T H E J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 1, Issue of January 5, pp. 468-472,1991 Printed in U. S. A .

Biotinylated Antisense Methylphosphonate Oligodeoxynucleotides INHIBITION OF SPLICEOSOME ASSEMBLY AND AFFINITY SELECTION OF U1 AND U2 SMALL NUCLEAR RNPs*

(Received for publication, August 2, 1990)

Jamal Temsamani, Sudhir Agrawal, and Thoru PedersonS From the Cell Biology Group, The Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

Methylphosphonate (PC) backbone oligodeoxynucle- otides complementary to the 5”terminal nucleotides of U1 and U2 small nuclear (sn) RNAs do not elicit RNase H action under conditions in which natural (phospho- diester) oligodeoxynucleotides yield extensive RNase H cleavage. We show here that antisense PC oligonu- cleotides can mask sites in U1 and U2 snRNPs that are required for spliceosome formation. We further report that biotinylated derivatives of antisense PC oligos can be used for affinity selection of U1 and U2 snRNPs.

At least five small nuclear (sn)’ RNAs (Ul, U2, U4, U5, and U6) participate as cofactors in mRNA splicing (Steitz et al., 1988). These RNAs function in the form of ribonucleopro- tein complexes, termed snRNPs (Luhrmann, 1988). A key technique in elucidating the functions of these snRNPs in mRNA splicing is the use of oligodeoxynucleotides comple- mentary to certain, available RNA sequences in the snRNPs (Kramer et al., 1984). The oligo-hybridized segment of RNA is then cleaved by RNase H, to produce a site specifically deleted forms of a given snRNP, followed by analysis of its capacity to support mRNA splicing (Kramer et al., 1984; Krainer and Maniatis, 1985; Black et al., 1985; Berget and Robberson, 1986; Black and Steitz, 1986).

An important variation on this theme has recently been introduced (Sproat et al., 1989; Lamond et al., 1989; Barabino et al., 1989). These investigators have synthesized U2 snRNP complementary oligoribonucleotides containing 2“O-methyl groups (2’-OMe RNA). These 2’-OMe RNAs are resistant to DNA- or RNA-specific nucleases (Dunlab et al., 1971; Sproat et al., 1989) and therefore offer certain experimental advan- tages, such as the ability to mask (but not excise) desired RNA sequences in snRNPs (Lamond et al., 1989; Barabino et al., 1989; Blencowe et al., 1989).

We and others have recently shown that methylphosphon- ate (PC) backbone oligodeoxynucleotides complementary to hybridization available regions of U1 or U2 snRNPs do not induce RNase H cleavage under conditions in which extensive cleavage is observed with phosphodiester backbone oligode-

* This investigation was supported by Grant GM-21595-16 (to T. P.) from the National Institutes of Health and funds provided by the Harold G. and Leila Y. Mathers Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. The abbreviations used are: sn, small nuclear; PO, phosphodiester

internucleoside linkage; PC, methylphosphonate internucleoside link- age; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul- fonic acid; G(5’)ppp(5’)G, guanosine-5’-triphospho-5’-guanosine.

oxynucleotides (Agrawal et al., 1990; Furdon et al., 1989). Here we report further studies on the use of antisense PC oligodeoxynucleotides to mask sites in U1 and U2 snRNPs that are critical for mRNA splicing. We also show that tetra- biotinylated PC oligodeoxynucleotides can be used to affinity select U1 and U2 snRNPs from nuclear extracts. An advan- tage of the present method is that, unlike 2’-OMe oligoribo- nucleotides (see above), PC oligodeoxynucleotides can be readily generated with commercially available reagents on standard DNA synthesizers.

MATERIALS AND METHODS

Oligodeoxynucleotide Synthesis-Oligodeoxynucleotides were syn- thesized using a Milligen/Biosearch synthesizer model 8700 (Burling- ton, MA). Reagents for oligodeoxynucleotide synthesis (nucleoside H-phosphonate and nucleoside P-cyanoethyl phosphoramidite) were purchased from Milligen/Biosearch. Nucleoside methylphosphon- amidites for synthesizing oligodeoxynucleotide methylphosphonates were purchased from Glen Research (Herndon, VA). The NHS-ester of caproylbiotin was obtained from Clonetech (Palo Alto, CA). N-l- Trifluoroacetylhexanediamine was prepared as reported earlier (Agra- wal and Tang, 1990).

Oligodeoxynucleotides containing phosphodiester internucleoside linkages were synthesized using P-cyanoethyl phosphoramidite chem- istry (Atkinson and Smith, 1984). Deprotection of oligonucleotides was carried out in concentrated ammonia for 8 h a t 55 “C. Oligonu- cleotides were purified on an ion exchange HPLC (Partisphere SAX) using a linear gradient of 1-300 mM KHzP04 containing 60% form- amide (Agrawal et al., 1987). Materials in the product peak were isolated by desalting on Sephadex G-25 with a solvent of 20% ethanol/ Hz0 (v/v), followed by evaporation.

Methylphosphonate analogues of oligodeoxynucleotides were as- sembled using nucleoside methylphosphonamidites (Agrawal and Goodchild, 1987). In the case of oligodeoxynucleotides C and D (Table I), the last coupling was carried out with the nucleoside P-cyanoethyl phosphoramidite to generate an unmodified phosphate internucleo- side linkage at this position. Deprotection of oligonucleoside meth- ylphosphonates were carried out in concentrated ammonia for 2 h followed by 6 h in ethylenediamine/ethanol (l : l , v/v). Deprotected oligonucleotides were purified on reversed-phase HPLC (Waters No- vapak CIS cartridge with RCM 100) using a gradient of 0-80% acetonitrile in 100 mM ammonium acetate (Agrawal, S., 1989). The product peak was desalted on Sephadex G-25 using 20% ethanol/HzO (v/v) as eluting solvent.

Assembly of Amine-junctionalized Oligodeoxynucleoside Methyl- phosphonates-Functionalization of oligonucleotides for attaching biotin was carried out by incorporating a suitably protected aminoal- kylphosphoramidite internucleoside linkage, as reported earlier (Agrawal and Tang, 1990). For introducing one primary amine group into the sequence, the first coupling was carried out using H-phos- phonate chemistry (Garegg et al., 1985; Froehler et al., 1986; Froehler, 1986; Agrawal et al., 1988), followed by oxidation with N-l-trifluo- roacetylhexanediamine/carbon tetrachloride. The next 15 couplings were carried out on the controlled pore glass-bound dinucleoside phosphoramidite, using nucleoside methylphosphonamidates and standard coupling reactions. For incorporating four amino groups into an oligodeoxynucleotide, four phosphoramidate internucleoside linkages were introduced. A pentamer, (T)5, was assembled using H-

468

Antisense Methylphosphonate Oligoa for snRNPs 469

phosphonate chemistry. The controlled pore glass-hound pentamer H-phosphonate wasoxidized with N-I-trifluoroacetvlhexanediamine/ rarhon tetrachloride to generate a phosphoramidate-(N-trifluoroac~ etylaminohexyl) internucleoside linkage. The next 15 couplings were carried out with nucleoside methylphosphonamidites using standard conditions.

Deprotection of oligonucleoside methylphosphonates carrying one or four amino groups was carried for 2 h at room temperature in roncentrated ammonia followed hy 6 h in ethylenediamine/ethanol (1:1. v/v). Purification of the crude amino-functionalized oligonucleo- side methylphosphonate was carried out on reversed-phase HPLC, using a linear gradient of 0 4 0 % acetonitrile as descrihed ahove. Product peaks were pooled, evaporated, and desalted on Sephadex C- 25.

Hiotinylntion o/ Amino-functionalizrd C)li~odrox~nuclrosid~~ Mrth- vlphosphonotes-The lyophilized oligonucleoside methylphosphonate (30 A21n units) carrying one or four amino groups was dissolved in 500 pl of 250 mM potassium phosphate huffer, pH 8, and NHS- caproyl hiotin (5-8 mg, 11-17.6 pmol. dissolved in 80 pl of dimethyl- formamide) was added to the solution. The mixture was vortexed and left at room temperature (24 " C ) for 8 h. The reaction was passed through Sephadex C-25 (20% ethanol/H?O, v/v) to remove excess hiotin ester and salt. Analysis hy reversed-phase HPLC showed an almost quantitative reaction, with the hiotinylated product eluting later than the unreacted material. The hiotinylated oligonucleoside rnethylphosphonates were purified in five runs each. The product peaks were desalted on Sephadex (;-25. The yields were typically 20- 23 A,,, units.

RNase H Assa.vs-I5 pI of HeLa cell nuclear extract (Dignam rt ai., 1983) were incuhated with the appropriate oligodeoxynucleotide (200 pg/ml, final concentration) at 30 "C for 30 min in reactions containing 30% (v/v) extract in huffer D. 3.2 mM MgCI,, 0.5 mM ATP, and 20 mM creatine phosphate. Reaction mixtures were treated with proteinase K (2 mg/ml, final Concentration) in 0.5"; SDS. 1 0 0 mM Tris-HCI, pH 7.6, 1 mM EDTA for 20 min at 30 "C, followed hy phenol-chloroform extraction and ethanol precipitation in the pres- ence of 20 pg/ml glycogen. The RNAs were analyzed by electropho- resis on 12% polyacryhmide, 7 M urea gels.

Northrrn Blots-RNA was purified from HeLa nuclear extract hy proteinase K treatment followed by phenol-chloroform extraction and ethanol precipitation. The RNAs were separated on a 12% polyacrylamide, 7 M urea gel, and electrophorectically transferred onto a nylon memhrane for 2 h a t 300 mA, 4 "C. in a solution containing 27 mM citric acid, 34 mM Na,HPO,. The membrane was then baked at 80 "C for 1 h and prehyhridized for 2 h at 37 "C in hybridization huffer (1 M NaCI, 1% SDS. 10% dextran sulfate, and 150 pg/ml sheared calf thymus DNA). Hybridization was carried out with 10'' cpm/ml ( = I O ng/ml) of the appropriate methylphosphonate oligodeoxynucleotide in the same huffer for 3 h a t 3 7 "C. The mem- hrane was then washed twice for 30 min in 1 X SSC (where SSC is standard sodium citrate), 0.1% SDS and once in 0.5 X SSC and 0.5% SDS at room temperature. The membrane was air dried and exposed to x-ray film a t -70 "C.

The PC oligodeoxynucleotides (C and D, Tahle I). containing a 5'- terminal phosphodiester internucleoside linkage, were 5' end-laheled with [y-:"P]ATI'and T4 polynucleotide kinase (Promega) (Murakami r t al., 1985). followed hy t.wo ethanol precipitations.

Spliceosome Assemh/y-Nuclear extract was incuhated in the pres- ence of the appropriate oligodeoxynucleotide as descrihed ahove. An adenovirus pre-mRNA (5 X IO' cpm, -0.5 ng) was added. and the reaction was continued for another 30 min. This pre-mRNA was transcribed from Sau3A-cleaved pHSAdl0 DNA (kindly provided by Maria Konarska and Philip Sharp, Massachusetts Institute of Tech- nology). Transcription was in the presence of 0.5 mM G(Fi')ppp(5')(;, each of the four nucleoside triphosphates (125 p ~ ) and 50 pCi of [ a - '"PIUTP. Reactions were terminated hy the addition of heparin (2 mg/ml, final concentration). Samples (10 p l ) were loaded directly onto a 3.5% polyacrylamide gel (acrylamide to hisacrylamide mass ratio of 80:l) containing 50 mM Tris-glycine. pH 8.8, (Konarska and Sharp, 1986) that had heen prerun for 30 min at 14-16 V/cm in the same huffer. Electrophoresis of the samples was carried out at the same voltage until the xylene cyano1 dye had just migrated off the hottom of the gel. Gels were dried and autoradiographed at -70 "C.

Streptauidin-Agarosr Af/inify Sdection-200 pI of streptavidin- agarose heads (Hethesda Research Laboratories) were preincuhated for 15 min at 4 "C with an equal volume of SR huffer (0.15 M KCI, 20 mM HEPES. pH 7.9, 3.2 mM MgCI,. 0.5 mM dithiothreitol) containing 200 pg/ml glycogen, 100 pg/ml hovine serum albumin, and

100 pg/ml tRNA. The heads were washed three tinws (0.8 1111 e.;~ch~ with SR huffer alone. The heads were then incuhted wi th M I p c ( 1 1

3"hiotinylated oligodeoxynucleotide in SI4 huffer for 1 h a t .1 (' with agitation. The nnhound olig(~(Ieox~n~~cleoti(1e was removed hy t h r w washings (0.8 ml each) with SH huffer. After the lin;ll wash. the heads were incuhated with 1.5 M I of Hel,a nurlear extrart antl I85 P I o f SI3 huffer in the presence ofO.5 mxf A'l'l'and 20 nlht rreatine phosphate. The hinding reaction proceeded lor 1 h at 4 "(' with agitation. The heads were pelleted and the supernatant RNA wits recovered hy treatment with proteinase K, J)henol-chlorofornl ext rtwtion. antl ethanol precipitation as descrihed ahove. The heads were washed lour times with SH huffer rontaining 0.5 M K('l 10.8 ml each) over 1 h at 4 "C. RNA was then released from the streptavidin-;1E;lros~, hy trcant- ment with proteinase K for 20 min. 30 "c'. followed hy incuhnt ion frlr 15 min at 90 "C. The heads were pelletrd and the supernatant was phenol-chloroform-extractetl and ethanol preripit;ltetl in the presence of 'LO pg/ml glycogen. The R S A s were analyzed on ;I 12"; p(1lyacry1- amide, 'i M urea gel.

In the event it may he useful information to other investigators. we can report that prehinding o f tetral)ic,tinyl;lted I ) ( * olicos streptavidin-agarose prior to incuhation with 1'1 and ('2 snRSI' in the nuclear extract was more eflirient than the ;Ilternativo method o f first incubating nuclear extrart with olico follc~wed hy rc.covery o n streptavidin-agarose (data not shown).

RESIILTS A N D D1SCI;SSION

W e initially investigated the capacity of methylphosphon- a te (PC) hackhone oligodeoxynucleotides to hase pair with complementary RNA sequences under standard DNA/HNA hybridization conditions. As shown in Fig. 1, "1'-laheled PC oligodeoxynucleotides (C and D, Tahle I ) complementary t o 14 5"terminal nucleotides of human U1 or U 2 small nuclear RNAs hybridized to the respective HNA species in a standarc: Northern blot experiment.

When the same (unlabeled) oligos were incuhated with HeLa nuclear extract under conditions permissive for enclog- enous RNase H activity (Agrawal rt al., 1990). they did not induce cleavage of U1 or U2 RNAs, even though extensive (80 to >95%) cleavage was ohserved with phosphodiester hackhone oligos (Fig. 2). T h e failure of the PC oligos to induce RNase H cleavage could conceivahly reflect RNase H resist- ance of PC oligodeoxynuc1eotide:RNA hyhrids or could he due to non-hybridization to U1 or U2 s n R N P s in the nuclear extract under these conditions. To address this point, we investigated the ahility of phosphodiester (PO) L'crsus meth- ylphosphonate backhone oligos complementary to the 5' ends of U 1 or U2 RNAs to inhihit the assemhly o f pre-mHNA splicing complexes.

Following preincuhat.ion of the various oligos in He1,a nu-

1 2 "- -

u2

UT- ,

FIG. 1. Northern blot. RNAs were purified from H c s l . a nuclear extract. separated on a polyacrylamide gel and transferred onto a nylon memhrane as descrihetl under "Materials and Xlethods." The membrane was prohed with "1'-laheled I T oligos I(' and I ) . Tahle I ) complementary to 111 RNA ( lanr I ) and t.2 HSA Ikrnrv 2 ) .

470 Antisense Methylphosphonate Oligos for snRNPs TAnI.!? I

Oligodro.~ynuclrr,lidr.s synthrsizrd for thr prrsrnt invrstigalion Shaded nucleotides have 8"phosphodiester linkages. Non-shaded nucleotides contain :~'-methvlphosphonate

linkages. Oligodeoxynucleotides C, D, and E each contain one hiotin molecule ( H = X , ) and oligodeoxyn~tcleotides F and G each contain four biotin molecules ( H = X ? ) .

Oligonucleotides Synthesized

Number Sequence Binding S i t e

A UI (1-14) 5' ~TGCCAGGTAAGTAT I 3' ' X1 :

B IAGGCCGAGAAGCGAT I U2 (1 -15)

c BTGCCAGGTAAGT-XI U I (3 -16)

D PGGCCGAGAAGCG-X I U2 (3- 16)

E AGCTTCCGGTCTCC-XI None

F CCTGCCAGGTAAGTA-X2 UI (2 -16 )

G AAGGCCGAGAAGCGA-X2 U2 ( 2 - 16)

H N N H I " y o

N O / \

"

uz - u1 -

I 2 3 4 5

FIG. 2. RNase H action with PO and PC oligodeoxynucleo- tides. PO ( A and H, Tahle I ) and I T (( ' and D, Tahle I ) oligomers complementary to the 5' end of U1 or U2 RNA were incuhated with nuclear extract for 30 min a t :IO "C. RNAs were extracted and ana- lyzed hy polyacrylamide gel electrophoresis. The gel was stained with ethidium hromide. IAne I, no oligo; lane 2, U1 oligo (PO); lanr 3 , 111 oligo (PC); lanr 4, U2 oligo (PO); lanr 5 , U2 oligo (PC).

cbar extract, a "'P-labeled adenovirus pre-mRNA was added and the subsequent formation of spliceosomes was monitored by electrophoresis of RNP complexes in non-denaturing poly- acrylamide gels (Konarska and Sharp, 1986). Preincubation of the nuclear extract with a PO oligo complementary to nucleotides 1-14 of U1 RNA (A, Table I ) inhibited the for- mation of both A and R splicing complexes (Fig. 3, lanes 2- 3 ) , in confirmation of previous investigations (Frendewey et al., 1987; Zillman et al., 1988; Mayrand and Pedersen, 1990). The formation of A and R complexes was also inhibited by a

1 2 3 4 5 6 7 8 9 1 0 1 1 . " - B"

A"

FIG. 3 . Inhibition of spliceosome assembly. PO and PC o l i - gomers (A-D, Tahle I ) were incubated in HeIda nuclear extract for 30 min at 80 "C followed hy addition CJf adenovirus pre-mHNA. Splicing complexes were then analyzed hy electrophoresis on a non-denaturing polyacrylamide gel. Lane I, no oligo; lanr 2. 5 pg of 1'1 oligo f 1 ' 0 ) ; lanr 9 , 15 pg of U1 oligo (PO); lanc 4 , -5 pg o f t i1 oligo ( I T ) ; lonr 5 , 15 pg of U1 oligo (PC); lanr 6, 5 pg of U2 oligo (1'0); Ianr 7, 15 pg o f U2 oligo (PO); lane 8, 5 pg of 112 oligo (PC) ; lanr 9. 15 pg o f 1'2 oligo (PC); lane IO. 5 pg of oligo (oligo E. Tahle I ) ; lanc 1 1 , 15 pg o f control oligo. The puaiiicns of A and H splicing complexes are indicated.

PC oligo (C, Table I ) complementary to the 5' end of U1 RNA (Fig. 3, lanes 4 and S), indicating that the failure of this oligo to induce RNase H cleavage of U1 snRNP (Fig. 2) is not due to an incapacity for hybridization under the condi- tions of these nuclear extract experiments.

Preincubation of nuclear extract with a PO oligo (R , Table I) complementary to nucleotides 1-15 of U2 RNA also inhib- ited the formation of both A and R splicing complexes (Fig. 3, lanes 6 and 7). again confirming previous reports (Fren-

Antisense Methylphosphonate Oligos for snRNPs 47 1

dewey et al., 1987; Zillman et al., 1988; Mayrand and Pederson, 1990). As can be seen in Fig. 3, lanes X and 9, the formation of B complexes was inhibited by the comparable PC oligo (D, Table I) at the lower concentration tested, and both A and B complexes were inhibited at the higher PC oligo concentra- tion. No inhibition of either A or B splicing complex formation was observed when nuclear extract was preincubated with a random sequence control PC oligo (E, Table I) (Fig. 3, lanes 10 and 1 1 ). Thus, these results demonstrate that PC oligos can mask functional sites in U1 and U2 snRNPs in a se- quence-specific manner under conditions where no RNase H cleavage occurs.

Because of the RNase H resistance of DNA/RNA hybrids formed between PC oligos and U1 or U2 snRNPs, we were prompted to see if this could be exploited for the isolation of snRNPs, in a manner formally analogous to the method employing 2’-OMe oligoribonucleotides. (Barabino et al., 1989; Blencowe et at., 1989). In initial experiments we coupled, via a 6-carbon linker, a biotin molecule to the 3’ ends of PC oligos complementary to nucleotides 3-16 of U1 or U2 RNAs (C and D, Table I). Incubation of these monobiotinylated PC oligos in nuclear extract followed by selection with streptavi- din-agarose did not lead to isolation of U1 or U2 snRNPs, even though this procedure did recover protein-free U1 and U2 RNAs in control experiments (data not shown). In a different strategy we coupled four, rather than one, biotin groups to the 3’ end of the oligos (F and G, Table I). In addition, we elected to prebind these tetrabiotinylated PC oligos to streptavidin-agarose prior to incubation with nuclear extract. This led to efficient and sequence-selective affinity selection of U1 and U2 snRNPs (Fig. 4, lanes 5 and 6). Densitometry indicated that 50-60% of the U1 or U2 snRNP was selected. No RNA was recovered when nuclear extract was incubated with streptavidin-agarose beads not coupled to an oligodeoxynucleotide (Fig. 4, lane 4 ) .

The fact that antisense PC oligos can block prespliceosome and spliceosome assembly (Fig. 3) suggests that these oligos may be of use in defining regions of other snRNPs that participate in RNA processing reactions. Moreover, the PC oligo method obviates the need to use exogenous RNase H

1 2 3 4 5 6

u2 - u1 -

FIG. 4. Affinity selection of U 1 and U2 snRNPs. Biotinylated PC oligos (E and F, Table I ) were incubated with streptavidin-agarose followed by incubation with HeLa nuclear extract. RNAs were ex- tracted and analyzed by gel electrophoresis. Lanes 1-3 show RNA remaining in the supernatant after selection with, no oligo (lane I ), U1 oligo (lane 2) . U2 oligo (lane 3 ) . Lunes 4-5 show RNA recovered from streptavidin-agarose beads after selection with, no oligo (lone 4 ) . U1 oligo (lane 5 ) , U2 oligo (lane 6 ) . The gel was stained with ethidium bromide.

when the in vitro RNA processing system lacks sufficient RNase H activity of its own, and adds the attractive feature of a sequence-specific blocking effect, rather than a K N A cleavage. Another possible advantage of antisense PC oligos relates to the fact that mammalian cells contain an activity that unwinds double-stranded RNA (Wagner and Nishikura, 1988), which could attenuate the effects of certain antisense oligoribonucleotides. Finally, the fact that the affinity selected U1 and U2 snRNPs could be released from the biotin-PC 0ligo:streptavidin matrix by reducing the ionic strength of the (0.15 M KCI) binding buffer to weaken RNA/DNA base- pairing, just as in oligo(dT)-cellulose purification of polv(A)’ RNA, (data not shown) indicates the feasibility of recovering intact snRNPs, although we have not yet conducted func- tional studies with such snRNPs.

The quantitative extent of affinity selection of U1 and 172 snRNPs we observed with tetrabiotinylated PC oligos was similar to that reported for U2 snRNP using ribose ‘L’-OMe oligoribonucleotides (Sproat et al., 1989). It deserves to he mentioned that, in both procedures, the efficiency of selection is not greater than 50-700/;, and, in fact, we have not succeeded in quantitatively depleting nuclear extracts of LJ1 or U2 snRNPs with our procedure (see also Rarabino et 01.. 1989). Thus, with neither method, at their present stages of devel- opment, can one envision carrying out studies of snRNP function based on extract depletion. One obvious limitation to quantitative snRNP depletion by either method is the fac: that -10% of the U1 and U2 snRNPs are engaged in spliceo- somes at any given time (Lerner et 01.. 1980; Calvet and Pederson, 1981; Calvet et al., 1982). with pre-mRNA base- pairing masking the otherwise oligo available sites.

Yet, beyond this, there are differences in the two methods. For example, PC oligodeoxynucleotides can be readily gener- ated on automated synthesizers whereas 2’-OMe oligoribo- nucleotides cannot due to the present lack of a commercial supply of 2’-OMe monomers. In addition, in the 2’-OMe oligoribonucleotide method, the guanosine residues in each oligomer are deliberately substituted with inosine (Lamond et al., 1989), a helix-destabilizing effect in the formed RNA/ RNA hybrids. On the other hand, the 2’-OMe oligoribonucle- otide method requires less material than our procedure using PC oligodeoxynucleotides. This presumably reflects a higher stability of RNA/RNA duplexes in the 2’-OMe oligoribonu- cleotide method relative to the DNA/RNA hybrids in our PC oligodeoxynucleotide procedure (Inoue et af . , 1987; Quartin and Wetmur, 1989).

A final point worth considering is the bearing of our results on the relative potencies of antisense PO versus PC oligode- oxynucleotides against viral gene expression in oioo, a major research field at present. Since their introduction (Miller et al., 1979), methylphosphonate oligodeoxynucleotides have been known to display, at near-physiological ionic strength, a more stable helix in DNA/DNA duplexes or DNA/RNA hy- brids than their natural, phosphodiester counterparts (Froeh- ler et al., 1988; Sarin et al., 1988; Quartin and Wetmur. 1989). Whether methylphosphonate backbone oligodeoxynucleo- tides form more or less stable hybrids with RNA than PO oligos in our experiments in nuclear extracts (60 mM KCI) is difficult to know (see Quartin and Wetmur. 1989). Although we have not investigated this point in detail, our results on inhibition of spliceosome formation show that PC oligomers form sequence-specific complexes with complementary se- quences in U1 and U2 RNAs in a nuclear extract that is permissive for mRNA splicing. At the lower tested concentra- tions, inhibition of splicing complex formation was more extensive with PO oligomers but, at a %fold higher concen-

472 Antisense Methylphosphonate Oligos for snRNPs

tration, the U1-complementary PC oligomer was more effi- cient than its PO counterpart in inhibiting splicing complex formation (Fig. 3). This suggests to us that significant differ- ences in DNA/RNA hybrid stability of PO versus PC oligo- mers can occur a t near-physiological conditions, indicating the need for detailed investigation of each particular situation, taking into consideration the possible influence of target RNA secondary structure (e.g. Agrawal et al., 1989), before adopting PO versus PC oligodeoxynucleotides as the preferable ap- proach in antisense DNA strategies aimed at viral inhibition.

Acknowledgments-We thank our colleague Paul Zamecnik for advice, Vipin Kohli for assistance, and Carol Tuttle for word proc- essing.

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