3578-3584 Nucleic Acids Research, 1995, Vol. 23, No. 17

Design, biochemical, biophysical and biologicalproperties of cooperative antisense oligonucleotidesEkambar R. Kandimalla, Adrienne Manning, Christopher Lathan1, Randal A. Byrn1 andSudhir Agrawal*

Hybridon, Inc., 1 Innovation Drive, Worcester, MA 01604, USA and lThe Robert Mapplethorpe Laboratory forAIDS Research, Division of Hematology/Oncology, Department of Medicine, The New England DeaconessHospital, 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 siteson target mRNA sequences are designed and evalu-ated for their binding affinity and biological activity.Sequence-specific binding of short tandem oligo-nucleotides is compared with a full-length singleoligonucleotide (21 mer) that binds to the same targetsequence. Two short oligonucleotides that bind with-out a base separation between their binding sites onthe target bind cooperatively, while oligonucleotidesthat have a one or two base separation between thebinding oligonucleotides do not. The binding affinity ofthe tandem oligonucleotides is improved by extendingthe ends of the two oligonucleotides with complemen-tary sequences. These extended sequences form aduplex stem when both oligonucleotides bind to thetarget, resulting in a stable temary complex. RNase Hstudies reveal that the cooperative oligonucleotidesbind to the target RNA with sequence specificity. Ashort oligonucleotide (9mer) with one or two mis-matches does not bind at the intended site, while longeroligonucleotides (21 mers) with one or two mismatchesstill bind to the same site, as does a perfectly matched21 mer, and evoke RNase H activity. HIV-1 Inhibitionstudies reveal an increase in activity of the cooperativeoligonucleotide combinations as the length of thedimerization domain increases.

INTRODUCTION

Progress in chemical synthesis of nuclease-resistant oligonucleo-tides (1) and developments in large scale solid phase synthesis ofoligonucleotides (1,2) have permitted antisense oligonucleotidesto advance to human clinical trials (3-5). Antisense oligonucleo-tides recognize target mRNA sequences through Watson-Crickhydrogen bonding between A and T and G and C. Thisrecognition is highly specific and should lead to the developmentof less toxic and more site-specific therapeutic agents (6). Thelength of the antisense oligonucleotide affects its specificity forthe target sequence. An oligonucleotide of 13 or more bases long

should bind to a unique sequence that occurs only once in aeukaryotic mRNA pool (7). Theoretically, sequence specificityshould increase as the length of oligonucleotide (>l5mer)increases, but, practically, increasing the length of an antisenseoligonucleotide beyond the minimum length that can hybridize tothe target (i.e. 11-14 bases) might decrease its specificity (8,9). Adecrease in hybridization specificity might lead to non-sequence-specific effects and subsequent increased toxicity (8,10).Cooperative interactions often serve to improve sequence

specificity, affmiity and biological activity ofmacromolecules (11).Several studies have demonstrated how cooperative interactionscan 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 duplexformation 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-1gag mRNA (3,22). This purine-rich sequence is highly conserved(23,24) and is necessary for dimerization of two RNA genomeswithin the virus particle (25,26). In the present study we used a 21base site as the target and compared different designs of shortoligonucleotides that can bind adjacently on the target with a 21meroligonucleotide that binds to the same site. The oligonucleotidesused in the study are shown in Table 1. We used thermal meltingand RNase H assays to determine the binding affinity andspecificity ofthe oligonucleotides to their target sequences and alsotested the ability of the new oligonucleotide designs to inhibitHIV-l in cell cultures.

MATERIALS AND METHODSOligonucleotide synthesis and purification

The oligodeoxyribonucleotides were synthesized on a Milligen8700DNA synthesizer using ,B-cyanoethylphosphoramidite chem-istry on a solid support. Monomer syntions and other DNAsynthesis reagents were obtained from Milligen Biosearch. Aftersynthesis and deprotection, oligonucleotides were purified onreverse phase (C18) HPLC, detritylated, desalted (Waters C18sep-pack cartridges) and checked for purity by PAGE (27).

* To whom correspondence should be addressed

(Kc) 1995 Oxford University Press

Nucleic Acids Research, 1995, Vol. 23, No. 17 3579

RNA was synthesized in a similar manner, but using an RNAsynthesis cycle, with 2'-t-butyldimethylsilyl-3'-p-cyanoethyl-N,N-diisopropyl phosphoramidites purchased from Millipore. Aftersynthesis RNA was deprotected with a 3:1 mixture of ammoniumhydroxide and ethanol at 55°C for -16 h and then withtetrabutylammonium fluoride at room temperature for another16 h. RNA was then purified on 20% denaturing PAGE, elutedfrom the gel and desalted using a C18 sep-pack cartridge (Waters).

Phosphorothioate oligonucleotides for RNase H and HIV-1inhibition studies were synthesized as above, but using sulfuizingagent as the oxidant, instead of iodine. Post-synthetic processingwas carried out as above, but desalting was performed by dialysisfor 72 h against double distilled water.

UV thermal melting studies

UV melting experiments were carried out in 150 mM NaCl, 10mM sodium dihydrogen phosphate, 2 mM MgCl2, pH 7.4, bufferusing a DNA target strand. The oligonucleotide concentrationwas 0.36 ,uM as a single strand. The oligonucleotides were mixedin buffer, heated to 95°C, cooled to room temperature and left at4°C overnight. Thermal denaturation profiles were recorded at260 nm at a heating rate of 0.5 °C/min on a Perkin-Elmer Lambda2 spectrophotometer equipped with a Peltier thermal controller andattached to a personal computer for data collection. The meltingtemperatures (Tm) were measured from the first derivative plots(dA/dTversus 7). Each value is an average oftwo separate runs andthe values are within ±1.0°C.

RNase H assay

RNA target was labeled at the 3'-end using T4 RNA ligase (NewEngland Biolabs) and [32P]pCp (New England Nuclear) usingstandard protocols (27). End-labeled RNA (3000-5000 c.p.m.)and -5-10 pmol of each oligonucleotide studied was mixed with90 pmol yeast tRNA in 30 g1 of a solution containing 20 mMTris-HCl, pH 7.5, 10mM MgCl2, 10mM KCI, 0.1 mM DTT, 5%w/v sucrose and 40 U RNasin (Promega) at 4°C overnight. Analiquot (7 gl) was removed as a control, 1 1 (0.8 U) Escherichiacoli RNase H (Promega) was added to the remaining reactionmixture and the mixture was incubated at room temperature.Aliquots (7 ,l) were removed at different time intervals and thesamples analyzed on a 7 M urea-20% polyacrylamide gel. Afterelectrophoresis the autoradiogram was developed by exposingthe gel to Kodak X-Omat AR film at -70°C.

HIV-1 inhibition assay

The effect ofthe antisense oligonucleotides on replication ofHIV- 1

during an acute infection was determined. The test system is a

modification of the standard cytopathic effect (CPE)-based MT-2cell assay (28-30). Briefly, serial dilutions of antisense oligo-nucleotides or combinations of oligonucleotides were prepared in50 ml volumes of complete medium (RPMI-1640, 10% fetalbovine serum, 2mM L-glutammine, 100 U/ml penicillin, 100 mg/mlstreptomycin) in triplicate in 96-well plates. Virus, diluted tocontain a 90% CPE dose of virus in 50 ml, was added, followed by100 ml 4 x 105/ml MT-2 cells in complete medium. The plateswere incubated at 37°C in5% CO2 for 5 days.MT dye was addedand quantitated at OD540-OD690 as described (28). Percentinhibition was calculated by the formula (experimental - virus

MT-2 cells (31) were provided by the AIDS Reference andResearch 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 ofVujcic (34).

RESULTS AND DISCUSSION

Design of oligonucleotides

Oligonucleotide 1 binds to the entire 21 base target sequence (Table1). Oligonucleotides 2 and3 are 9- and 12mers, respectively (Table1) which bind to the target sequence at adjacent sites (Fig. 1A) andcover the same 21 base target as oligonucleotide 1. Oligonucleo-tides 4 and 5 bind to the same site as 3 but are separated by one andtwo bases on the target sequence respectively from the binding siteof oligonucleotide 2. Oligonucleotides 6-11 have an extendedsequence on either the 5'- or 3'-end of the binding sequence, whichforms a duplex stem between the two cooperative oligonucleotideswhen they both bind to the target at adjacent sites (Fig. IB).Oligonucleotide pair 6 & 9 forms a 3 bp stem. Oligonucleotidepairs 7 & 10 and 8 & 11 bind to the same length of the target asoligonucleotide pair 6 & 9, but with 5 and 7 bp extendeddimerization domains, respectively. These oligonucleotides bind tothe target with one base separation between them. Oligonucleo-tides 12-15 contain one or two mismatches, as shown in Table 1.

Table 1. Oligonucleotide sequences used in the study

Sequence no. Sequencea

RNA (target) 5'-GGAGGCUAGAAGGAGAGAGAUGGGUG-CGAGAGCGU

DNA (target) 5'-CTAGAAGGAGAGAGATGGGTGCGAGAG

1 5'-CTCGCACCCATCTCTCTCCTT

2 5'-CTCGCACCC

3 5'-ATCTCTCTCCTT

4 5'-TCTCTCTCCTTC

5 5'-CTCTCTCCTTCT

6 5'-CGGTCTCTCTCCTTC7 5'-GCCGGTCTCTCTCCTTC

8 5'-GCGCCGGTCTCTCTCCTTC

9 5'-CTCGCACCCCCG10 5'-CTCGCACCCCCGGC1 1 5'-CTCGCACCCCCGGCGC12 5'-CTCtCACCCATCTCTCTCCTT

13 5'-CTCtCAaCCATCTCTCTCCTT

14 5'-CTCtCACCC

15 5'-CTCtCAaCC

GEM 91 5'-CTCTCGCACCCATCTCTCTCCTTCT

aSequence shown in bold is the 21 base target, underlined bases represent theextended dimerization domain and lower case letters indicate mismatches.

Thermal melting studies

Thermal melting data of the oligonucleotides are summarized inTable 2. Thermal melting experiments were carried out using acontrol)/(medium control virus control) x loo.

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 oligonucleotidesto tandem sites. (B) Binding of oligonucleotides that have extended dimerization domains and their dimerization.

DNA target strand and phosphodiester oligonucleotides. The duplexof the DNA target and oligonucleotide 1 shows a Tm of67.7°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 oligonucleo-tides 2 & 4 and 2 & 5 with the target sequence have Tm values of44.4 and 46°C, respectively. The Tm of the duplex fonmed byoligonucleotides 2 & 3 together is greater than that of the average ofthe duplexes formed by 2 and 3 individually with the target sequence(Table 2). In contrast, the Tm values of the oligonucleotidecombinations 2 & 4 and 2 & 5 are less than the average of the Tmvalues of the two individual oligonucleotides in the experiment.These data suggest that the two short oligonucleotides 2 and 3targeted to two adjacent sites bind in a cooperative fashion.Furthermore, we observed a single, sharp, cooperative transition inthe Tm of the duplex of oligonucleotides 2 & 3 with the targetsequence (Fig. 2B). The cooperative interactions (2 & 3) areprobably driven by stacking interactions between the terminal bases(35). Thennal melting studies of the duplexes of oligonucleotides6-11 suggest that the stability of the terary complex formedincreases as the number of base pairs in the dimerization domainincreases (Table 2). The double helical complexes with 3 (6 & 14),5 (7 & 10) and 7 (8 & 11) bp dimerization domains show Tm valuesof 45.9, 48.4 and 53.2°C, respectively. In the absence of the targetsequence these combinations of oligonucleotides show no definedmelting transition, indicating no stable complex fornation. Furtherincreases in the length ofthe dimerization domains (more than sevenbases), however, result in the formation ofa stable complex betweenthe two tandem oligonucleotides even in the absence of the targetsequence. In all cases we observed a single, sharp, cooperativemelting transition (Fig. 2C). These data suggest that the cooperativeinteractions are further facilitated by extended dimerizationdomains, which form a duplex stem when the tandem oligonucleo-tides bind to the target at adjacent sites.The duplex of oligonucleotide 12, which contains a mis-

matched base, and the target sequence has a Tm of 61.4°C. Theduplex of the target sequence with oligonucleotide 13, with two

mismatches, has a Tm of 55.6°C. The duplex of mismatchedoligonucleotide 14 with the target shows a broad transition below30°C, without a defined Tm (data not shown). We observed notransition in the case of oligonucleotide 15, indicating that thisoligonucleotide does not interact with the target. These Tm valuessuggest that the short oligonucleotides are more sensitive tomismatches than the longer oligonucleotides (8,9,36). The Tmvalues obtained for the duplexes of mismatched oligonucleotides12 and 13 (21mers) with the target sequence clearly suggest thatlonger oligonucleotides can bind to any sequence containing upto two mismatches under physiological temperature and saltconditions (36).

RNase H assay

RNase H is an enzyme that recognizes RNA-DNA hetero-duplexes and hydrolyzes the RNA component of the heterodu-plex (37). Earlier studies indicate that a4-6 bp hybrid is sufficientto evoke RNase H activity (38). We investigated the RNase Hactivation properties ofphosphorothioate analogs ofoligonucleo-tides, using a 35mer RNA target sequence (Table 1). This assayprovides additional information on the sequence-specific bindingof cooperative oligonucleotides.We compared the RNase H activation properties of oligo-

nucleotides 14 and 15 with those of oligonucleotides 12 and 13to establish the sequence specificity of short tandem versus longeroligonucleotides. Figure 3 shows the RNase H hydrolytic patternof the targetRNA in the presence ofthe mismatched oligonucleo-tides. Oligonucleotide 12, with one mismatch (experiment 5 inFig. 3), showed a similar RNase H degradation pattern to thecompletely 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 arelocated. The degradation pattern, however, on either side of themismatches is very similar to that observed with oligonucleotide1 without mismatches. These results indicate that in spite of the

Nucleic Acids Research, 1995, Vol. 23, No. 17 3581

0.8-

'u0.6

0.4-

z0.2-

0

l.0-c

6+9 8+11

0.8-7+10

0.6-

0.4-

0.2-

0

20 30 40 50 60 70 80

Temperature, °C

Figure 2. Thermal melting profiles (dAWdT versus 7) of representativeoligonucleotides with their DNA target. Numbers correspond to oligonucleo-tide numbers in Table 1.

two mismatches, oligonucleotide 13 binds to the RNA stronglyenough to activate RNase H.

In the case of oligonucleotides 14 and 15, with one and twomismatches, respectively, (experiments 2 and 3 in Fig. 3), we

observed little or no RNA degradation as compared with oligo-nucleotide 2 (experiment 1 in Fig. 3). These results demonstratesequence-specific binding of the cooperative oligonucleotides tothe target. The results with oligonucleotides 12 and 13 suggestthat in an in vivo situation antisense oligonucleotides of .20 basesmight bind to a number of sequences in the mRNA poolcontaining 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 oligonucleotideswithout mismatches is shown in Figure 4. In experiments 2 and5 in Figure 4 RNase H hydrolytic activity occurs towards the3'-end of the target RNA (the lower half of the autoradiogram) inwhich oligonucleotides 2 and 11 are present. Similarly, RNAdegradation bands are present only in the upper half of the

Table 2. Tm of oligonucleotides with DNA target sequencea

Oligonucleotide Complexb Tm, OCc

1 TTCCTC TCT CTACCCACGC TC 67.7 (s)

2 CCCACGCTC 49.1 (s)

3 TTCCTCTCTCTA 43.4(s)

4 CTrCCTCTCTCT 43.6(s)

5 TCTTCCTCTCTC 45.0(s)

2+3 TTCCTC TCT CTA-CCCACGC TC 47.8 (s)

2+4 CTTCCTC TCT CT CCCACGC TC 44.4 (b)

2+5 T CTT CCTC TCT C CCCACGC TC 45.9 (b)

6+9 CTTCCTC TCT CT CCCACGCTC 45.9 (b)G CG CC G

7+10 CTTCCTC TCT CT CCCACGCTC 48.4 (s)GCG CC GCGG C

8+11 CUT CCTC TCT CT CCCACGCTC 53.2 (s)G CG CC GC GG CC GG C

12 TTCCTC TCT CTACCCACTC TC 61.4(b)

13 TTCCTC TCT CTACCAACIC TC 55.6 (b)

aSee experimental section for buffer conditions.bDNA target strand is shown as a thin line for complex structure (see Table1 for sequence); underlined bases represent mismatches; X represents numberof base separations between the two binding oligonucleotides.cLetters s and b in parentheses indicate sharp and broad transitions, respect-ively.

autoradiogram, indicating binding of oligonucleotides 3 and 8 onthe 5'-side ofthe target (Fig. 4, experiments 3 and 6, respectively).When combinations of oligonucleotides 2 & 3 and 8 & 11 arepresent (Fig. 4, experiments 4 and 7, respectively) the RNase Hdegradation pattern is very similar to that observed with controloligonucleotide 1 (Fig. 4, experiment 1). These results suggestthat the short tandem oligonucleotides bind to the target RNA asexpected, with sequence specificity, and evoke RNase H activity.The intactRNA target is digested over time as a result ofRNase

H action on the hybrid duplex. The extent (efficiency) of RNaseH hydrolysis depends on dissociation ofthe hybrid duplex formed(9). Dissociation of the duplex depends on the length of theantisense oligonucleotide and the mismatches present in thehybrid. In the presence of oligonucleotides greater than 9mersRNA is degraded by >80% in 5 min andlO0% in 15 min. Inthepresence of oligonucleotide 2 (9mer) RNA is not completelyhydrolyzed after up to 15 min. We observed similar hydrolysispatterns in the presence of oligonucleotides 9-11 (11- to l5mers).These results suggest that the extended domain remains as adangling end in solution and does not interfere with binding of the

3582 Nucleic Acids Research, 1995, Vol. 23, No. 17

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Figure 3. RNase H hydrolysis pattern ofRNA target in the absence and presence of the mismatched oligonucleotides compared with the control oligonucleotide 1.The control lane is at the 15 min time point in the absence of antisense oligonucleotide. *Labeled end of the RNA target.

antisense domain to the target sequence. Two short oligonucleo-tides binding to the target at adjacent sites have similar RNase Hactivation properties (target RNA hydrolysis patterns) as oligo-

nucleotide 1, which binds to the same length of the target RNA.In the presence of the mismatched oligonucleotides RNA

hydrolysis rates show a different pattern for longer oligonucleo-tides than for shorter ones. The RNA hydrolysis rate is notsignificantly altered by the presence of mismatches in the case oflonger oligonucleotides (Fig. 3, experiments 4-6 for oligonucleo-

tides 1, 12 and 13) (9,36). In the presence of the 9mer with one

mismatch (14), however, RNA degradation is reduced by >50%(Fig. 3) and with oligonucleotide 15, which has two mismatches,degradation is reduced by >90% (Fig. 3). Similar rates of RNAhydrolysis by RNase H in the presence of mismatched oligo-nucleotides 12 and 13 compared with oligonucleotide 1 suggeststrong interactions between the target RNA and oligonucleotides12 and 13, in spite of mismatches (vide infra thermal meltingdata), considering that the rate of RNase H hydrolysis is

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Nucleic Acids Research, 1995, Vol. 23, No. 17 3583

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Figure 4. RNase H hydrolysis pattern of the RNA target sequence in the absence and presence of the oligonucleotides at different time points. The control lane is atthe 15 min time point in the absence of antisense oligonucleotide. *Labeled end of the RNA target.

proportional to the binding affinity of the oligonucleotide for thetarget RNA (8,9,36).

HIV-1 inhibition studies

We studied the inhibition of HIV-1 replication by representativephosphorothioate analogs of the cooperative oligonucleotides incell cultures. The reference oligonucleotide, the 25mer GEM 91(see Table 1 for sequence), showed dose-dependent inhibition ofHIV-1 replication and 50% inhibition was obtained at -0.55 IMconcentration (Fig. 5). Oligonucleotide 1, a 21mer, also showeddose-dependent inhibition of HIV-1 replication, however, it wassignificantly less effective than GEM 91. This is in agreementwith our and other earlier studies in which inhibition of HIV-1

replication by oligodeoxynucleotide phosphorothioates is foundto be length dependent (39,40).The combination of oligonucleotides 2 (9mer) & 3 (12mer),

which bind to the same sequence on the target as oligonucleotide1, failed to show inhibition of HIV-1 replication (Fig. 5). Theoligonucleotide combination 8 & 11, with a 7 bp dimerizationdomain, showed a significant increase in inhibition of HIV-1replication over oligonucleotide 1 or the combination of oligo-nucleotides 2 & 3, which was dose-dependent. The increase ininhibition of HIV-1 replication by oligonucleotide combination 8(19mer) & 11 (16mer) is probably due to cooperative interactions,rather than the increased length, as the length of these oligonucleo-tides remains shorter than a 21mer (oligonucleotide 1). Significantloss of inhibition of HIV-1 replication was observed by reducing

r-

3584 Nucleic Acids Research, 1995, Vol. 23, No. 17

100-

80-

2 60-

,r 40-

0-

10000 1000 100 10

Oligonucleotide Concentration, nM

Figure 5. Dose-dependent inhibition of HIV-1 replication by representativecooperative oligonucleotides. At each dose experiments were carried out intriplicate and from the mean value percent inhibition was calculated. Thestandard deviation was in the range 0.8-10%. A dot in the middle of thesequence represents combination of oligonucleotides 2 & 3.

the number of bases in the dimerization domain. The combinationsof oligonucleotides 7 & 10 and 6 & 9, containing 5 and 3 bpdimerization domains, showed less activity (data not shown).

CONCLUSIONS

We have compared the binding and biological properties ofa singlelong oligonucleotide with those of two short oligonucleotides thatbind adjacently on the same target strand, thereby covering thesame length of the target sequence. The results demonstrate that thecombination of two short oligonucleotides binds to the targetcooperatively with sequence specificity. RNase H experimentalresults suggest that longer antisense oligonucletides have a greaterchance of binding to a number of target sequences containing upto two mismatches than do short oligonucleotides and then evokeRNase H activity similar to that observed with a perfectly matchedoligonucleotide. Short oligonucleotides might bind more specifi-cally to the target than do longer oligonucleotides, therebypreventing RNase H degradation of non-targeted RNAs. Specifi-cally, this approach might be valuable for targeting sequences withpoint mutations. In addition, undesirable non-specific effects mightbe reduced by using two short oligonucleotides rather than one

long oligonucleotide. For example, long oligonucleotides thatcontain a modified backbone, such as phosphorothioates, activatecomplement (41), with adverse hemodynamic effects (42). Thecombination oligonucleotides might represent an alternativetherapeutic strategy to the use of a single oligonucleotide in casesin which use of the latter is limited by concentration and chainlength constraints and the associated problems of toxicity.

ACKNOWLEDGEMENT

We thank Ms Allison Roskey for establishing RNase H experi-mental conditions.

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TCT TCCTCTCT CTACCCACGCTCTC -a GEM 91

TTCCTC TCr CTACCCACGCTC ----- 1

TTCCTCTCT CTACCCACGCTC 0 2+3

CITCCTC TCT CT CCCACGCTC ---- 8+11G C

G CCC GG CC GGCC

1T