Bioconjugate Chem. 1993, 4, 467-472
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2’,5’- 0ligoadenylate:Antisense Chimeras-S ynthesis and Properties Krystyna Lesiak,+ Shahrzad Khamnei, and Paul F. Terrence* Section on Biomedical Chemistry, Laboratory of Medicinal Chemistry, Building 8, Room B2A02, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. Received May 17, 1993”
We have synthesized a novel bioconjugate which joins an antisense oligonucleotide to a unique and potent inhibitor of translation, pn5’A2’(p5’A2’)mp5’A (2-5A). Two residues of 4-hydroxybutyl phosphate were employed as linkers to attach the 2’,5’-oligoadenylate moiety through its 2’-terminus to the 5’terminus of the chosen antisense sequence, (dT)zo. The syntheses were carried on a solid support according to the phosphite triester method of DNA synthesis (Letsinger, R. L., and Lunsford, W. B. (1976) J. Am. Chem. SOC.98, 3655-3661; Beaucage, S. L., and Caruthers, M. H. (1981) Tetrahedron Lett. 22, 1859-1862). The generated 2-5A antisense chimeras retained both the ability of the 2-5A molecule to activate the 2-5A-dependent RNase as well as the ability of the oligo(dT) moiety to hybridize to the complementary poly(A). Moreover, the chimera, when annealed to its target nucleic acid sequence, was still effectively bound to the 2-5A-dependent nuclease. The methodology described represents a new approach to the selective modulation of mRNA expression.
INTRODUCTION Regulation of the availability and/or stability of mRNA may be one avenue to the regulation of gene expression by exogenous agents which could find possible use in the therapy of cancer, cardiovascular diseases, and infection by parasites, viruses, and other microorganisms. Two very different approaches to such regulation at the translational level include modulation of RNA lability by the 2-5A system (1-3) and control of RNAavailability and stability by antisense oligonucleotides (4, 5). The small and unique 2’,5’-phosphodiester-bond-linked oligonucleotidereferred to as 2-5A [pn5’A2’(~5’A2’)mp5’AI has been shown to play an important role in the antiencephalmyocarditis virus action of interferon (6,7).2-5A is biosynthesized by 2-5A synthetase [ATP:(2’-5’)oligo(A) adenylyl transferase, EC 2.7.7.191 from ATP after the synthetase is activated by the double-stranded RNA presumably arising from initial viral replication (1-3). The synthesized 2-5A in turn activates a 2-5A-dependent endonuclease (RNase L) which is capable of degrading viral mRNA as well as cellular mRNA and rRNA (1-3). For this reason, the 2-5A molecule is a potent inhibitor of translation, blocking cell-free protein synthesis at nanomolar concentrations. 2-5A also can block translation and viral replication in intact cells, but only when any of a variety of manipulations (hypertonic salt treatment, calcium phosphate coprecipitation, liposome encapsulation, etc.) have been employed to enhance the uptake of this highly charged oligonucleotide (2). The poor uptake of the 2-5A molecule would have to be regarded as a liability in any effort to employ it as an exogenous regulator of mRNA stability. Another potential limitation of any attempt to capitalize on the 2-5A system to modulate the stability of a specific RNA would be the relatively nonspecific endonucleolytic action of its target RNase L, which cleaves RNA after UNp residues (8, 9).
* Author to whom correspondence should be addressed.
+ Present address: Laboratory of Biophysics, Division of Allergenic Products and Parasitology, Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892. * Abstract published in Advance ACS Abstracts, September 1, 1993. Not subject to US. Copyright.
Oligonucleotide antisense agents have been found to be highly specific inhibitors of mRNA expression through the mechanisms of passive hybridization or via degradation of the generated hybrid by RNase H (4, 5). However, certain modifications destined to enhance the potency of such antisense reagents abolish the RNase H degradation pathway since the corresponding hybrids are no longer substrates (10). To circumvent this problem and to enhance the versatility and potency of antisense oligonucleotides as potential therapeutic agents, a number of modified oligodeoxynucleotideshave been prepared which would cause target nucleic acid chain scission through chemical, photochemical, or nuclease-induced cleavage (11-19). Recently we have reported (20) on a new approach to the specific and targeted cleavage of RNA by combining the extreme specificity of the antisense concept with the potent RNA-degrading activity of the 2-5A-dependent endonuclease RNase L. Herein, we report on the synthesis, structure confirmation, and properties of such 2-5Aantisense chimeras. The structures of oligonucleotides prepared for this study and their corresponding abbreviations used throughout this report are presented in Scheme I. EXPERIMENTAL PROCEDURES Chemicals. Most reagents used in this study were from Aldrich (Milwaukee, WI), including anhydrous solvents (Sureseal packing). Snake venom phosphodiesterase used for characterization of synthesized oligonucleotides was obtained from Cooper Biomedical, and bacterial alkaline phosphatase was from US Biochemicals (Cleveland, OH). Long-chain alkylamino controlled-pore glass solid support (CPG-LCA, pore size 500A) and 5’-0-(4,4’-dimethoxytrityllthymidine 3’-0-[2-cyanoethyl NJV-diisopropylphosphoramidite] were from Sigma (St. Louis, MO). Physical Measurements. Concentrations of oligonucleotides were measured spectrophotometrically using extinction coefficients for aaenosine and thymidine (and their oligomers) reported in the literature (21). UV absorption spectra were recorded on a Varian DMS-200 spectrophotometer. lH NMR and 3lP NMR spectra were recorded on either Varian XL-300 or GE GN-300 instruments in the solvents indicated below.
Published 1993 by American Chemical Society
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Lesiak et al.
Scheme I
pSA2'@SA2')2pSA2'p 1 [OCH2(CH2hCH20]4p1[OCH2(CH2)2CH20]4pSd?3'@5'dT3')iap5'dT
PA4:T18 A2'(pSA2')2pSA2'p 1 [OCH2(CH2)2CH20]4p1[OQI2(CH2)2CH20]4pSdT3'(p5'dT3') @'dT A4:T18
pSA2'(pSA2'hpSA2'p 1 [OCH2(CH2)2CH20]4p1 [OCH2(CH2)2CH20]4pSdT3'(pSdT3')2pSdT
pA4:T4 A2'p 1 [OCH2(CH2)2CH20]4p1[OCH2(CH2)2CHzO]4pSdT3'@SdT3')2pSdT
A:T4 A2'p 1 [OCH2(CH2)2CH20]4p1 [OCH2(CH2hCH20]4pS'dT
A:T HPLC Methods. Two HPLC systems were used throughout this study. The first (system A) included two Beckman llOB solvent delivery modules and a 167 UV/ vis variable-wavelength detector (set to operate at 260 and 280 nm) controlled with Beckman System Gold software. The second system (system B) consisted of two Beckman llOB solvent-delivery modules (controlled by a NEC controller) and a Beckman 153UV detector operating at 254 nm. The following HPLC columns were used: a semipreparative Ultrasphere ODS [Altex, reversed-phase CIS, 10 X 250 mm, flow rate of 2 mL/min, linear gradients of mobile phase B in buffer A, where A was 50 mM ammonium acetate, pH 7.0, and B consisted of 50% (v/v) methanol in water], an analytical Ultrasphere ODS column [Altex, reversed-phase CIS, 4.9 X 250 mm, flow rate of 1 mL/min, linear gradients of B (as defined above) in 20 mM ammonium phosphate, pH 7.01, a Vydac 3040L [Separations Group, 4.6 X 250 mm, DEAE ion exchange, flow rate of 1 mL/min, linear gradients of ammonium phosphate, pH 6.7, in 20% (v/v) aqueous acetonitrilel, and a TSK-GEL DEAE-NPR (TosoHaas, nonporous ion exchange, 4.6 X 35 mm, flow rate of 1 mL/min, linear gradients of 0.5 M NaCl in 20 mM Tris, pH 8.0). High-PerformanceCapillary Electrophoresis. Analyses were performed on an Applied Biosystems 270A-HT capillary electrophoresis instrument using MICRO-GELm (Applied Biosystems Inc., Foster City, CA) gel-filled capillaries (50 pm id., effective length 27 cm; running buffer, 75 mM Tris phosphate, pH 7.6, 10% methanol). Detection was at 260 nm. Synthesis of Oligonucleotides. Syntheses were performed manually on DNA synthesis columns (1.5 cm, American Bionetics, Inc.) loaded with approximately 1.5 pmol of CPG-bound 5'-0-(dimethoxytrityl)thyidine (221, using adaptors and gas-tight syringes (23). The procedure was based on the phosphite triester method of DNA/RNA synthesis (24-28) using for chain elongation 2-cyanoethyl phosphoramidite derivatives of 5'-0-(4,4'-dimethoxytrityl)thymidine, 4-0-(4,4'-dimethoxytrity1)-1,4-butanediol (as protected linker in the chain extension), or 5'-0-(4,4'dimethoxytrityl)-3'-0- (tert-butyldimethylsily1)-NG-benzoyladenosine (29). The complete synthetic cycle is presented in Table I. When needed, oligonucleotides were 5'-end phosphorylated with bis(2-cyanoethoxy)(diisopropy1amino)phosphine (30). Syntheses were monitored by quantitating spectrophotometrically the release of the dimethoxytrityl cation. The synthesized oligonucleotides were cleaved from the solid support with a mixture of concentrated (28% ) aqueous ammonia and ethanol (3:l) by 2-h incubation at room temperature. N6-Benzoyl
Table I. Solid-Phase Synthesis of OligonucleotidesCouDlina Cycle ~~
step 1. detrilylation 2. washing 3. washing 4. drying 5. coupling
6. washing 7 . drying 8. capping
9. washing 10. drying 11.oxidation
12. washing 13. drying
~~
solvents/reagents time 3 % DCA in CHzClz 90 s 2% Py in acetonitrile acetonitrile nitrogen 3 min 0.2 M monomer in 8 min for Ado, 3 min for Thd 0.5 M tetrazole/ acetonitrile and 5'-end phosphitylation acetonitrile nitrogen 2 min A + B, 1:l 2 min A 30% AczO in THF B: 0.6 M DMAP in PyiTHF (3:2 v/v) acetonitrile nitrogen 2 min 0.1 M 12 in lutidine: 45 s THF:water, 20:80:1 acetonitrile nitrogen 3 min
volume, mL 1 1 3 0.15
3 1
3
1
3
groups, protection for adenosine residues, were removed by incubating the resulting solutions for 6 h at 55 OC. Finally, the 3'-0-(tert-butyldimethylsilyl)protecting groups were removed by treatment with 1M tetrabutylammonium fluoride in THF for at least 12 h at room temperature. Coupling efficiencies for oligonucleotide synthesis were as follows: for d T coupling, 99%; for butanediol linker coupling, 89-90 % ;for 2',5'-linkages, 89-95 % Final yields, after HPLC purification and desalting, were as follows: for A:T, 63 5% ; for A:T4,50%;for pA4:T4,255% ;for pAd:T18, 23%. 4-0-(4,4'-Dimethoxytrityl)-l,4-butanediol. 1,4-Butanediol (9 g, 10 mmol) was dried by repeated coevaporation with anhydrous pyridine and then dissolved in 50 mL of the same solvent containing triethylamine (2 g, 20 mmol). 4,4'-Dimethoxytrityl chloride (3390 mg, 10 mmol) was added to this solution and the mixture was kept for 2 h at room temperature. The reaction was terminated by pouring the mixture into a beaker containing ice (100 g), and products were extracted with ethyl acetate. The extract was dried with magnesium sulfate, concentrated, and purified on a silica gel column, which then was eluted with methylene chloride containing 1% methanol. The yield was 870 mg (22 5% ). 'H NMR (CDCls, 1% deuteriopyridine) 6 [ppm]: 1.68 (m,4H, CH2), 3.10 (t, J = 5.7 Hz, 2H, CHzO), 3.62 (t, J = 5.8 Hz, 2H, CHzOH), 3.76 (9,6H, CHsO), 6.79-7.46 (m, aromatic protons). High-resolution mass spectrum (electron impact: calculated for C25H2804
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Bloconjugate Chem., Vol. 4, No. 6, 1993 469
2-5A Antlsense Chimeras
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Figure 1. Electropherogram of p&:TI8. The region of the chromatogramfrom 0-10 min containedno peaks and is therefore not shown.
392.1988, found 392.1981. This product was converted to 2-cyanoethyl 4-(4,4’-dimethoxytrity1)butylN,N-diisopropylphosphoramidite. (2-Cyanoethyl)-N,N-diisopropylphophonamidic chloride (237 mg, 1mmol) was added slowly to a solution of 4-0-(4,4’-dimethoxytrityl) - 1,4butanediol(390 mg, 1mmol) and ethyldiisopropylamine (510 mg, 4 mmol) in anhydrous methylene chloride (3 mL) under anhydrous conditions with cooling (ice bath). The mixture was kept at room temperature for 1h, the solvent was evaporated, and the product was purified on a silica gel column (1.8 X 14 cm, eluted with benzene-petroleum ether-triethylamine (6:3:1). The yield was 330 mg (90%). lH NMR (CDC13,1% deuteriopyridine) 6 [ppml: 1.17 (t, J = 7.0 Hz, 12H, CHsC), 1.70 (m, 4H, CHzC), 2.60 (t, J = 6.5 Hz, 2H, CHzCN), 3.08 (t,J = 5.7 Hz, 2H, CHzO); 3.80 (m, 4H, CHzOP), 3.78 (s, 6H, CH30), 3.80 (m, 2H, CHI, 6.80-7.49 (m, aromatic protons). 3lP NMR (CDC13, 1% deuteriopyridine) 6 [ppml: 147.6. High-resolution mass spectrum (FAB): calculated for C ~ ~ H ~ ~ N 593.3144, ZO~P found 593.3112. Preparation of Oligonucleotide A4:Tla. This oligonucleotide was prepared by alkaline phosphatase digestion (2 units, 0.1 M Tris chloride, pH 8.0,37 “C, overnight) of the crude pA4:Tle and was purified on a DEAE-NPR anionexchange column. Oligonucleotide Purification. Oligonucleotide pA4: Tla was purified on the Vydac column using HPLC system B with an elution gradient of 0.02 to 0.28 M ammonium phosphate, in 30 min. Approximately 80% of applied sample was recovered. The compound showed only one major peak when analyzed on the DEAE-NPR ionexchange column (HPLC system A, with a 30-min gradient of 0.1 to 0.4 M NaC1, tR = 28 min) and by capillary electrophoresis (Figure 1). Oligonucleotide &:TIS was purified under similar conditions on the DEAE-NPR column. Oligonucleotides pA4:T4, AT4, and A:T were purified on the semipreparative Ultrasphere ODS column (system B, a 30-min gradient of 20 to 100% of B). Oligonucleotide Characterization. The oligonucleotides were characterized by digestion with snake venom phosphodiesterase. Substrate (0.1-0.2 OD) was incubated with 0.05-0.1 unit of SVPD in 50 mM Tris chloride, pH 8.0,0.5 mM MgClZ, 1-3 h, 37 “C. When oligonucleotide AT4 was digested under these conditions, there resulted
two products formed in a 4 1 ratio. The product appearing in greater quantity was 5’-TMP. The other product corresponded to adenosine coupled to the butanediol linkers (referred to hereafter as A w r ) since upon overnight reaction with 0.5 M KOH, it gave adenosine 2’(5’)monophosphate. Digestions of oligonucleotides p&:Tle, A4:T18, and p&:T4 with snake venom phosphodiesterase under the above conditions gave the following products in the indicated ratios: 5’-TMP, 5’-AMP, P5’Alinker (18:3:1); 5’-TMP, A, 5’-AMP, P6’Alinker (18:1:2:1); and 5’-TMP, 5’AMP, P5’Alinker (4:3:1). The structures of oligonucleotides pA4:T4, AT4, and A:T were corroborated further by proton NMR. Oligonucleotide pA4:T4 ‘H NMR (DzO) 6 [ppml: 1.84 and 1.87 (both s, total of 12 H, 4 thymidine CHs’s), 6.056.25 (m, 4 H, 4 adenosine H-1’ protons), 6.45-6.55 (m, 4 H, 4 thymidine H-1’ protons), 7.80-8.40 (m, 12 H, adenine H-2 and H-8, thymine H-6 protons). OligonucleotideA:T4 ‘H NMR (Dz0) 6 [ppml: 1.83 and 1.85 (12 H, 4 thymidine CH3’s), 6.13 (d, J = 7.2 Hz, 1H, adenosine H-1’ proton), 6.22 (m, 4 H, thymidine H-1’ protons), 7.63 to 7.65 (m, 4 H, thymine H-6 protons), 8.21 and 8.31 (both s, adenine H-2 and H-8). Oligonucleotide A:T ‘H NMR (DzO) 6 [ppml: 1.91 ( 8 , 3H, thymidine CH3), 6.29 (d, l H , adenosine H-1’, J = 6.6 Hz), 6.34 (t, l H , thymidine H-1’, J = 7.0 Hz), 7.74 (8,l H , thymine H-6), 8.36 and 8.58 (s, l H , adenine H-2 and H-8). RESULTS AND DISCUSSION
Joining 2-5A in covalent linkage to an antisense 3’,5’linked oligonucleotide conceivably could produce a chimeric molecule with the advantages of both of the two distinct approaches to inhibition of mRNA expression. A high degree of specificity, apparently missing from RNase L-directed cleavages, could be imparted by the antisense domain of the chimera, whereas the 2-5A domain could provide a localized [by virtue of the 2’,5’-phosphodiesterase (31)]activation of the potent 2-5A-dependent RNase L. The antisense partner in the cojoined molecule also may facilitate the uptake of the 2-5A since it is believed that antisense DNA oligomers are taken up by cells, perhaps, by a specific transport mechanism (32). For the design of a first-generation of 2-5A-antisense chimeras, we chose to prepare an oligo(dT) Wmer to simplify both synthesis and characterization of this molecule. We added the tetrameric 2-5A derivative p5’A2’~5’A2’pVA2’~5’A to the 5’-terminus of the antisense oligomer. Only a 5’-monophosphate was required at the terminus of the 2-5A component since the human RNase L from CEM and Daudi lymphoblastoid cells can be activated by the monophosphate and does not require a 5’-triphosphate, as does RNase L from most lines of mouse cells (33,34). The two biologically active molecules were linked with two 1,cbutanediol units joined to each other and to the antisense and 2-5A by phosphodiester bonds. This allowed the synthesis of both components and the linkage reaction also to be carried out in one reaction sequence on a solid support using phosphoramidite oligonucleotide synthesis technology. Although we linked the 2’4erminus of the 2-5A domain through butanediol linkers to the 5‘-end of the antisense oligonucleotide,other modes of linkage would be equally viable options. The only exception would be linkages that would incorporate the 5‘-terminus of the 2-5A partner in the chimera. Previous studies (35) have shown that incorporation of this 5’-phosphate into an internucleotide linkage results in significantly decreased binding to RNase L. Our choice of two butanediol units as linkers was somewhat arbitrary. Our chief reason to use linkers at all
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Time (min) Figure 2. Kinetics of the hydrolysis of 2-5A monophosphate as compared to the 2-5A:AS chimeras. Oligonucleotides AT4 (0) and p5‘A2’p5’A2‘p5‘A2‘p5‘A (m) were both evaluated at initial concentrationsof 0.25 mM whereas the initial concentration of oligonucleotide p&:Tla( e) was 0.13 mM. Reaction mixtures were constituted in 50 mM Tris HC1, pH 8.0, 1 mM MgClz, containing 2.5 X 103 units of snake venom phosphodiesterase (Cooper Biomedical). Incubation was at 37 O C . Aliquots were removed at the indicated times and analyzed by HPLC. In the case of 2-5A tetramer monophosphate,the formation of 5’AMP was used as a marker of degradation whereas, for the other two oligomers,5’TMP formation was used to follow degradation since no AMP was released during the time of reaction. In addition, the parent (n)oligonucleotide peak could not be employed for exact quantitation during degradation since its integration was interfered with by the n - 1 peak formed by action of the phosphodiesterase. See the text for further details. was governed by a concern that the 2-5A-dependent endonuclease might distrub hybrid helix formation if it were proximal to the antisense component, or that the resulting hybrid helix formation might interfere with binding of the 2-5A component to the 2-5A-dependent endonuclease. Current studies are addressing the effect, if any, of the nature and length of linker needed for optimal chimera activity. The structures of the synthesized 2-5A:antisense chimeras were confirmed by the enzymatic digestion and NMR (vide supra). Snake venom phosphodiesterase digestion permitted determination of the ratio of adenylate residues to thymidylate residues and in the case of compounds pA4:Tla and p&:T4 allowed identification of the 5’-terminal residue. Proton NMR confirmed the presence of the requisite numbers of thymidine methyl groups and pyrimidine H6 and anomeric (Hl’) protons as well as adenosine H2, H8, and anomeric protons. The oligonucleotide, 2-5A, is subject to facile degradation by phosphodiesterases including one with specificity toward 2’,5’-linkages (36, 37). We examined enzymic degradation of chimeric constructs by monitoring HPLC’s of aliquots removed at various times during digestion by snake venom phosphodiesterase. Although, we monitored both the starting oligonucleotide peak and intermediate peaks (n- 1, etc.), as well as formation of product dTMP and/or AMP peaks, it was most descriptive to present the results of the degradation of 2-5A itself as AMP formation and the degradation of chimeric molecules by TMP formation, since in the latter instance no AMP was detectable during degradation (Figure 2). This is to be expected since snake venom phosphodiesterase acts from the 3’(2’)-terminus of the oligonucleotide. Addition of the oligothymidylate antisense component to the 2’-terminus of the 2‘,5‘-oligoadenylate completely protected the 2-5A
,
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-M OLIGOMER Figure 3. Radiobinding assays to determine the affinity of the chimera p&:Tls (A)for the 2-5A-dependent RNase of mouse liver cytoplasmic extract as compared to 2-5A (ppp5’A2’p5’A2’p5’A, 0 )itself. This procedure employed the radiolabeled probe p5’A2’p5’A2’p5’A2fp5’A3’[~2P]p5’C3’pand a nitrocellulose filter binding assay (39) to determine the concentration of 2-5A or chimera required to displace a constant concentration of radiolabeled probe from the 2-5A-dependent RNase. 100%binding refers to the binding of radiolabeled probe in the absence of any other added oligonucleotide. The data for p&:Tls represent the means and standard deviationsfromsix assays whereas the 2-5A data were gathered from seven determinations. moiety from degradation by snake venom phosphodiesterase under conditions that led to rapid degradation of unmodified 2-5A itself (Figure 2). Observation of this effect required the use of a 100-foldsmaller concentration of enzyme than was employed for the enzyme characterization studies (vide supra). Presumably this antisense “tailing” (38) of 2-5A also would provide resistance to degradation by the 2’,5’-phosphodiesterase. Other chemical modifications at the 2’-terminus of 2-5A have been shown to impart considerabIe resistance to phosphodiesterases and to potentiate translational inhibition and antiviral activity (2). The ability of the 2-5A:antisense constructs to bind to the 2-5A-dependent RNase L was evaluated using a radiobinding assay (39)which measures the ability of 2-5A or an analogue to compete with a 32P-labeled probe, p5’A2’p5’A2’p5‘A2’p5fA3’[32Plp5’C3’p,for the endonuclease. Chimeric 2-5A:antisense congeners were undiminished in their ability to bind to the 2-5A-dependent RNase. Thus the tetramer-tetramer adductp&:dTd, when examined for its affinity for the 2-5A-dependent RNase of mouse liver, gave an IC50 (concentration needed to inhibit probe binding by 50%) of 1 X 10-9 M, not significantly different from the IC50 of 7 X 1@l0 M obtained for 2-5A itself, in agreement with previous reports (40, 41). The length of the antisense sequence of the chimera did not have any negative effect on endonuclease binding since as is clear from the data of Figure 3, there was very little, if any, significant decrease in the ICs0 of 5’A2’p5’A2’p5‘A as compared to ppp5’A2‘p5’A2’p5’A itself. As noted earlier (20), UV absorbance-temperature profiles revealed that the 2-5A moiety of the p&:dTlg chimera did not prevent the ability of the T l g region to anneal with its complementary sequence as represented in poly(A) (Figure 4). When this p&:dTla-poly(A) complex was denatured in such a melting experiment and the solution was allowed to cool down to room temperature, the complex reformed, thereby indicating that the complex formation was fully reversible and that no significant decompositionoccurred during the melting process. Under the same conditions, the complex d(T)zo:poly(A) had a T,
2-5A Antisense Chimeras
1’35 1.25
Bioconjugate Chem., Vol. 4, No. 6, 1993 471
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to an antisense-targeted sequence within a modified HIV-1 RNA in a cell-free system from human lymphoblastoid cells (20).
1
LITERATURE CITED
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V0C) Figure 4. Absorbance-temperature profile of an equimolar mixture of poly(A) and p&Tla as determined in KHzPOl buffer (40 mM, pH 7.0). In this experiment, lo00 r L of 3.2 X 1o-B M poly(A) in KHzPOl buffer (pH 7.0,40 mM) was mixed with 1000
WL of 2.9 X 1o-BM p&:Tla in KH~POIbuffer, and the resulting
mixture was incubated at 4 OC overnight before determination of melting temperature. AJAi refers to the ratio of absorbance (At) at a given temperature compared to the absorbance (Ai) at the initial temperature (25-30 OC). No attempt was made to determine complex stoichiometry (2- or 3-stranded). Under similar conditions, an equimolar mixture of poly(A) and oligo(dT)m gave a complex with a T , of 40 OC.
of 40 O C , in good agreement with literature valuesreported under similar conditions (21). To date, we have not determined whether or not the p&:dTle possesses selfcomplementary structure. When the antisense region of the chimera was annealed to its complementary sequence, there was no significant effect on the ability of the construct to bind to 2-5Adependent RNase of mouse liver. For example, when pA4: dTl8 was mixed in various concentrations with excess poly(A), there was no significant change in the binding affinity as compared with the unannealed pA4:dTla or as compared to 2-5A itself (Figure 3). In addition, when a radiobinding assay was performed on an aliquot of the above solution that had been used to determine the T, of the pA4:Tl~ complex, an identical binding curve was obtained (data not shown). We have prepared a novel bioconjugate; specifically, a chimera formed by the linkage of an antisense oligonucleotide to 2’,5’-oligoadenylate, thereby linking the antisense strategy for control of gene expression with the interferon-associated 2-5A system. Such conjugate molecules retained their ability to bind to both relevant receptors, i.e., the nucleic acid sequence complementary to the antisense sequence present in the conjugate as well as the binding of the 2-5A domain to the 2-5A-dependent RNase (421, which would be responsible for targer RNA degradation. Moreover, the hybrid complex between the 2-5A.antisense chimera and the target RNA also retained undiminished 2-5A-dependent RNase binding ability. We also have demonstrated that the antisense oligonucleotide component of the 2-5A:antisense conjugate targets a specific RNA sequence to which it binds while the accompanying 2-5A component activates the latent constitutive 2-5A-dependent RNase thereby causing the cleavage of the RNA (20).This strategy and methodology was demonstrated to produce specific cleavages proximal
(1) Kerr, I., and Brown, R. E. (1978) pppA2’p5‘A2‘p5‘A
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