffer kit) without urea was used for the CGE analysis. The duplexes were loaded electrokinetically at 10 kV for 30 s and separated at 15 kV in 10 min. Cell Culture and Transfection of Cells. HeLa cells (American Type Tissue Culture Collection, Manassas, VA) were cultured in a culture flask in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, CA) and liquid (high glucose) supplemented with 10% heat inactivated fetal bovine serum (FBS). The cells were not allowed to exceed 75-80% confluency. Prior to treatment (24 h before treatment), the cells were detached from the flask using trypsin (Invitrogen, CA) and plated in 96-well plates at a density of 5000 cells/well. The cells were transfected with siRNAs complexed with 6 µg mL-1 Lipofectin (Invitrogen, CA) in serum-free Opti-MEM I reducedserum medium. The cells were incubated in the transfection medium for 4 h, and the transfection medium was removed from the cells and replaced with fresh DMEM and 10% fetal calf serum and incubated at 37 °C in 5% CO2 for 16 h. RNA Expression Analysis. Total RNA was harvested after 16 h using the RNeasy process from Qiagen (Valencia, CA) according to the manufacturer’s protocol. Gene expression was determined via real time quantitative RT-PCR on an ABI Prism 7900 system (Applied Biosystems, Foster City, CA) as described in the literature.47,48 The following primer probe set (Qiagen, Valencia, CA) was used: hu PTEN (accession no. U92436.1), forward primer 5′-AATGGCTAAGTGAAGATGACAATCAT, reverse primer 5′-TGCACATATCATTACACCAGTTCGT, and FAM/TAMRA probe 5′-TTGCAGCAATTCACTGTAAAGCTGGAAAGG. The total RNA for each well was measured using RiboGreen (Molecular Probes, Eugene, OR), and these values were used for sample-to-sample normalization.49

Acknowledgment. We thank Frank Bennett for his enthusiastic support of this research and Nancy Meskan for her assistance in preparing this manuscript. We are grateful to Dr. B. Ross for synthesizing 2′-modified nucleoside phosphoramidites and for helpful discussions and valuable comments. Supporting Information Available: ES-MS and CGE profiles of modified oligonucleotides and the Tm data of selected siRNA duplexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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