Bioconjugate Chem. 2005, 16, 1081−1087
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Properties of Isonucleotide-Incorporated Oligodeoxynucleotides and Inhibition of the Expression of Spike Protein of SARS-CoV Zhanli Wang, Jifeng Shi, Hongwei Jin, Liangren Zhang,* Jingfeng Lu, and Lihe Zhang National Research Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China. Received September 21, 2004; Revised Manuscript Received April 11, 2005
Antisense oligonucleotides are recognized to be very efficient tools for the inhibition of gene expression in a sequence specific way. For the discovery of a novel efficient way to modify oligonucleotides, a series of single isonucleotide-incorporated antisense oligodeoxynucleotides have been synthesized, in which an isonucleotide was introduced at different positions of the sequences. The binding behaviors of modified oligodeoxynucleotides to the complementary sequence were studied by UV, CD, and molecular dynamics simulation. The results showed that although the incorporated isonucleotides at certain positions of the sequence interfere with the binding ability to a different extent, B-form duplexes were maintained and the binding abilities of the 3′-end-modified duplexes were better than the corresponding mismatched duplexes. The digestion of modified oligodeoxynucleotides by snake venom phosphodiesterase showed that an isonucleotide strongly antagonizes hydrolysis. The DNA/RNA hybrid formed by a modified oligodeoxynucleotide and its target RNA could activate RNase H. The 3′-endmodified antisense oligodeoxynucelotides inhibited S-glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. This study indicated the possibility of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3′-end of the sequence.
INTRODUCTION
Since the first report of the inhibition of Rous sarcoma viral replication by an antisense oligonucleotide in a cell culture in 1978 (1), antisense technology has been rapidly developed either in studying gene function or in developing genetic therapy. As a class of potential therapeutic agents, antisense oligonucleotides have been developed for the treatment of diseases such as cancer, inflammation, and virus infection (2, 3). The first antisense drug (Vitravene/formivirsen) was approved by FDA for the treatment of CMV retinitis in patients with AIDS in 1998, and there are also dozens of antisense therapeutics in clinical trials (4). As antisense technology progressed to become a therapeutic option, several drawbacks have emerged that negatively affect its therapeutic efficacy. For example, natural oligodeoxynucleotides are readily degraded in the serum or in vivo and show poor uptake by the cell membrane. To improve the biostability of oligonucleotides, chemical modification is widely used. Among the modifications, the phosphorothioate oligodeoxynucleotide is most frequently used, which is resistant to nuclease digestion, is recognized by RNase H, and is easyily mass-produced. Though phosphorothioate oligodeoxynucleotides are currently used as the standard choice of chemically modified oligodeoxynucleotides, its non-sequence-specific protein binding leads to significant side effects, including complement activation, thrombocytopenia, inhibition of cell-matrix interaction, or reduction of cell proliferation (5). To overcome these limitations, intensive efforts were focused on the development of other kinds of chemically modified oligonucleotides, which were named as a second generation of oligonucleotides. For example, modifications of the 2′ position of riboses with electronegative substituents such * To whom correspondence should be addressed. Tel: +8610-82802567, Fax: +86-10-82802724. E-mail: liangren@ bjmu.edu.cn.
as the 2′-O-(2-methoxy)ethyl (MOE) group (6) or a 2′-O,4′C-methylene bridge (locked nucleic acid; LNA) were performed (7). Since such ribose modifications confer an RNA-like conformation to the oligonucleotide, they increase the target RNA binding affinity and cellular uptake. But on the other hand, the RNA-like conformation leads to the disappearance of the oligonucleotide’s ability to activate RNase H which is considered to be an important pathway of antisense action. Therefore, the 2′ribose-modified oligonucleotide has been successfully introduced only in the format of mixed-backbone analogues, in which the modified nucleotides are placed at the ends to leave an RNase H-compatible DNA gap (8). Isonucleosides represent a novel class of nucleoside analogues in which the nucleobase is linked to various positions of ribose other than C-1′. Because of the shift of N-glycosidic bond, the chemical and enzymatic stabilities of the nucleoside are increased. Previous studies showed that homo-oligoisonucleotides antagonized the hydrolysis of snake venom phosphodiesterase. Furthermore, some kinds of homo-oligoisonucleotides also showed strong binding abilities to their complementary sequences though they were a little weaker than the native counterpart (Figure 1a, b, c) (9-12). The results suggested that antisense oligonucleotide constructed from isonucleotide could antagonize the hydrolysis of nuclease and give acceptable binding ability. A practical way is to build a mixed backbone, in which the isonucleotide is placed at the critical position of the sequence. 3′-Exonuclease is the most common nuclease in vivo. The isonucleotide-modified oligodeoxynucleotide at the 3′-end will provide a model for studying the effects of isonucleotide-modified antisense oligonucleotide. In this study, an 18-mer antisense sequence 5′- AAC ATC TCC TGA GGG AAC -3′, which was complementary to the mRNA region encoding spike (S) glycoprotein of the SARS-CoV (22398-22415bp) (13, 14), was designed based on the reported strategy (15, 16). An isonucleotide was incorporated at the 3′-end or
10.1021/bc049769h CCC: $30.25 © 2005 American Chemical Society Published on Web 08/30/2005
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Figure 1. Structures of isonucleotides and the sequences of isonucleotide-incorporated oligodeoxynucleotides (I-V), complementary sequence (VI), and mismatched sequences (VII-XII, the mismatched bases are underlined).
center of the sequence (Figure 1). The effects of the isonucleotide-modified antisense oligonucleotides on the binding ability, nuclease degradation, and RNase H activity were investigated. Molecular dynamics simulation was used to evaluate the effects of incorporation of isonucleotide on the regularity of duplex formation. Meanwhile, the oligodeoxynucleotide with an isonucleotide incorporated at the center of the sequence and mismatched oligonucleotides were also used as controls. It was reported that the existence of hydroxymethyl group on the isonucleotide could affect the binding ability of duplex formation very obviously (11), the allyl groupprotected isonucleotide was also used for studying the effect of a hydroxyl group on the duplex formation of modified oligonucleotides. EXPERIMENTAL SECTION
General. Chemical reagents were purchased from Acros or Sigma Co. Column chromatography was performed on silica gel (200-300 mesh; Qingdao Chemical Co.). UV spectra were recorded with a Varian Cary 300 Bio UV-visible spectrophotometer. NMR spectra were recorded on a Varian VXR-300 or Varian Inova-500 with TMS as internal standard. FAB-MS was determined with a VG-ZAB-HS. Synthesis. 6′-O-Benzoyl-1′-dimethoxytrityl-4′-deoxy-3′O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-4-(N6benzoyladenin-9-yl) -2′,5′-anhydro-L-mannitol (3). All reagents were deoxygenated before use. 6′-O-Benzoyl-1′dimethoxytrityl-4′-deoxy-4-(N6-benzoyladenin-9-yl)-2′,5′anhydro-L-mannitol (2) (460 mg, 0.58 mmol) was dissolved in dry THF (8 mL) under an atmosphere of argon. Diisopropylethylamine (DIPEA) (0.41 mL, 2.5 mmol) was added at 0 °C, followed by adding dropwise 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite (0.28 mL, 0.88 mmol). Ten minutes later, a white precipitate appeared. After the mixture was kept at room temperature for 30 min, methanol (1 mL) was added and white precipitate disappeared in 10 min. The solution was diluted with EtOAc (25 mL) and extracted with 5% NaHCO3 (10 mL × 2), saturated aqueous NaCl (10 mL × 1), and deionized H2O (10 mL × 1) in turn. The aqueous phase was extracted with EtOAc (15 mL), and the combined organic layer was dried with anhydrous Na2SO4. After filtration and concentration, the residue was applied to flash silica
Wang et al.
gel chromatography eluting with cyclohexane/ dichloromethane/acetic acetate (10:1:1-4:1:1, 0.5% Et3N) to give compound 3 (485 mg, white foam, 84%). 6′-O-Allyl-1′-O-dimethoxytrityl-4′-deoxy-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-4′-(N6-benzoyladenin-9-yl)-2′,5′-anhydro-L-mannitol (6). Compound 6 was prepared from 6′-O-allyl-1′-O-dimethoxytrityl-4′-deoxy4′-(N6-benzoyladenin-9-yl)-2′,5′-anhydro-L-mannitol (5) in a similar approach to the preparation of compound 3 from 2 in 84.0% yield as white foam. Solid-Phase Synthesis of Oligodeoxynucleotides. Oligodeoxynucleotide synthesis was carried out in 1 µmoL scale with a DNA synthesizer model 392 (Applied Biosystem) applying regular phosphoramidite chemistry. The isonucleotide monomer (compound 3 or 6) was incorporated into the proper position based on the requirement of the oligodeoxynucleotide sequence. Cleavage and deprotection of the oligomers were performed in concentrated ammonium at 50 °C for 24 h. The crude product was purified by 15% PAGE (7 moL‚L-1 urea) and desalted by C18 column. The pure oligonucleoties were lyophilized and stored at -20 °C. Thermal Denaturation and Circular Dichroism Studies. Oligodeoxynucleotide and its complementary sequence at an equivalent concentration (6 µmoL‚L-1) were heated to 80 °C in the buffer solution (0.14 M NaCl, 0.01 M Na2HPO4, pH 7.2, 1.0 M EDTA) and then cooled to room-temperature slowly. The hyperchromicity curves of the hybridized duplex were recorded on a Varian Cary 300 Bio UV-visible spectrophotometer. CD spectra were recorded by a JASCO J-715 spectropolarimeter at 15 °C in a thermostatically controlled 1 cm cuvette. Molecular Modeling. All calculations were performed on an SGI OCTANE 2 workstation. The initial structures of DNA duplexes were built from the canonical B-form structure using Insight II simulation program. The AMBER 7.0 suite of molecular simulation programs and ff99 force field were used in the computations (17). The molecular mechanical parameters of isonucleotides were obtained using Gaussian 98 program under HF/6-31G** level of theory. The constructed oligodeoxynucleotide duplex was immersed in a periodic TIP3P water box, which extended 10 Å away from any solute atom. This yielded about 4400 water molecules used for solvation. To neutralize the negative charges of simulated molecules, Na+ counterions were placed next to each phosphate group. Molecular dynamics (MD) simulations were carried out by using the SANDER module of AMBER 7.0 with SHAKE algorithm applied to hydrogen atoms. Each simulation began with 1000 steps of minimization followed by 25 ps of equilibration dynamics at 300 K with full constraints on duplex and a 9-Å nonbonded Lennard-Jones cutoff. The duplex then was gradually allowed to relax by sequentially lowering restraints. The final production simulations of 1.5 ns for duplexes I/VI, II/VI, III/VI, or IV/VI and 1.0 ns for duplex V/VI were performed at constant pressure (1 atm) and temperature (300 K). The final structure of each duplex was obtained from the averaged structure of the last 800 ps of molecular dynamics calculation followed by 1000 steps of minimization. All duplexes were analyzed with CURVES 5.3 program. Nuclease Resistance Analysis. The modified and unmodified oligodeoxynucleotides were 5′-32p labeled with [γ-32p]ATP using T4 polynucleotide kinase. Labeled material was purified by gel electrophoresis on 20% 7M urea polyacrylamide gels. 32p-labeled DNA was incubated with 100 ng of snake venom phosphodiesterase I in buffer,
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Isonucleotide-Incorporated Oligodeoxynucleotides Scheme 1. Synthesis of Isonucleotide-Incorporated Oligodeoxynucleotidesa
a Reagents and conditions: (i) DIPEA, 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, THF, rt; (ii) solid-phase DNA synthesis.
which contained 50 mM Tris-HCl (pH 8), 5 mM MgCl2 at 21 °C. Total reaction volume was 50 µL. After 10 min, aliquots were mixed with 100 µL of stop solution which contained 0.05% (w/v) bromophenol blue and 0.05% (w/ v) xylene cyanol in 95% formamide. Aliquots were subjected to 20% polyacrylamide gel electrophoresis and visualized by autoradiography. In Vitro Transcript of SARS Fragment. The 75 nt of SARS-CoV DNA fragment corresponding to nucleotides 22380 to 22455 was generated by PCR amplification using the DNA template (gift from Beijing Institute of Genomics) and primers 5′-ATT TAG GTG ACA CTA TAG AAC CTC TAA TTT CAG GG-3′ (sense primer) or 5′-CTC CAA AAG GAC ACA AGT TTG TAA-3′(antisense primer). Sp6 promotor sequence was introduced at the 5′-end by the use of primers. All PCR products were sequenced prior to in vitro transcription. Transcription was performed directly from PCR-amplifying DNA fragments using sp6 RNA polymerase. RNAs were purified by gel electrophoresis on 20% 7 M urea polyacrylamide gels. RNase H Cleavage. The in vitro transripted RNAs of SARS fragment were 5′-32p labeled with [γ-32p]ATP as described above. 32p-labeled RNA and the modified or unmodified oligonucleotides were incubated with 0.5 units of RNase H and 50 units of RNase inhibitor in buffer for 30 min at 21 °C. The buffer contained 50 mM Tris-HCl (pH 8), 10 mM MgCl2, and 5 mM DTT. Loading buffer (30% glycerol with 0.14 nM bromophenol blue, and 0.19 nM xylene cyanol FF) was added, and samples were loaded on a 20% polyacrylamide gel and run at room temperature and recorded by autoradiography. Infection of Sf9 Cells with Viruses and Antisense Oligonucleotides Treatment. Sf9 cells (fall armyworm ovary, S. frugiperda) were cultured in Sf-900 II SFM medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 27 °C. The recombinant baculoviruses routinely propagated and titrated by plaque assay in Sf9 cells and working virus stocks were stored frozen in the dark at -80 °C. Sf9 cells were subcultured prior to antisense oligodeoxynucleotide treatment and maintained in logarithmic growth phase with viability of 98%. The oligodeoxynucleotide was added to Sf9 cells after the recombinant baculoviruses inoculated for 24 h. Each monolayer of Sf9 cells at 7 × 104 cells/well in six-well plates were transfected with oligodeoxynucleotide by liposome-mediated transfection with lipofectamine (Invitrogen). After 4 h, the medium was replaced with SFM medium supple-
mented with 10% heat-inactivated fetal bovine serum, and culture was continued with fresh medium containing the oligodeoxynucleotide. Plates of each transfected group were treated with recombinant baculoviruses. The same amounts of viruses were inoculated at 27 °C as control. RT-PCR Analysis. Total RNA was extracted from control and antisense oligonucleotide-treated cells using Trizol (Invitrogen). RNA concentrations were determined spectrophotometrically from the absorbencies of samples at 260 nm. RT-PCR was performed using the SuperScript One Step RT-PCR System (Invitrogen) following the instructions of the manufacturer. Forward and reverse primers for SARS gene were 5′-CCA CCT CTG CTC ACT GAT GAT AT-3′ and 5′-CCT TGT TAA ATT GGT TGG CGA TTT GTT-3′, respectively, which amplified a fragment of 205 bp. GAPDH mRNA was used as an internal control, and the primers were 5′-CGG GAA ACT GTG GCG TGA TGG-3′ (forward) and GTC GCT GTT GAA GTC AGA GGA GA-3′ (reverse). The expected product was 292 bp. RT-PCR was performed using 100 ng of total RNA. Each reaction contained 0.2 µM forward and reverse gene-specific primers in addition to reagents present in the SuperScript PCR buffer. RNA was reversetranscribed for 50 min at 42 °C and then subjected to 30 cycles of PCR (94 °C, 30 s; 55 °C, 40 s; 72 °C, 50 s). Samples were analyzed on 2% agarose gels. Western Blot Analysis. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked with 5% skim milk in TBST (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20), reacted with anti-His antibody (Qiagen) at a 1:1000 dilution. Proteins were detected using horseradish peroxidase-conjugated anti-mouse immunoglobulin G (Sigma) and an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech). RESULTS AND DISSCUSSIONS
Synthesis. The phosphoramidite 3 and 6 were synthesized from the corresponding epoxides 1 and 4, respectively (Scheme 1) (18). Compound 3. 1H NMR (500 MHz, DMSO-d6) 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), 3.27 (m, 2H, H-1′), 3.45 (m, 2H, -POCH2), 3.70 (s, 6H, OCH3), 4.30 (m, 1H, H-2′), 4.53 (m, 2H, 6′-CH2), 5.01 (m, 1H,
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Figure 2. The melting behaviors of duplexes formed by isonucleotide-modified or mismatched oligodeoxynucleotides with their complementary sequence. Table 1. Tm Values of the Duplexes of Modified and Unmodified Oligonucleotides with the Complementary DNA Sequence duplexes
Tm (°C)
∆Tm (°C)
I/VI II/VI III/VI IV/VI V/VI VII/VI VIII/VI IX/VI X/VI XI/VI XII/VI
59.3 54.2 58.6 54.1 61.5 58.0 58.0 49.1 56.2 44.3 37.1
-2.2 -7.7 -2.9 -7.4 -3.5 -3.5 -12.4 -5.3 17.2 24.4
H-5′), 5.20 (m, 1H, 4′-H) 5.32 (m, 1H, 3′-H), 6.89 (d, J ) 8.5 Hz, 4H, Ph-H), 7.21-7.40 (m, 8H, Ph-H), 7.55-7.65 (m, 7H, Ph-H), 7.78 (m, 2H, Ph-H), 8.05 (m, 2H, Ph-H), 8.67 (s, 1H, H-2), 8.69 (s, 1H, H-8), 11.20 (s, 1H, 6-NH). 31 P NMR: 151.7, 152.5. Compound 6: 1H NMR (500 MHz, DMSO-d6): δ 0.56 (d, 3H, CH3), 0.74 (d, J ) 7.0 Hz, 3H, CH3), 0.92 (d, J ) 6.5 Hz, 6H, CH3), 1.18 (t, J ) 7.0 Hz, 2H, CH2CN), 2.41 (m, 2H, 2CH), 3.20-3.38 (m, 4H, POCH2, H-1′), 3.60 (m, 2H, H-6′), 3.75 (s, 6H, OCH3), 3.99 (m, 2H, OCH2 (allyl)), 4.21 (m, 1H, H-2′), 4.77 (m, 1H, H-5′), 5.02-5.20 (m, 2H, H-3′, H-4′), 5.22 (m, 2H, CH2d), 5.85 (m, 1H, dCH), 6.90 (m, 5H, Ph-H), 7.207.34 (m, 7H, Ph-H), 7.48-7.65 (m, 9H, Ph-H), 8.05 (t, J ) 5 Hz, 2H, Ph-H), 8.63 (s, 1H, H-2), 8.71 (s, 1H, H-8), 11.19 (s, 1H, 6-NH). 31P NMR: 150.5, 150.1. The isonucleotide-incorporated oligodeoxynucleotides were synthesized by using phosphoramidite solid-phase strategy. The syntheses were performed in an automated DNA/RNA synthesizer on a 1 µmol scale according to the regular phosphoramidite protocol except for a prolonged coupling time to ensure an adequate coupling yield when using phosphoramidite 3 or 6. For convenience, the synthesis was started with commercially available cytosine-loaded controlled pore glass (CPG). The coupling efficiency was determined by the release of DMT, and the yield was over 92% for coupling isonucleotide phosphoramidite. After cleavage from the solid support and removal of the protecting groups with concentrated aqueous ammonia, the oligodeoxynucleotides were purified by PAGE and desalted by a C18 column. Four kinds of isonucleotide-incorporated oligonucleotides were synthesized, in which the isonucleotide was at the 3′-end or in the center of the sequence, and the 6′-OH of isonucleotide was free or protected by an allyl group. All oligonucleotides were confirmed by MALDI-TOF mass spectroscopy, and the masses were in good agreement with
Figure 3. CD spectra of duplexes formed by the isonucleotidemodified oligodeoxynucleotides and their complementary sequence. Red, I/VI; green, II/VI; dark yellow, III/VI; blue: IV/ VI; black, V/VI.
the calculated values. MALDI-TOF MS: I, 5590.2 (M + Na, calcd 5590.4); II, 5589.7 (M + Na, calcd 5590.4); III, 5548.5 (M + Na, calcd 5549.8,); IV, 5549.1 (M + Na, calcd 5549.8). Binding Behaviors with Oligodeoxynucleotide. Thermal denaturation study was used to evaluate the duplex stability formed by isonucleotide-incorporated oligodeoxynucleotide and its complementary sequence (Figure 2). The result showed that the single isonucleotide incorporation interfered with the stability of the formed duplex, which depends on the position of incorporation. When isonucleotide was at the 3′-end of the sequence (I, III), the Tm value decreased about 3 °C compared with its natural counterpart (Table 1). When isonucleotide was in the center of the sequence (II, IV), the Tm value decreased about 7 °C. The different stabilities of the hybrid duplex might result from the different neighbor influence. When isonucleotide was at the end of the sequence, the effect would mainly involve one side of base pair, whereas when isonucleotide was in the central position of the sequence, the effects involve the base pairs of two sides. Comparing to the consecutive isonucleotide-incorporated oligonucleotide, which leads to a decrease of Tm of about 0.4 °C (11), single isonucleotide incorporation resulted in a more intensive decrease of duplex stability. The previous finding showed that the 6′-OH of the isonucleotide could stabilize the duplex formed by the homo-oligoisonucleotide and its complementary sequence. In this study no obvious difference was observed when the 6′-OH of the isonucleotide was free or protected, which indicated herein that the single 6′-OH did not affect the stability of duplex significantly. For comparison, one to three nucleoside-mismatched sequences (VII-XII) were also investigated. The results showed that when the isonucleotide was located at 3′end, the duplex was more stable than the corresponding mismatched sequences, whereas when isonucleotide was located at the center of the sequence, the duplex was less stable than the corresponding single-nucleoside-mismatched sequence but was more stable than multinucleoside-mismatched sequence. Circular dichroism (CD) spectra showed that the hybrid duplex formed by modified oligodeoxynucleotides and their complementary sequence possessed very similar conformations (Figure 3). Different from the consecutive isonucleotide-incorporated oligonucleotide, which formed a distorted B-form duplex when hybridized with
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Figure 4. Overlap of simulated structures of isonucleotide-modified oligodeoxynucleotide-formed duplex with the native DNA duplex. The structures are from the part of the averaged structure of the last 800 ps molecular dynamics simulation. White arrows point to the position of the isonucleotide. (a) I/VI, (b) II/VI, (c) III/VI, (d) IV/VI.
its complementary sequence, the single isonucleotidemodified oligodeoxynucleotides formed typical B-form duplexes such as natural oligodeoxynucleotides. The computer simulation results also showed that B-form duplexes were maintained during the entire course of simulation. The result indicated that the incorporation of isonucleotide into the oligodeoxynucleotide might only lead to the local conformational change of the duplex. The averaged structure of the native DNA duplex was overlapped with the isonucleotide-modified duplexes, respectively. It clearly indicated that the helical parameters for the modified oligonucleotide duplexes were very similar to the native DNA duplex. The presence of the isonucleotides A1 or A2 mainly affected the local conformation of phosphorodiester backbone (Figure 4). Strand-strand interactions of the five simulated DNA duplexes (I/VI, II/VI, III/VI, IV/VI, and /V/VI ) were investigated (Table 2). The interaction energy (Einter) was obtained from the total energy of the duplex (Eduplex) subtracted by the sum of two individual strands (Estrand1, Estrand2), i.e., Einter ) Eduplex - (Estrand1 + Estrand2), and here the energy is composed of nonbonded van der Waals interaction and electrostatic interaction. The amount of interaction energy reflected the binding ability of oli-
Table 2. The Interaction Energy (kcal/mol) of the DNA Duplexes from the MD Trajectoriesa duplex
Eduplex
Estrand1b
Estrand2c
Einter
I/VI II/VI III/VI IV/VI V/VI
-1156.1 -973.6 -1084.1 -1121.2 -1190.2
-441.0 -325.7 -418.2 -394.9 -452.5
-386.4 -323.9 -337.7 -404.0 -393.1
-328.7 -324.0 -328.2 -322.3 -344.6
a Energy being from the averaged structure over the last 800 ps of the molecular dynamics simulation. b Energy of isonucleotide incorporated strand. c Energy of native complementary sequence.
godeoxynucleotide with its complementary sequence. The results indicated that the native DNA duplex V/VI possessed the strongest nonbonded interaction, and the duplexes I/VI and III/VI possessed stronger nonbonded interaction than duplexes II/VI or IV/VI. The calculated results were in good agreement with the thermal denaturation results. Nuclease Hydrolysis and RNase H Cleavage. The hydrolysis behaviors of 5′-32P-labeled isonucleotideincorporated oligodeoxynucleotides by snake venom phosphodiesterase (SVPDE) were observed by gel electrophoresis (Figure 5). The results showed that the controlled
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Figure 5. Autoradiogram of SVPDE digestion profiles of oligodeoxynucleotides. Lanes 1-6: marker, V, II, I, IV, III, respectively. Lanes 7-9: undigested oligonucleotide V, I, III, respectively.
natural oligodeoxynucleotide was completely hydrolyzed to small fragments after 10 min digestion, whereas the isonucleotide-incorporated oligodeoxynucleotide I or III was hydrolyzed to the 17-mer residues, and II or IV was hydrolyzed to 12-mer residue under the same conditions. The size of the hydrolyzed residues of the modified oligodeoxynucleotides was in accordance with the incorporation position of isonucleotide, i.e., the hydrolysis was inhibited at the point of isonucleotide substitution. The hydroxyl free or protected isonucleotides (I or III) have similar properties. Compared with some modified oligodeoxynucleotide, such as 2′-O-[2-(guanidium)ethyl]modified oligonucleotide, which needs to have at least two consecutive modified nucleotides at the 3′-terminus for maintaining acceptable stability against exonuclease digestion (19), isonucleotide modification showed a much better antagonistic property. The result indicated that either hydroxymethyl free or protected isonucleotide could antagonize the activity of phosphodiesterase. It implied that an enzymatic stable oligonucleotide could be constructed by incorporating isonucleotide to the ends of oligodeoxynucleotide. RNA cleavage by RNase H in the DNA/RNA hybrid was investigated (Figure 6). After the hybrid duplexes were digested by RNase H for 30 min, DNA/RNA hydrids showed different digestion patterns. Though the digested RNA fragments have not been investigated in this study, the efficiency of RNase H digestion in the presence of different oligodeoxynucleotides could be determined by measuring the intact RNA band in each lane. In general, the extent of the target RNA cleavage by RNase H in the hybrids formed by isonucleotide-modified DNA/RNA duplexes is higher than in the natural DNA/RNA. Both kinds of isonucleotide-modified oligodeoxynucleotides yielded similar RNA cleavage patterns. The results showed that the efficiency of RNase H cleavage in the presence of free or allyl-protected hydroxymethy isonucleotide-modified or unmodified oligodeoxynucleotides were in the order of allyl (III, IV) > free (I, II) > normal (V). It was reported that the efficiency of RNase H digestion was dependent on the feasibility of the formation of the DNA/RNA/RNase H complex, which was associated with many factors, such as the necessity of an A-form duplex, the width of the minor groove, and the duplex stability (20). Our result implied that the modified oligodeoxynucleotide sequences III and IV were more suitable for the formation of the DNA/RNA/RNase H complex. It was in inverse accordance with their binding abilities to the complementary sequences. The
Figure 6. Autoradiogram of 5′-end-[γ-32P]-labeled RNA treated with RNase H in the presence of isonucleotide-incorporated antisense oligodeoxynucleotide or mismatched antisense oligonucleotide. Control 1: RNA, control 2: RNA + RNase H, control 3: RNA + V.
Figure 7. Effects of modified or mismatched antisense oligodeoxynucleotides on SARS coronavirus spike protein gene mRNA expression upon treatment at 50 µmol/L for 36 h. (a). Lanes 1-4: negative control, V, I, III, respectively; (b and c) Lanes 1-4: negative control, VII, VIII, IX, respectively; Lane 5: I (b), III (c).
sequences with nucleotides mismatched at the 3′-end (VII, VIII, IX, and X) also showed some efficient digestions. Antisense-Induced Down-Regulation of S Protein. To determine the effect of 3′-isonucleotide-modified antisense oligodeoxynucleotides on gene expression of SARS coronavirus spike protein, Sf9 cells were transfected with 50 µM of oligodeoxynucleotides I, III, and V or mismatched controls VII-IX, and total RNA was isolated after 36 h. The level of SARS RNA was determined by RT-PCR. A remarkable decrease in gene expression of spike protein was observed in the cells treated by modified oligodeoxynucleotide I or III, whereas no obvious change was observed after the cell was treated by controlled native or mismatched antisense oligonucleotide (Figure 7). The concentration dependence of gene expression in samples incubated with various concentrations of oligodeoxynucleotide I or III was investigated (Figure 8).
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Figure 8. Effects of various concentrations of isonucleotideincorporated antisense oligodeoxynucleotide on SARS coronavirus spike protein gene mRNA or on GAPDH mRNA after 36 h of treatment. Lanes 1-4: 0, 1, 10, 100 µmol/L, respectively. (a) (SARS-CoV, I), (b) (SARS-CoV, III), (c) (GAPDH, I), (d) (GADPH, III).
Figure 9. Effects of oligonucleotide on SARS coronavirus spike protein gene protein levels upon treated at 50 µmol/L for 36 h. Lane 1: negative control, lane 2-4: VII-IX, lane 5: I (a), III (b).
These data indicated that the modified oligodeoxynucleotide was able to inhibit transcription of the endogenous spike protein gene and was more effective than the native counterpart. We also measured the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As expected, the expression of the GAPDH was not affected in these treated samples (Figure 8c,d). Western blot showed that modified oligodeoxynucleotides I and III decreased the protein expression remarkably and that III was stronger than I, whereas one to three mismatch controls (VII, VIII, IX) had almost no effect on the gene expression protein levels (Figure 9). In conclusion, isonucleotide-modified oligodeoxynucleotides strongly antagonized the hydrolysis of snake venom phosphodiesterase. Furthermore, the modified antisense oligodeoxynucleotide also activated the RNase H though the incorporation of an isonucleotide into the oligodeoxynucleotide which led to a change of local conformation of the duplex. It was found that the 3′-isonucleotide-modified antisense oligodeoxynucelotides inhibited S glycoprotein expression of SARS-CoV at the mRNA levels in the insect Sf9 cells. The inhibition was dependent upon the concentration of the antisense oligomer. Therefore, this study provided a new strategy of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3′end of the sequence. ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation of China (20132030, SFCBIC 20320130046). Insect cell Sf9 and the recombinant baculoviruses used in this study were kindly provided by Professor Jianguo Chen (Peking University, Beijing, China). LITERATURE CITED (1) Zamecnik, P. C., and Stephensen, M. L. (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. U.S.A. 75, 280-284.
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