JULY/AUGUST 2007 Volume 18, Number 4 © Copyright 2007 by the American Chemical Society
COMMUNICATIONS Studies on Aminoisonucleoside Modified siRNAs: Stability and Silencing Activity Zong-Sheng Li,†,# Ren-Ping Qiao,†,# Quan Du,‡ Zhen-Jun Yang,*,† Liang-Ren Zhang,† Pei-Zhuo Zhang,§ Zi-Cai Liang,‡ and Li-He Zhang*,† State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100083, Institute of Molecular Medicine, Peking University, Beijing, 100871, and Shanghai GenePharma Co. Ltd., Z-J High Tech Park, Shanghai, 201203, China. Received December 26, 2006; Revised Manuscript Received May 11, 2007
A novel class of aminoisonucleoside was synthesized and incorporated into a luciferase gene-targeting siRNA. Structural and functional analyses of such a kind of siRNAs indicated that sense strand modifications with aminoisonucleoside at the 3′ or 5′ terminal, such as ssIso-1 and ssIso-2, have less effect on RNA duplex thermal and serum stabilities, and their functional activities are also comparable to their native siRNAs. In contrast, antisense strand modifications with aminoisonucleoside at the corresponding positions, such as asIso-2 or asIso-1, bring a striking negative effect on RNA duplex stability but still maintain around 40-50% of gene knockdown.
INTRODUCTION RNAi1 was first reported by A. Fire and co-workers in Caenorhabditis elegans (1) and represents a ubiquitous pathway of post-transcriptional gene silencing in nearly every eukaryote studied thus far. The real trigger of RNAi is a small doublestranded RNA (siRNA) that is ∼19 bases in length which * To whom correspondence should be addressed. E-mail: zdszlh@ bjmu.edu.cn,
[email protected]; Tel: +86- 10-82802503, Fax: +8610-82802503. † School of Pharmaceutical Sciences, Peking University. ‡ Institute of Molecular Medicine, Peking University. § Shanghai GenePharma Co. Ltd. # The authors contributed equally to this paper. 1 Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; dsRNA, double-stranded RNA; RISC, RNA-induced silencing complex; LNA, locked nucleic acid; DMF, N,N-dimethyl foramide; THF, tetrahydrofuran; DIPEA, diisopropylethylamine; TMSCl, chlorotrimethylsilane; DMT, 4,4-dimethoxytrityl chloride; T, thymidine; A, adenosine; U, uridine; EDTA, ethylenediaminetetraacetic acid, disodium salt; SVPDE, snake venom phosphodiesterase; FBS, fetal bovine serum; ATP, adenosine triphosphate.
assembles with RISC in the cytoplasm and initiates a complicated cascade for the ultimate degradation of sequencehomologous RNAs, The sequence-selective gene knockdown by siRNA and its rapid adoption as a powerful tool for gene regulation in mammalian cells has generated the expectation for its use to improve target therapeutics for cancer, metabolic, inflammatory, infectious, neurological, and other types of diseases (2). Although a variety of methods have been developed to generate siRNA, such as plasmid vectors for transient transfection and viral vectors for stable transduction (2), chemical synthesis of siRNAs represents a more attractive resource for clinical purpose and has great advantages in accommodating chemical modifications and dosing changes (3-6). However, like the hindrances in development of antisense technology, which is the previous generation of RNA-targeting small RNA molecules, siRNA-based therapeutics is also obstructed by its intrinsic qualities, such as poor intracellular uptake, limited blood stability, and nonspecific immune stimulation, and so forth.
10.1021/bc060398+ CCC: $37.00 © 2007 American Chemical Society Published on Web 06/01/2007
1018 Bioconjugate Chem., Vol. 18, No. 4, 2007
Li et al.
Table 1. Sequences of Native siRNA (ssNC/asNC) and Aminoisonucleoside-Modified Sense Strand siRNA (ssIso-1-ssIso-5) or Aminoisonucleoside-Modified Antisense Strand siRNA (asIso-1-asIso-5)a
a Top strand depicts the sense strand in the 5′-3′ direction (same as the target sequence). Bottom strand depicts the antisense strand in the 3′-5′ direction (complementary to the target sequence). A and T letters indicate the aminoisonucleotide.
Chemical modification has been extensively explored to improve antisense serum stability and pharmacokinetics so as to extend circulation in blood for a sufficiently long time (7-12). Many modified antisense oligos are being tested in clinical trials with fomivirsen (Vitravene; Isis) as the first oligo drug approved by the U.S. FDA for the treatment of eye cytomegalovirus infection. Currently, such chemical modifications are also being applied to synthetic siRNAs, and some modified siRNAs have been reported to be more potent and stable than their native siRNAs (6, 13-15). For example, phosphorothioate modification throughout the siRNA duplexes significantly enhanced gene-silencing activities (16, 17). Alternative modification, 2′-position methylation within the pentose sugar, was generally well tolerated with siRNA silencing activity being comparable with the native form. Modification on base moieties is relatively less attempted with N3-methyluridine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, and inosine being reported so far (3, 16). However, all three modifications resulted in a slight decrease of siRNA activity. Recently reported backbone modification, boranophosphate linkage, seems to be more attractive, since it makes siRNA 300-fold more active than the native form and 2-fold more stable than the classic phosphorothioate (18). Several of the siRNA reagents have already made their way into clinical trials, one with initially positive indications reported (19). Isonucleosides represent a novel class of carbohydratemodified nucleoside in which the nucleobase is linked to various positions of ribose other than C-1′, and some of these nucleosides have shown interesting biological activities. In our previous reports for synthesis and incorporation of isonucleosides into antisense oligos, we found that modified antisense oligos not only possess strong nuclease resistance but more interestingly become good substrates of RNase H (20). CD spectra and molecular modeling demonstrated that isonucleoside-modified DNA duplexes still adopt a B-like conformation, but the formation of Watson-Crick hydrogen bonding was perturbed by the torsion of the backbone (21, 22), while isonucleosidemodified oligonucleotide/RNA duplex formed an A-like conformation in solution (23). An introduction of an amino group into the isonucleoside may increase the thermal stability of the isonucleoside-modified oligonucleotide with its complementary sequence. Here, we report the chemical synthesis and incorporation of aminoisonucleosides into the siRNA duplex and analyze the effect of such a modification at different positions on siRNA structural stability and functional activity.
Members of the wee1 kinase family control the timing of cell cycle events by acting as a negative regulator of entry into mitosis (G2 to M transition) by protecting the nucleus from cytoplasmically activated cyclin B1-complexed CDC2 before the onset of mitosis. These kinases have been evaluated as promising anticancer target genes. siRNA is used in the current experiment as designed against the 3′ end of the coding sequence of the wee1 mRNA (Table 1) (24).
MATERIALS AND METHODS General. All solvents were dried and distilled prior to use. Chemical reagents were purchased from Acros and Sigma Co. Thin layer chromatography was performed on silica gel GF254 (Qing-Dao Chemical Co., China) plates with detection by UV or by heating. Silica gel (200-300 mesh; Qing-Dao Chemical Co.) was used for short column chromatography. NMR spectra were recorded on a Varian VXR-300, Varian Inova-500 instrument. 1H NMR spectra were referenced using internal standard TMS and 31P NMR spectra using external standard 85% H3PO4. Mass spectra (ESI-TOF+ MS) and highresolution mass spectra (ESI-TOF+ HRMS) were obtained at MDS SCIEX QSTAR and Bruker DALTONICS APEX IV 70e instruments. MALDI-TOF mass spectra for oligonucleotides were obtained at SIMADZU AXIMA CFR plus, and the data are reported in m/e (intensity to 100%). Optical rotations were recorded on Perkin-Elmer 243B Polarimeter. 4-Deoxy-4-(thymin1-yl)-2,5-anhydro-L-mannofuranose dimethylacetal (1) and 4-deoxy-4-(adenine-9-yl)-2,5-anhydro-L-mannofuranose dimethylacetal (7) were synthesized according to Yang et al. (22) (Scheme 1). Synthesis. 4,6-Dideoxy-4-(thymin-1-yl)-6-azido-2,5-anhydroL-mannofuranose dimethyl acetal (2). To a mixture of compound 1 (4.41 g, 13.95 mmol), triphenylphosphine (4.02 g, 15.35 mmol), and sodium azide (5.00 g, 76.92 mmol) in dry DMF (100 mL) was added carbon tetrabromide (4.90 g, 14.80 mmol) at room temperature. The mixture was stirred at room temperature for 48 h, and then methanol was added until the mixture turned into clear solution. Stirring was continued for another hour. The solvent was removed under vacuum, and the residue was purified by a short-column chromatography using a gradient of MeOH in CH2Cl2 (2-2.5%) to yield 2 (3.57 g, 75%) as white foam. UV (CH3OH): λmax ) 270.5 ( ) 8804). [R]20 D ) -34.50 (C 0.004 g/mL, CH3OH). 1H NMR (DMSO-d6, 500 MHz): δ 1.79 (s, 3H, 5-CH3), 3.29-3.54 (m, 2H, H-6′), 3.34 (s, 3H, 1′-OCH3), 3.35 (s, 3H, 1′-OCH3), 3.81 (t, J2′,1′ ) J2′,3′
Communications Scheme 1 . Synthesis of 6 and 13a
a Reagents and conditions: (i) Ph3P/CBr4/NaN3/DMF/rt; (ii) a. 1% HCl, reflux; b. 2N NaOH/NaBH4, rt; (iii) TMSCl/Py/BzCl/Py, rt; (iv) 10% Pd/C/H2 /EtOH, rt; (v) CF3COOC2H5/Et3N/CH3OH, rt; (vi) DMTrCl/Py, rt; (vii) 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite/ Et(Pri)2N/THF, rt.
) 5.5 Hz, 1H, H-2′), 4.16 (m, 1H, H-5′), 4.40 (m, 1H, H-3′), 4.49 (d, J1′,2′ ) 5.5 Hz, 1H, H-1′), 4.65 (dd, J ) 8 Hz, J ) 9 Hz, 1H, H-4′), 5.58 (d, J ) 5.5 Hz, 1H, 3′-OH), 7.54 (d, J ) 1 Hz, 1H, H-6), 11.30 (s, 1H, N-3). 13C NMR(DMSO-d6, 125.7 MHz): δ (5-CH3), 51.2 (C-6′), 53.8 (1′-OCH3), 54.3 (1′-OCH3), 64.0 (C-4′), 73.5 (C-3′), 77.0 (C-5′), 81.6 (C-2′), 103.9 (C-1′), 109.5 (C-5), 138.1 (C-6), 151.1 (C-4), 163.6 (C-2). HRMS (ESITOF+) calcd for C13H20N5O6 (M+ + H): 342.1408. Found: 342.1424. 4,6-Dideoxy-4-(thymin-1-yl)-6-azido-2,5-anhydro-lL-mannitol (3). Compound 2 (0.15 g, 0.44 mmol) was dissolved in 1% hydrochloric acid in 50% water/50% THF (10 mL) and refluxed overnight. After cooling to 0 °C and neutralization with 2 M NaOH, sodium borohydride (0.032 g, 0.86 mmol) was added. The solution was stirred at room temperature for 4 h, then neutralized with 1% HCl at 0 °C. The solvent was removed under vacuum, and the residue was dissolved in C2H5OH. After filtration, the mixture was purified by a short-column chromatography using a gradient of MeOH in CH2Cl2 (5%) to yield 3 (0.11 g, 84%) as white foam. UV (CH3OH): λmax ) 271 ( ) 1 8802). [R]20 D ) -48.25 (C 0.004 g/mL, CH3OH). H NMR (DMSO-d6, 500 MHz) δ 1.77 (s, 3H, 5-CH3), 3.32-3.63 (m, 2H, H-6′), 3.74-3.77 (m, 1H, H-2′), 4.09-4.12 (m, 1H, H-5′), 4.09-4.27 (m, 1H, H-3′), 4.71 (t, J ) 8.5 Hz, 1H, H-4′), 4.81 (t, J ) 5 Hz, 1′-OH), 5.57 (d, J ) 5.5 Hz, 1H, 3′-OH), 7.56 (s, 1H, H-6), 11.27 (s, 1H, N-3). 13C NMR (DMSO-d6, 125.7 MHz): δ 12.0 (5-CH3), 51.8 (C-6′), 61.4 (C-1′), 63.1 (C-4′), 72.4 (C-3′), 77.0 (C-5′), 83.0 (C-2′), 109.5 (C-5), 137.8 (C-6), 151.2 (C-2), 163.7 (C-4). HRMS (ESI-TOF+) calcd for C11H16N5O5 (M+ + H): 298.1145; Found: 298.1150. 4,6-Dideoxy-4-(thymin-1-yl)-6-trifluoroacetylamino-2,5-anhydro-L-mannitol (4). To a solution of compound 3 (0.37 g, 1.25 mmol) in 10 mL anhydrous methanol was added 10% Pd/C (0.18 g), then the mixture was reduced by hydrogen (4 kg/cm2) for 16 h. After filtration, the solvent was removed under vacuum and the residue was dissolved in 25 mL methanol; triethylamine (0.96 mL, 6.90 mmol) and ethyl trifluoroacetate (0.5 mL, 4.14 mmol) were added, and the mixture was reacted overnight at room temperature. The solvent was removed under vacuum and the residue was purified by a short-column chromatography using a gradient of MeOH in CH2Cl2 (7%) to yield 4 (0.39 g, 85%) as white foam. UV (CH3OH): λmax ) 271 ( ) 7965). 1 [R]20 D ) -3.00 (C 0.004 g/mL, CH3OH). H NMR(DMSO-d6, 500 MHz) δ 1.78 (s, 3H, 5-CH3), 3.32-3.64 (m, 4H, H-1′ and H-6′), 3.75-3.78 (m, 1H, H-2′), 4.13-4.17 (m, 1H, H-5′), 4.26-4.30 (m, 1H, H-3′), 4.53 (t, J ) 7.5 Hz, 1H, H-4′), 4.81 (t, J ) 5.0 Hz, 1′-OH), 5.59 (d, J ) 5.5 Hz, 1H, 3′-OH), 7.54 (d, J ) 1.0 Hz, 1H, H-6), 9.51 (t, J ) 5.0 Hz, 1H, 6′-NH-), 11.32 (s, 1H, N-3). 13C NMR (DMSO-d6, 125.7 MHz): δ 12.0
Bioconjugate Chem., Vol. 18, No. 4, 2007 1019
(5-CH3), 41.9 (C-6′), 61.1 (C-1′), 65.3 (C-4′), 72.9 (C-3′), 76.1 (C-5′), 83.1 (C-2′), 109.5 (C-5), 138.2 (C-6), 151.0 (C-2), 156.3 and 156.5 (CF3CO), 163.6 (C-4). Anal. Calcd. for C13H16F3N3O6 (367.1) C 42.51, H 4.39, N 11.44. Found: C 42.28, H 4.60, N 11.20. 1-O-Dimethoxytrityl-4,6-dideoxy-4-(thymin-1-yl)-6-trifluoroacetylamino-2,5-anhydro-L-mannitol (5). Compound 4 (0.41 g, 1.1 mmol) was dissolved in dry pyridine (10 mL), and dimethoxytrityl (DMT) chloride (0.38 g, 1.1 mmol) was added at 0 °C. The solution was stirred at room temperature for 23 h. After evaporation, the mixture was purified by a short-column chromatography using a gradient of MeOH in CH2Cl2 (1.5%, 1% Et3N added) to yield 5 (0.49 g, 66%) as white foam. 1H NMR (DMSO-d6, 500 MHz) δ 1.70 (s, 3H, 5-CH3), 3.08-3.47 (m, 4H, H-1′ and H-6′), 3.73 (s, 6-H, Ph-OCH3), 3.97-4.00 (m, 1H, H-2′), 4.24-4.29 (m, 2H, H-3′ and H-5′), 4.53 (t, J ) 8.0 Hz, 1H, H-4′), 5.60 (d, J ) 5.5 Hz, 3′-OH), 6.87-7.43 (m, 13H, Ph), 7.51 (s, 1H, H-6), 9.66 (t, J ) 5.5 Hz, 1H, 6′-NH-), 11.34 (s, 1H, N-3). 13C NMR (DMSO-d6, 125.7 MHz): δ 11.9 (5-CH3), 41.9 (C-6′), 54.9 (Ph-OCH3), 64.3 (C-1′), 65.3 (C-4′), 72.8 (C-3′), 75.4 (C-5′), 81.1 (C-2′), 109.4 (C-5), 113.3, 126.6, 127.7, 129.7, 135.6 (Ph), 138.3 (C-6), 151.0 (C-2), 158.0 (CF3CO), 163.6 (C-4). Anal. Calcd. for C34H34F3N3O8 (669.2) C 60.98, H 5.12, N 6.27. Found: C 60.96, H 5.17, N 6.24. 1-O-Dimethoxytrityl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-4,6-dideoxy-4-(thymin-1-yl)-6-trifluoroacetylamino-2,5-anhydro-L-mannitol (6). Compound 5 (0.47 g, 0.7 mmol) was dissolved in anhydrous THF (5 mL) under argon. To this solution was added diisopropylethylamine (DIPEA, 0.5 mL, 2.8 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.3 mL, 1.4 mmol). The mixture was stirred at 0 °C for 10 min, then at room temperature for 2 h. The reaction mixture was quenched by addition of MeOH (0.6 mL). After stirring for 15 min, EtOAc (40 mL) was added, and the organic layer was washed with 5% aqueous NaHCO3 (10 mL × 2), followed by H2O (10 mL × 2) and saturated NaCl solution (10 mL × 2). The solution was dried (Na2SO4), then evaporated under vacuum to oil, and the oil was purified by a short-column chromatography eluting with petroleum ether/EtOAc (6:1-3: 2, 0.5% Et3N) to afford compound 6 as a white foam solid (0.43 g, 70%). 1H NMR (CDCl3, 500 MHz): δ 0.58 (d, J ) 7.0 Hz, 3H, CH3), 0.74 (d, J ) 6.5 Hz, 3H, CH3), 0.92 (d, J ) 7.0 Hz, 6H, CH3), 1.18 (t, J ) 7.0 Hz, 2H, CH2CN), 1.70 (s, 3H, 5-CH3), 2.41 (m, 2H, CH), 3.08-3.47 (m, 6H, H-1′, H-6′ and -POCH2), 3.72 (s, 6-H, Ph-OCH3), 3.98-4.01 (m, 1H, H-2′), 4.22-4.28 (m, 2H, H-3′ and H-5′), 4.53 (t, J ) 8.0 Hz, 1H, H-4′), 6.867.42 (m, 13H, Ph), 7.50 (s, 1H, H-6), 9.67 (t, J ) 5.5 Hz, 1H, 6′-NH-), 11.30 (s, 1H, N-3). 31P NMR (CDCl3, 121.5 MHz): δ 154.18 and 154.84. 4,6-Dideoxy-4-(adenin-9-yl)-6-azido-2,5-anhydro-L-mannofuranose dimethyl acetal (8). Compound 8 (white foam, 2.15 g, 94%) was prepared from compound 7 (2.12 g, 6.52 mmol) in the same way as compound 2 from compound 1. UV (CH3OH): λmax ) 262 ( ) 13452). [R]20 D ) -27.50 (C 0.004 g/mL, CH3OH). 1H NMR (DMSO-d6, 500 MHz): δ 3.36 (s, 3H, 1-OCH3), 3.37 (s, 3H, 1-OCH3), 3.29-3.56-3.47 (m, H-6′), 3.91 (t, J ) 5.5 Hz, 1H, H-2′), 4.56-4.59 (m, 2H, H-1′ and H-5′), 4.72-4.80 (m, 2H, H-3′ and H-4′), 5.73 (d, J ) 6.0 Hz, 1H, 3′-OH), 7.26 (br. s, 2H, 6-NH2), 8.15 (s, 1H, H-2), 8.23 (s, 1H, H-8). 13C NMR (DMSO-d6, 125.7 MHz): δ 51.1 (C-6′), 53.8 (1′-OCH3), 54.4 (1′-OCH3), 63.4 (C-4′), 74.7 (C-3′), 77.6 (C-5′), 82.0 (C-2′), 103.7 (C-1′), 119.1 (C-5), 139.9 (C-8), 149.6 (C-4), 152.4 (C-2), 156.0 (C-6). HRMS (ESI-TOF+) calcd for C13H19N8O4 (M+ + H): 351.1523. Found: 351.1519. 4,6-Dideoxy-4-(adenin-9-yl)-6-azido-2,5-anhydro-L-mannitol (9). Compound 9 (white foam, 0.40 g, 60%) was prepared from compound 8 (0.74 g, 2.1 mmol) in the same way as
1020 Bioconjugate Chem., Vol. 18, No. 4, 2007
compound 3 from compound 2. UV (CH3OH): λmax ) 260.5 1 ( ) 12802). [R]20 D ) -13.8 (C 0.004 g/mL, CH3OH). H NMR (DMSO-d6, 500 MHz): δ 3.31-3.69 (m, 4H, H-1′ and H-6′), 3.84-3.87 (m, 1H, H-2′), 4.54-4.57 (m, 1H, H-5′), 4.64-4.68 (m, 1H, H-3′), 4.77 (t, J ) 8.5 Hz, 1H, H-4′), 4.88 (t, J ) 5.5 Hz, 1H, 1′-OH), 5.68 (d, J ) 5.5 Hz, 1H, 3′-OH), 7.24 (br. s, 2H, 6-NH2), 8.13 (s, 1H, H-2), 8.24 (s, 1H, H-8). 13C NMR (DMSO-d , 125.7 MHz): δ 51.7 (C-6′), 61.3 (C-1′), 6 62.8 (C-4′), 73.3 (C-3′), 77.4 (C-5′), 83.4 (C-2′), 119.1 (C-5), 139.9 (C-8), 149.6 (C-4), 152.4 (C-2), 156.0 (C-6). HRMS (ESITOF+) calcd for C11H15N8O3 (M+ + H): 307.1261. Found: 307.1263. 4,6-Dideoxy-4-(N6-benzoyladenin-9-yl)-6-azido-2,5-anhydroL-mannitol (10). Compound 9 (0.33 g, 1.1 mmol) was dissolved in dry pyridine (10 mL) and TMSCl (1.4 mL, 11 mmol) was added at 0 °C. The solution was stirred at room temperature for 2 h. Then, benzyl chloride (0.7 mL, 6 mmol) was added at 0 °C and the solution was stirred at room temperature for 4 h. Then, the pH of the solution was adjusted to 8-9 by NH4OH at 0 °C. After evaporation, the mixture was dissolved in CH2Cl2. After filtration, the solvent was evaporated under vacuum, and the residue was purified by short-column chromatography using a gradient of MeOH in CH2Cl2 (3.5%) to yield 10 (0.39 g, 88%) as white foam. UV (CH3OH): λmax ) 280.5 ( ) 13051). [R]20 D ) -14.5 (C 0.004 g/mL, CH3OH). 1H NMR (DMSO-d , 500 MHz): δ 3.32-3.71 (m, 4H, H-1′ 6 and H-6′), 3.88-3.91 (m, 1H, H-2′), 4.59-4.63 (m, 1H, H-5′), 4.69-4.73 (m, 1H, H-3′), 4.90-4.95 (m, 2H, H-4′ and 1′-OH), 5.77 (d, J ) 6.0 Hz, 1H, 3′-OH), 7.52-8.04 (5H, Ph), 8.62 (s, 1H, H-2), 8.74 (s, 1H, H-8), 11.16 (s, 1H, 6-NH-). 13C NMR (DMSO-d6, 125.7 MHz): δ 51.7 (C-6′), 61.2 (C-1′), 62.9 (C-4′), 73.4 (C-3′), 77.4 (C-5′), 83.3 (C-2′), 125.8 (C-5), 128.4, 132.4 and 133.3 (Ph), 143.5 (C-8), 150.2 (C-4), 151.4 (C-2), 152.6 (C-6), 165.5 (Ph-CO-). HRMS (ESI-TOF+) calcd for C18H19N8O4 (M+ + H): 411.1523. Found: 411.1514. 4,6-Dideoxy-4-(N6-benzoyl-adenin-9-yl)-6-trifluoroacetylamino2,5-anhydro-L-mannitol (11). Compound 11 (white foam, 0.40 g, 89%) was prepared from compound 10 (0.39 g, 0.94 mmol) in the same method as compound 4 from compound 3. UV (CH3OH): λmax ) 279 ( ) 13764). [R]20 D ) -17.00 (C 0.004 g/mL, CH3OH). 1H NMR (DMSO-d6, 500 MHz): δ 3.39-3.71 (m, 4H, H-1′ and H-6′), 3.87-3.90 (m, 1H, H-2′), 4.61-4.64 (m, 1H, H-5′), 4.69-4.73 (m, 1H, H-3′), 4.79 (t, J ) 8.0 Hz, 1H, H-4′), 4.91 (t, J ) 5.5 Hz, 1H, 1′-OH), 5.77 (d, J ) 6.0 Hz, 1H, 3′-OH), 7.54-8.05 (5H, Ph), 8.53 (s, 1H, H-2), 8.73 (s, 1H, H-8), 9.56 (t, J ) 5.5 Hz, 1H, 6′-NH-), 11.16 (s, 1H, 6-NH-). 13C NMR (DMSO-d6, 125.7 MHz): δ 41.7 (C-6′), 61.0 (C-1′), 64.5 (C-4′), 73.5 (C-3′), 76.3 (C-5′), 83.3 (C-2′), 125.8 (C-5), 128.4, 132.4 and 133.3 (Ph), 143.7 (C-8), 150.3 (C-4), 151.3 (C-2), 152.3 (C-6), 156.4 and 156.6 (CF3CO), 165.5 (Ph-CO-). HRMS (ESI-TOF+) calcd for C20H20N6O5F3 (M+ + H): 481.1441. Found: 481.1463. 1-O-Dimethoxytrityl-4,6-dideoxy-4-(N6-benzoyladenin-9-yl)6-trifluoroacetylamino-2,5-anhydro-L-mannitol (12). Compound 12 (white foam, 0.35 g, 79%) was prepared from compound 11 (0.27 g, 0.56 mmol) in the same method as that of compound 5 from compound 4. 1H NMR (DMSO-d6, 500 MHz): δ 3.213.56 (m, 4H, H-1′ and H-6′), 3.74 (s, 6H, Ph-OCH3), 4.074.10 (m, 1H, H-2′), 4.78-4.90 (m, 3H, H-3′, H-4′ and H-5′), 5.78 (d, J ) 6.0 Hz, 1H, 3′-OH), 6.89-8.06 (18H, Ph), 8.51 (s, 1H, H-2), 8.74 (s, 1H, H-8), 9.66 (t, 1H, 6′-NH-), 11.16 (s, 1H, 6-NH-). 13C NMR (DMSO-d6, 125.7 MHz): δ 41.7 (C-6′), 54.9 (Ph-OCH3), 63.7 (C-1′), 64.5 (C-4′), 73.0 (C-3′), 75.6 (C-5′), 81.1 (Ph3C), 85.2 (C-2′), 111.3, 126.0, 126.6, 127.7, 128.4, 129.8, 132.4, 133.3, 135.6 (Ph), 144.0 (C-8), 150.3 (C-4), 151.2 (C-2), 152.3 (C-6), 156.4 and 156.7 (CF3CO), 165.5
Li et al.
(Ph-CO-). Anal. Calcd. for C41H37F3N6O7 (782.2) C 62.91, H 4.76, N 10.74. Found C 62.77, H 4.83, N 10.51. 1-O-Dimethoxytrityl-3-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite-4-dideoxy-4-(N6- benzoyladenin-9-yl)-6-trifluoroacetylamino-2,5-anhydro-L-mannitol (13). Compound 13 (white foam, 0.31 g, 77%) was prepared from compound 12 (0.32 g, 0.41 mmol) in the same method as compound 6 from compound 5. 1H NMR (CDCl3, 500 MHz): δ 0.58 (d, J ) 7.0 Hz, 3H, CH3), 0.74 (d, J ) 6.5 Hz, 3H, CH3), 0.92 (d, J ) 7.0 Hz, 6H, CH3), 1.18 (t, J ) 7.0 Hz, 2H, CH2CN), 2.41 (m, 2H, CH), 3.21-3.56 (m, 6H, H-1′, H-6′ and -POCH2), 3.74 (s, 6H, Ph-OCH3), 4.08 (m, 1H, H-2′), 4.78-4.90 (m, 3H, H-3′, H-4′ and H-5′), 5.78 (d, J ) 6.0 Hz, 1H, 3′-OH), 6.89-8.06 (18H, Ph), 8.51 (s, 1H, H-2), 8.73 (s, 1H, H-8), 9.65 (t, 1H, 6′-NH-), 11.14 (s, 1H, 6-NH). 31P NMR (CDCl3, 121.5 MHz): δ 153.98 and 154.56. Oligonucleotides. siRNAs (21 nt, Table 1) were chemically synthesized by automatic DNA synthesizer (model NW3900) using commercial phosphoramidities as building blocks (Shanghai Gene Biotech Co.). Aminoisonucleoside-modified siRNAs were synthesized by the same method but replacing the regular T and A unit with building blocks 6 and 13 at the defined positions (Table 1) (synthesis scale, 0.2 µmol; coupling time, 10 min). Cleavage and deprotection of RNA oligos were carried out according to the standard protocol (treated with 29% NH3‚H2O in sealed vessel at 55 °C for 16 h; the trifluoroacetyl group was removed at this stage (25); then, treated with Bu4NF; TBDMS group was removed). The synthetic oligoribonucleotides were then purified by reverse-phase HPLC (Shimadzu LC-6AD, C-18 column, 10 × 250 mm) eluting with 0.1 M TEAA/MeCN, and the pure oligonucleotides were lyophilized and stored at -20 °C. siRNA duplexes were prepared by mixing complementary sense- and antisense-strand RNAs at equal 50 µm in MQ water, and then incubated in boiling water for 1 min, followed by gradual temperature decrease to room temperature and then 4 °C, allowing the efficient formation of siRNA duplex. The quality of the RNA duplexes was assessed on a 15% PAGE gel. Tm Measurement. UV melting experiments were recorded with a Pharmacia LKB Biochrom 4060 spectrophotomer. Samples were dissolved in a buffer solution containing 0.14 M NaCl, 0.01 M Na2PO4, and 0.1 mM EDTA, pH 7.2. The solution containing the sense-strand siRNA and complementary antisense-strand siRNA were heated to 95 °C for 3 min, then cooled gradually to 4 °C and used for the thermal denaturation studies. Thermally induced transitions of each mixture were monitored at 260 nm. Sample temperature was increased at 0.5 °C/min between 15 and 85 °C. In all experiments, the concentration of each oligonucleotide strand was 2 µM. Serum Stability. Duplexes of single-stranded RNA or siRNA (330 ng/µl) were incubated at 37 °C in 500 µL of 50% fetal bovine serum containing 7.5 µL TBST. Aliquots of 5 µL were withdrawn at different time points and immediately frozen in 15 µL × TBE-loading buffer. Samples were subjected to electrophoresis in 15% polyacrylamide-TBE under nondenaturing conditions and visualized by staining with SYBR Gold nucleic acid staining reagents and quantified by Storm 840 hardware and Imagequant software (Amersham Biosciences, Uppsala, Sweden). Transfection and Dual-Luciferase Assay. Human embryonic kidney cells (HEK293) were grown in DMEM (Life Technologies, Gibco) and seeded in 24-well plates. After the cells reached about 50% confluence, the culture medium was changed to OPTIMEM (Gibco) and then transfected with plasmids and siRNA duplex in the presence of 0.17% Lipofectamine 2000 (Invitrogen). For each well, 0.17 g of recombination plasmid and 0.017 g pRL-TK were used. The final
Bioconjugate Chem., Vol. 18, No. 4, 2007 1021
Communications Table 2. MALDI-TOF Mass Spectrum DATA for Modified Oligonucleotides
calcd. found
calcd. found
ssNC
ssIso-1
ssIso-2
ssIso-3
ssIso-4
ssIso-5
6652 6650
6679 6678
6679 6679
6665 6662
6708 6713
6734 6732
asNC
asIso-1
asIso-2
asIso-3
asIso-4
asIso-5
6628 6627
6641 6642
6641 6643
6641 6643
6670 6671
6670 6670
concentration of siRNA is 13 nM. The transfection medium was changed to culture medium (1 mL) after 4 h. All experiments were performed in triplicate and repeated at least twice. Cells were harvested 24 h after transfection and lyzed by passive cell lysis. Dual-luciferase assay was conducted. Luciferase activities were determined with 10 µL cell lysate using the DualLuciferase Assay System (Promega) by NOVOStar (BMG Labtechnologies Gmbh, Germany). The ratio between firefly and Renilla luciferase readings was generated for each well, and the inhibition efficiency of each siRNA was calculated by normalizing to respective buffer control.
RESULTS Chemical Synthesis. Building blocks 6 and 13 for solidphase oligonucleotide synthesis were synthesized from starting materials 1 and 7 (22), respectively (Scheme 1). The general method for synthesis of azido isonucleoside intermediates is to introduce a leaving group first and then substituted by azido group. Here, we developed a one-step method (step I) in which, at room temperature, compound 1 or 7 was directly reacted with sodium azide in the presence of triphenyl phosphine and carbon tetrabromide, giving the key intermediate 2 or 8 in good yield (26). Treatment of compound 2 or 8 with 1% HCl followed by sodium hydroxide and sodium borohydride in a second onepot reaction at 0 °C (step II) gave unprotected azido isonucleoside 3 or 9 in 84% or 76% yield, respectively. Conversion of azidoisonucleosides into aminoisonucleosides was carried out by catalytic hydrogenation (step III). Prior to this reaction, compound 9 was first preprotected by a benzoyl group at the 6-amino group of the adenine moiety. After hydrogenation, the amino groups within the aminoisonucleosides were protected by trifluoroacetyl (step IV), yielding compounds 4 and 11, which were then protected by a DMT group and a 2-cyanoethyl-N,Ndiisopropylphosphoramidite group (steps V and VI) at the 1-hydroxyl and 3-hydroxyl positions for solid-phase chemistry. Oligonucleotides. The obtained building blocks 6 and 13 were finally incorporated into the sequences of sense or antisense strand of designed siRNAs according to the solid-phase protocols. The coupling efficiency was determined by the release of DMT, and the efficiency was over 90% for coupling 6 and 13. After the usual workup and purification by C-18 reverse HPLC, ten kinds of aminoisonucleoside-incorporated oligonucleotides were synthesized (Table 1). On the basis of the MALDI-TOF MS, the molecular weights of the aminoisonucleoside-modified sense strand and antisense strand of siRNA are concordant with the calculated value of the designed sequence (Table 2). The assessment results on a 20% PAGE gel showed the high quality of the RNA duplexes (Figure 1). Thermal and Serum Stability of siRNA Duplexes. It has been reported that isonucleoside modification confers high nuclease resistance to oligonucleotides, but decreases the oligonucleotide binding ability to the complementary strand (22). In order to overcome the poor binding issue, we incorporated aminoisonucleosides instead of isonucleosides into siRNA duplexes. We found that incorporation of one aminoisonucleoside at the 3′ or 5′ terminal of sense strand of siRNA exhibited no effect on the duplex thermal stability with Tm ) 66 °C for
Figure 1. PAGE analysis of siRNAs: ssNC/asNC and ssIsoX/asNC. 20% polyacrylamide denaturing (7 M urea) gel electrophoresis and visualized by staining with SYBR gold and quantified by Model & Storm 860 hardware and Imagequant software (Amersham Biosciences, PKU, China). Lanes 1 to 6 represent: (1) ssNC/asNC; (2) ssIso-1/ asNC; (3) ssIso-2/asNC; (4) ssIso-3/asNC; (5) ssIso-4/asNC; (6) ssIso5/asNC. Table 3. Tm and Serum Stability (T1/2) of siRNAs: NC (ssNC/asNC) and ssIso-X/asNC (X ) 1-5)
name
NCss NC/asNC
a ssIso-1/ asNC
b ssIso-2/ asNC
c ssIso-3/ asNC
d ssIso-4/ asNC
e ssIso-5/ asNC
Tm (°C) T1/2 (h)
66 16.53
66 14.96
66 13.79
62 13.51
56 4.06
36 5.86
both ssIso-1/asNC and ssIso-2/asNC, which were equal to native siRNA form, ssNC/asNC. However, internal incorporation of one aminoisonucleoside into siRNA duplexes decreased the thermal stability with Tm ) 62 °C for ssIso-3/asNC. Furthermore, when two or more aminoisonucleosides were incorporated into siRNAs, Tm values decreased strikingly with Tm ) 56 °C for ssIso-4/asNC and 36 °C for ssIso-5/asNC (Table 3). Incorporation of aminoisnucleoside at a different position of the antisense strand of siRNA showed the same effect as the case of ssIso1-5/asNC (data were not shown). After incubation of siRNAs with 50% FBS at 37 °C, the results indicated that siRNAs with a single aminoisonucleoside modification at the sense strand, such as ssIso-1/asNC, ssIsio-2/asNC, and ssIso-3/asNC, exhibited a slight decrease of serum stability compared with their native siRNA with all degraded in 24 h (Figure 2, I, II, and III) (Table 3). Modified siRNAs with two or more aminoisonucleosides at the sense strand, such as ssIso-4/asNC and ssIso-5/asNC, showed further weaker serum stability compared with the native siRNA and rapidly degraded within 1.5 h (Figure 2, IV and V) (Table 3). Gene-Silencing Activities of Aminoisonucleoside-Modified siRNA. The firefly luciferse-targeting modified siRNAs with aminoisonucleosides in either the sense strand or antisense strand were evaluated on the basis of a dual-luciferase assay in which renillar luciferase acts as an internal control. We found that sense strand-modified siRNAs with a single aminoisonucleoside at the 3′- or 5′-end, such as ssIso-1/asNC and ssIso-2/asNC, exhibited comparative and even better activity than the native siRNA (NC) (Figure 3, Table 4). However, such an advantage was not observed when 3′- and 5′-terminal modifications combined together, such as ssIso-4/asNC; instead, the silencing activity somehow decreases. Alternative modifications with aminoisonucleoside at the siRNA internal region, such as ssIso-3/asNC, bring some negative effect on siRNA activity compared with the terminal modification, and such a negative effect further becomes apparent when internal and terminal modification in the sense strand were combined together, such as ssIso-5/asNC, which only maintains about 40% of genesilencing activity (13 nM) (Figure 3, Table 4). In contrast to the tolerance toward sense strand modifications, antisense strand modification brings a strikingly negative effect on the siRNA activity. We found that replacement of a single nucleotide with an aminoisonucleoside at the 5′-end of the antisense strand, such as ssNC/asIso-2, reduced siRNA activity by almost 60%. A similar modification at the 3′-end of the antisense strand, such as ssNC/asIso-1, reduced siRNA activity by around 50%, somehow better than the 5′-end modification.
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Figure 2. Serum stability of siRNAs: NC (ssNC/asNC) and ssIso-X/asNC (X ) 1-5). The different ssIso-X/asNC were incubated in 50% FBS at 37 °C and withdrawn at indicated time points. The oligos were separated by PAGE and visualized with SYBR gold. ds depicts double-stranded siRNA marker and ss single-stranded. (a) ssIso-1/asNC (I); (b) ssIso-2/asNC(II); (c) ssIso-3/asNC (III); (d) ssIso-4/asNC(IV); (e) ssIso-v/asNC(V).
Figure 3. Firefly renilla luciferase activities of siRNAs: NC (ssNC/asNC), ssIso-X/asNC, and ssNC/asIso-X (X ) 1-5). All experiments were performed in triplicate and repeated at least twice.
DISCUSSION
Table 4. Firefly renilla Luciferase Activities of siRNAs: NC (ssNC/asNC), ssIso-X/asNC, and ssNC/asIso-X (X ) 1-5) NC ssNC/ asNC
18%
a ssIso-1/ asNC
b ssIso-2/ asNC
c ssIso-3/ asNC
d ssIso-4/ asNC
e ssIso-5/ asNC
15%
17%
22%
38%
61%
f ssNC/ asIso-1
g ssNC/ asIso-2
h ssNC/ asIso-3
i ssNC/ asIso-4
j ssNC/ asIso-5
61%
50%
98%
77%
126%
Simultaneous modifications of the 3′- and 5′-ends of the antisense strands by aminoisonucleosides lead to a further negative effect with just 20% activity remaining for ssNC/asIso-4. We also found that internal modification on siRNA antisense strand by replacing a single nucleoside with an aminoisonucleoside, such as ssNC/asIso-3, or combination of the terminal modification with internal modification on the antisense strand almost abolished the silencing activity, such as ssNC/asIso-5 (Figure 3, Table 4).
RNA interference (RNAi) is the process of using a doublestranded RNA (dsRNA) with specific sequence to regulate the expression of a sequence-homologous gene. According to the RNAi pathway, the siRNA duplex is first incorporated into a large multiprotein known as RNAi-induced silencing complex (RISC), by which the sense strand is excluded via an undefined mechanism, and then the antisense strand together with RISC recognizes and cleaves the target mRNA (27-30). Therefore, the sense strand is more like a courier that delivers the antisense strand, the main effective component of RNAi, into the RISC complex. As a result, many different chemical modifications can be accommodated in the sense strand but not in the antisense strand. Schwarz et al. reported that siRNA duplexes function asymmetrically with two strands not equally eligible for assembly into RISC (31). By statistical analyses of the internal stability of siRNA, Khvorova et al. found that the functional siRNAs display lower stability at 5′ end of the antisense strand than nonfunctional dsRNAs, suggesting that altering the nature of the dsRNA, chemically or structurally, might be a means for optimization of siRNA activity (32).
Bioconjugate Chem., Vol. 18, No. 4, 2007 1023
Communications
Among the chemical modifications, aminoisonucleoside may represent a novel direction in which the nucleobase is linked to the 4-position rather than C1 of the sugar ring. It has been shown that incorporation of isonucleotides into a oligonucleotide causes a change in the conformation of 2,5-anhydro-Lmannitol, bringing torsion angles in the sugar-phosphate backbones. This alternation might make the modified oligonucleotide less recognized by nucleases and thus increase its stability toward various enzymes (20-22). However, the conformational change within isonucleotise-modified oligonucleotides decreases RNA duplex thermal stability with a lower Tm value. For aminoisonucleotide-modified oligonucleotides, the introduction of amino groups may increase their thermal stability via the strong binding of cationic amino groups with negatively charged phosphate backbones of the complementary sequences, as shown from the identical Tm values by comparisons of ssIso-1/asNC and ssIso-2/asNC with their native siRNA, ssNC/asNC (Table 1). On the other hand, the alternations in torsion angles of the backbone of the oligonucleotide might promote the strand separation of the siRNA duplex that could potentially increase silencing activity. It might provide an explanation for the data above in which the siRNA duplex ssIso-1/asNC and ssIso-2/ asNC with aminoisonucleoside substitution at the 3′-end or 5′-end of the sense strand exhibited better activity than the native siRNA (NC). The comparable gene-silencing activities of ssIso-1/asNC, ssIso-2/asNC, and ssIso-3/asNC with their wild-type siRNA (Figure 3) indicate that introduction of a single aminoisonucleoside in the sense strand, at either the 3′-end or 5′-end or internally, is quite tolerant for maintaining siRNA activity. However, introduction of multiple aminoisonucleosides into the siRNA sense strand, such as ssIso-4/asNC and ssIso-5/asNC, seriously reduced its activity (Figure 3), potentially due to the lower serum and thermal stability (Table 1 and Figure 3). Even so, the combination of internal and terminal modifications at the sense strand, such as ssIso-5/asNC (Tm ) 36 °C), still retains 40% (at 13 nM) gene-silencing activity. On the contrary, introduction of a single aminoisonucleoside at the antisense strand, either 3′- or 5′-end, such as ssNC/asIso-2 or ssNC/asIso-1, strikingly affects siRNA function with 40% to 50% of genesilencing activity left. Modification of the antisense strand at both ends, such as ssNC/asIso-4, further reduces siRNA silencing activity with just ∼20% left. Hohjoh et al. have investigated the relationship of siRNA activity versus sense-antisense strand terminal mismatches, and found that the strand whose 3′-end is relatively easily unwound would be employed as the guider strand to incorporate into RISC for gene knockdown (33). Here, we conclude that siRNA sensestrand modification has less effect on siRNA activity, while antisense-strand modification significantly reduces siRNA activity. Our data also suggests the following: (i) the bases in modified siRNAs, despite their lower activity, retain their hybridization properties with complementary sequences; (ii) the torsion angles in the sugar-phosphate backbones would not perturb the entry of the modified siRNA into RISC; and (iii) the two strands of the siRNA duplex are not equally eligible for assembly into RISC. The siRNA stability depends on the thermal stability of the siRNA duplex and the serum stability of each individual strand. We have found that single-stranded RNA oligos containing aminoisonucleoside were degraded rapidly when incubated with serum (data was not shown), but the siRNA duplex with aminoisonucleoside at either the 3′- or 5′-end of the sense strand exhibited a slight serum stability decrease compared with the unmodified siRNA. However, incorporation of two or four aminoisonucleosides into the sense strand reduces the duplex
thermal and serum stability, which were potentially due to the combined torsion on siRNA backbone and the loose structure on the siRNA duplex. Many efforts have been made to increase siRNA stability at the cost of a slight reduction of siRNA efficacy. Here, the aminoisonucleoside modification is superior to other chemical modifications, since such modified RNAs are good substrates of RNase H (20), and isonucleoside triphosphates can be recognized by many different DNA polymerases (34). Furthermore, siRNA sense-strand modification with aminoisonucleoside at the 3′- or 5′-end showed comparable gene-silencing activity and serum stability as their native siRNA. Taken together, aminoisonucleoside-modified oligonucleotide represents a novel direction and is a good mimic of natural oligonucleotide with the required biological and chemical properties.
SUMMARY Ten modified siRNAs with aminoisonucleoside at various positions were synthesized and their thermal and serum stabilities and gene-silencing activities were studied. The sense-strand modification with aminonucleoside at the 3′- and 5′-end siRNAs showed similar duplex thermal and serum stability as the natural one. The identical gene activities indicated that such a modified siRNA is compatible with the intracellular siRNA machinery. It was also found that antisense strand modification with aminoisonucleoside, despite the reduction of gene-silencing activity, is still somehow tolerated. These findings are worthy of the further study.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (20332010 and SFCBIC 20320130046), the Ministry of Science and Technology of China (2005BA711A04), and a Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD200265).
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