Mar 14, 1995 - HIV-1 IIIB was originally obtained from Dr Robert Galo. (NCI) (32) ..... Arneson,M.A., Pirruccello,S.J., Ruddon,R.W., Kessinger,A., Zon,G. and.
3578-3584 Nucleic Acids Research, 1995, Vol. 23, No. 17
(Kc) 1995 Oxford University Press
Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides Ekambar R. Kandimalla, Adrienne Manning, Christopher Lathan1, Randal A. Byrn1 and Sudhir Agrawal* Hybridon, Inc., 1 Innovation Drive, Worcester, MA 01604, USA and lThe Robert Mapplethorpe Laboratory for AIDS Research, Division of Hematology/Oncology, Department of Medicine, The New England Deaconess Hospital, Harvard Medical School, Boston, MA 02215, USA Received March 14, 1995; Revised and Accepted June 26, 1995
ABSTRACT Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21 mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable temary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21 mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21 mer, and evoke RNase H activity. HIV-1 Inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases. INTRODUCTION Progress in chemical synthesis of nuclease-resistant oligonucleotides (1) and developments in large scale solid phase synthesis of oligonucleotides (1,2) have permitted antisense oligonucleotides to advance to human clinical trials (3-5). Antisense oligonucleotides recognize target mRNA sequences through Watson-Crick hydrogen bonding between A and T and G and C. This recognition is highly specific and should lead to the development of less toxic and more site-specific therapeutic agents (6). The length of the antisense oligonucleotide affects its specificity for the target sequence. An oligonucleotide of 13 or more bases long *
To whom correspondence should be addressed
should bind to a unique sequence that occurs only once in a eukaryotic mRNA pool (7). Theoretically, sequence specificity should increase as the length of oligonucleotide (>l5mer) increases, but, practically, increasing the length of an antisense oligonucleotide beyond the minimum length that can hybridize to the target (i.e. 11-14 bases) might decrease its specificity (8,9). A decrease in hybridization specificity might lead to non-sequencespecific effects and subsequent increased toxicity (8,10). Cooperative interactions often serve to improve sequence specificity, affmiity and biological activity of macromolecules (11). Several studies have demonstrated how cooperative interactions can be used to develop small molecule-based drugs (12,13). Cooperative binding of oligonucleotides (14) or their conjugates (15) to single-stranded DNA or RNA (16) through duplex formation or double-stranded DNA through triplex formation (17-21) has been reported. GEM 91 is a 25mer phosphorothioate oligonucleotide that binds to a complementary sequence in the initiation codon of the HIV-1 gag mRNA (3,22). This purine-rich sequence is highly conserved (23,24) and is necessary for dimerization of two RNA genomes within the virus particle (25,26). In the present study we used a 21 base site as the target and compared different designs of short oligonucleotides that can bind adjacently on the target with a 21mer oligonucleotide that binds to the same site. The oligonucleotides used in the study are shown in Table 1. We used thermal melting and RNase H assays to determine the binding affinity and specificity ofthe oligonucleotides to their target sequences and also tested the ability of the new oligonucleotide designs to inhibit HIV-l in cell cultures.
MATERIALS AND METHODS Oligonucleotide synthesis and purification The oligodeoxyribonucleotides were synthesized on a Milligen 8700 DNA synthesizer using ,B-cyanoethylphosphoramidite chemistry on a solid support. Monomer syntions and other DNA synthesis reagents were obtained from Milligen Biosearch. After synthesis and deprotection, oligonucleotides were purified on reverse phase (C18) HPLC, detritylated, desalted (Waters C18 sep-pack cartridges) and checked for purity by PAGE (27).
Nucleic Acids Research, 1995, Vol. 23, No. 17 3579 RNA was synthesized in a similar manner, but using an RNA synthesis cycle, with 2'-t-butyldimethylsilyl-3'-p-cyanoethyl-N,Ndiisopropyl phosphoramidites purchased from Millipore. After synthesis RNA was deprotected with a 3:1 mixture of ammonium hydroxide and ethanol at 55°C for -16 h and then with tetrabutylammonium fluoride at room temperature for another 16 h. RNA was then purified on 20% denaturing PAGE, eluted from the gel and desalted using a C18 sep-pack cartridge (Waters). Phosphorothioate oligonucleotides for RNase H and HIV-1 inhibition studies were synthesized as above, but using sulfuizing agent as the oxidant, instead of iodine. Post-synthetic processing was carried out as above, but desalting was performed by dialysis for 72 h against double distilled water. UV thermal melting studies UV melting experiments were carried out in 150 mM NaCl, 10 mM sodium dihydrogen phosphate, 2 mM MgCl2, pH 7.4, buffer using a DNA target strand. The oligonucleotide concentration was 0.36 ,uM as a single strand. The oligonucleotides were mixed in buffer, heated to 95°C, cooled to room temperature and left at 4°C overnight. Thermal denaturation profiles were recorded at 260 nm at a heating rate of 0.5 °C/min on a Perkin-Elmer Lambda 2 spectrophotometer equipped with a Peltier thermal controller and attached to a personal computer for data collection. The melting temperatures (Tm) were measured from the first derivative plots (dA/dTversus 7). Each value is an average of two separate runs and the values are within ±1.0°C.
RESULTS AND DISCUSSION Design of oligonucleotides Oligonucleotide 1 binds to the entire 21 base target sequence (Table 1). Oligonucleotides 2 and 3 are 9- and 12mers, respectively (Table 1) which bind to the target sequence at adjacent sites (Fig. 1A) and cover the same 21 base target as oligonucleotide 1. Oligonucleotides 4 and 5 bind to the same site as 3 but are separated by one and two bases on the target sequence respectively from the binding site of oligonucleotide 2. Oligonucleotides 6-11 have an extended sequence on either the 5'- or 3'-end of the binding sequence, which forms a duplex stem between the two cooperative oligonucleotides when they both bind to the target at adjacent sites (Fig. IB). Oligonucleotide pair 6 & 9 forms a 3 bp stem. Oligonucleotide pairs 7 & 10 and 8 & 11 bind to the same length of the target as oligonucleotide pair 6 & 9, but with 5 and 7 bp extended dimerization domains, respectively. These oligonucleotides bind to the target with one base separation between them. Oligonucleotides 12-15 contain one or two mismatches, as shown in Table 1. Table 1. Oligonucleotide sequences used in the study
Sequence no. RNA (target)
RNase H assay
RNA target was labeled at the 3'-end using T4 RNA ligase (New England Biolabs) and [32P]pCp (New England Nuclear) using standard protocols (27). End-labeled RNA (3000-5000 c.p.m.) and -5-10 pmol of each oligonucleotide studied was mixed with 90 pmol yeast tRNA in 30 g1 of a solution containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM KCI, 0.1 mM DTT, 5% w/v sucrose and 40 U RNasin (Promega) at 4°C overnight. An aliquot (7 gl) was removed as a control, 1 1 (0.8 U) Escherichia coli RNase H (Promega) was added to the remaining reaction mixture and the mixture was incubated at room temperature. Aliquots (7 ,l) were removed at different time intervals and the samples analyzed on a 7 M urea-20% polyacrylamide gel. After electrophoresis the autoradiogram was developed by exposing the gel to Kodak X-Omat AR film at -70°C.
HIV-1 inhibition assay The effect ofthe antisense oligonucleotides on replication of HIV- 1 during an acute infection was determined. The test system is a modification of the standard cytopathic effect (CPE)-based MT-2 cell assay (28-30). Briefly, serial dilutions of antisense oligonucleotides or combinations of oligonucleotides were prepared in 50 ml volumes of complete medium (RPMI-1640, 10% fetal bovine serum, 2 mM L-glutammine, 100 U/ml penicillin, 100 mg/ml streptomycin) in triplicate in 96-well plates. Virus, diluted to contain a 90% CPE dose of virus in 50 ml, was added, followed by 100 ml 4 x 105/ml MT-2 cells in complete medium. The plates were incubated at 37°C in 5% CO2 for 5 days. MT dye was added and quantitated at OD540-OD690 as described (28). Percent inhibition was calculated by the formula (experimental virus -
control)/(medium control
MT-2 cells (31) were provided by the AIDS Reference and Research Program (Division of AIDS, NIAID, NIH, Bethesda, MD). HIV-1 IIIB was originally obtained from Dr Robert Galo (NCI) (32) and propagated in H9 cells (33) by the method of Vujcic (34).
virus
control) x loo.
Sequencea
5'-GGAGGCUAGAAGGAGAGAGAUGGGUGCGAGAGCGU
5
5'-CTAGAAGGAGAGAGATGGGTGCGAGAG 5'-CTCGCACCCATCTCTCTCCTT 5'-CTCGCACCC 5'-ATCTCTCTCCTT 5'-TCTCTCTCCTTC 5'-CTCTCTCCTTCT
6
5'-CGGTCTCTCTCCTTC
7
5'-GCCGGTCTCTCTCCTTC
8 9 10 11 12 13 14 15 GEM 91
5'-GCGCCGGTCTCTCTCCTTC 5'-CTCGCACCCCCG 5'-CTCGCACCCCCGGC
DNA (target)
1
2 3 4
5'-CTCGCACCCCCGGCGC 5'-CTCtCACCCATCTCTCTCCTT 5'-CTCtCAaCCATCTCTCTCCTT 5'-CTCtCACCC 5'-CTCtCAaCC 5'-CTCTCGCACCCATCTCTCTCCTTCT
aSequence shown in bold is the 21 base target, underlined bases represent the extended dimerization domain and lower case letters indicate mismatches.
Thermal melting studies Thermal melting data of the oligonucleotides are summarized in Table 2. Thermal melting experiments were carried out using a
3580 Nucleic Acids Research, 1995, Vol. 23, No. 17
A
B
I I'
5,
Figure 1. Schematic representation of the two designs of short oligonucleotides that bind to adjacent sites cooperatively. (A) Binding of two short oligonucleotides to tandem sites. (B) Binding of oligonucleotides that have extended dimerization domains and their dimerization.
DNA target strand and phosphodiester oligonucleotides. The duplex of the DNA target and oligonucleotide 1 shows a Tm of 67.7°C (Fig. 2). The Tm of the double helical complex of oligonucleotides 2 & 3 with the target sequence is 47.8°C. The duplexes of oligonucleotides 2 & 4 and 2 & 5 with the target sequence have Tm values of 44.4 and 46°C, respectively. The Tm of the duplex fonmed by oligonucleotides 2 & 3 together is greater than that of the average of the duplexes formed by 2 and 3 individually with the target sequence (Table 2). In contrast, the Tm values of the oligonucleotide combinations 2 & 4 and 2 & 5 are less than the average of the Tm values of the two individual oligonucleotides in the experiment. These data suggest that the two short oligonucleotides 2 and 3 targeted to two adjacent sites bind in a cooperative fashion. Furthermore, we observed a single, sharp, cooperative transition in the Tm of the duplex of oligonucleotides 2 & 3 with the target sequence (Fig. 2B). The cooperative interactions (2 & 3) are probably driven by stacking interactions between the terminal bases (35). Thennal melting studies of the duplexes of oligonucleotides 6-11 suggest that the stability of the terary complex formed increases as the number of base pairs in the dimerization domain increases (Table 2). The double helical complexes with 3 (6 & 14), 5 (7 & 10) and 7 (8 & 11) bp dimerization domains show Tm values of 45.9, 48.4 and 53.2°C, respectively. In the absence of the target sequence these combinations of oligonucleotides show no defined melting transition, indicating no stable complex fornation. Further increases in the length of the dimerization domains (more than seven bases), however, result in the formation of a stable complex between the two tandem oligonucleotides even in the absence of the target sequence. In all cases we observed a single, sharp, cooperative melting transition (Fig. 2C). These data suggest that the cooperative interactions are further facilitated by extended dimerization domains, which form a duplex stem when the tandem oligonucleotides bind to the target at adjacent sites. The duplex of oligonucleotide 12, which contains a mismatched base, and the target sequence has a Tm of 61.4°C. The duplex of the target sequence with oligonucleotide 13, with two
mismatches, has a Tm of 55.6°C. The duplex of mismatched oligonucleotide 14 with the target shows a broad transition below 30°C, without a defined Tm (data not shown). We observed no transition in the case of oligonucleotide 15, indicating that this oligonucleotide does not interact with the target. These Tm values suggest that the short oligonucleotides are more sensitive to mismatches than the longer oligonucleotides (8,9,36). The Tm values obtained for the duplexes of mismatched oligonucleotides 12 and 13 (21mers) with the target sequence clearly suggest that longer oligonucleotides can bind to any sequence containing up to two mismatches under physiological temperature and salt conditions (36). RNase H assay RNase H is an enzyme that recognizes RNA-DNA heteroduplexes and hydrolyzes the RNA component of the heteroduplex (37). Earlier studies indicate that a 4-6 bp hybrid is sufficient to evoke RNase H activity (38). We investigated the RNase H activation properties of phosphorothioate analogs of oligonucleotides, using a 35mer RNA target sequence (Table 1). This assay provides additional information on the sequence-specific binding of cooperative oligonucleotides. We compared the RNase H activation properties of oligonucleotides 14 and 15 with those of oligonucleotides 12 and 13 to establish the sequence specificity of short tandem versus longer oligonucleotides. Figure 3 shows the RNase H hydrolytic pattern of the target RNA in the presence of the mismatched oligonucleotides. Oligonucleotide 12, with one mismatch (experiment 5 in Fig. 3), showed a similar RNase H degradation pattern to the completely matched oligonucleotide 1 (experiment 1 in Fig. 3). Oligonucleotide 13, with two mismatches (experiment 6 in Fig. 3), showed little or no RNA hydrolysis where the mismatches are located. The degradation pattern, however, on either side of the mismatches is very similar to that observed with oligonucleotide 1 without mismatches. These results indicate that in spite of the
Nucleic Acids Research, 1995, Vol. 23, No. 17 3581 Table 2. Tm of oligonucleotides with DNA target sequencea Oligonucleotide
Complexb
Tm, OCc
1
TT CCTC TCT CTACCCACGC TC
67.7 (s)
2
CCCACGCTC
49.1 (s)
3
4 5
43.4(s)
TTCCTCTCTCTA
43.6(s)
CTrCCTCTCTCT
45.0(s)
TCTTCCTCTCTC
2+3
TT CCTC TCT CTA-CCCACGC TC
47.8 (s)
2+4
CTT CCTC TCT CT CCCACGC TC
44.4 (b)
CCCACGC TC
45.9 (b)
CTT CCTC TCT CT CCCACGCTC G C
45.9 (b)
2+5
T CTT CCTC TCT C
0.8-
6+9
GC C G
'u0.6
0.4-
7+10
z
0.2-
CCCACGCTC GC G C
CTT CCTC TCT CT
48.4 (s)
C G
CG G C
0
l.0-c
8+11
8+11
6+9
CUT CCTC TCT CT CCCACGCTC G G C C G C G
0.87+10
0.6-
0.4-
0.2-
C C G G
53.2 (s)
C G C
12
TT CCTC TCT CTACCCACTC TC
61.4(b)
13
TT CCTC TCT CTACCAACIC TC
55.6 (b)
0
20
30
40
50
60
70
80
Temperature, °C
Figure 2. Thermal melting profiles (dAWdT versus 7) of representative oligonucleotides with their DNA target. Numbers correspond to oligonucleotide numbers in Table 1.
two mismatches, oligonucleotide 13 binds to the RNA strongly enough to activate RNase H. In the case of oligonucleotides 14 and 15, with one and two
mismatches, respectively, (experiments 2 and 3 in Fig. 3), we observed little or no RNA degradation as compared with oligonucleotide 2 (experiment 1 in Fig. 3). These results demonstrate sequence-specific binding of the cooperative oligonucleotides to the target. The results with oligonucleotides 12 and 13 suggest that in an in vivo situation antisense oligonucleotides of .20 bases might bind to a number of sequences in the mRNA pool containing up to two mismatches and evoke RNase H activity (8,36) (vide infra thermal melting studies). An autoradiogram showing the RNase H hydrolysis pattern of the RNA target in the absence and presence of oligonucleotides without mismatches is shown in Figure 4. In experiments 2 and 5 in Figure 4 RNase H hydrolytic activity occurs towards the 3'-end of the target RNA (the lower half of the autoradiogram) in which oligonucleotides 2 and 11 are present. Similarly, RNA degradation bands are present only in the upper half of the
aSee experimental section for buffer conditions. bDNA target strand is shown as a thin line for complex structure (see Table 1 for sequence); underlined bases represent mismatches; X represents number of base separations between the two binding oligonucleotides. cLetters s and b in parentheses indicate sharp and broad transitions, respectively.
autoradiogram, indicating binding of oligonucleotides 3 and 8 on the 5'-side of the target (Fig. 4, experiments 3 and 6, respectively). When combinations of oligonucleotides 2 & 3 and 8 & 11 are present (Fig. 4, experiments 4 and 7, respectively) the RNase H degradation pattern is very similar to that observed with control oligonucleotide 1 (Fig. 4, experiment 1). These results suggest that the short tandem oligonucleotides bind to the target RNA as expected, with sequence specificity, and evoke RNase H activity. The intact RNA target is digested over time as a result of RNase H action on the hybrid duplex. The extent (efficiency) of RNase H hydrolysis depends on dissociation of the hybrid duplex formed (9). Dissociation of the duplex depends on the length of the antisense oligonucleotide and the mismatches present in the hybrid. In the presence of oligonucleotides greater than 9mers RNA is degraded by >80% in 5 min and lO0% in 15 min. In the presence of oligonucleotide 2 (9mer) RNA is not completely hydrolyzed after up to 15 min. We observed similar hydrolysis patterns in the presence of oligonucleotides 9-11 (11- to l5mers).
These results suggest that the extended domain remains as a dangling end in solution and does not interfere with binding of the
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