508
Bioconjugate Chem. 1994, 5, 508-512
Synthesis of Uridine Phosphoramidite Analogs: Reagents for Site-Specific Incorporation of Photoreactive Sites into RNA Sequences Kavita Shah, Hongyan Wu, and Tariq M. Rana* Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854. Received December 17, 1993@
The synthesis of three new photoactive RNA phosphoramidites, 5-bromouridine, 5-iodouridine, and 04-triazolouridine, is reported. The 5’ OH of bromouridine and iodouridine were protected as dimethoxytrityl ether using dimethoxytrityl chloride and pyridine. Selective protection of 2‘ OH was achieved as the corresponding tert-butyldimethylsilyl ether. Protected ribonucleosides were converted to phosphoramidites using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. 04-Triazolouridine phosphoramidite monomer was prepared in one step from uridine phosphoramidite. These phosphoramidites were used to incorporate photoprobes a t any chosen sites in the RNA sequences during chemical syntheses. The modified monomers were incorporated into RNA oligomers with coupling yields >98%. After chemical synthesis, 04-triazolouridine was converted to 4-thiouridine by the addition of thiolacetic acid during standard deprotection methods. The extent of thiation and incorporation of modified nucleotides into RNA sequences were confirmed by nuclease digest, HPLC, and gel electrophoresis.
RNA plays a central role in cellular processes, including regulation and catalysis. Since the discovery of RNA enzymes, there has been a n increasing interest in RNA structure ( I ) . The multiple functions of RNA must reflect diversity in its three-dimensional structure. Despite the importance of RNA function, very little is known about its structure. Knowledge of the three-dimensional structure and general rules for RNA folding will be valuable to infer a more detailed mechanism of RNA function. X-ray crystallography provides high-resolution structures, and a few crystal structures of RNA have been determined (2, 3). Recently, high-resolution NMR techniques have been developed to elucidate RNA structures in solution ( 4 ) . There is a need for new methods to determine higher order RNA structure under physiological conditions (5, 6). Recent advances in chemical synthesis of DNA have opened a new field of DNA applications as structural probes and inhibitors. Oligonucleotides can be covalently linked to a dye, enzyme, or other biological macromolecule (for a recent review on chemically modified DNA see ref 7). Important biomedical applications of modified oligonucleotides include the detection and localization of messenger RNA, detection of bacterial or viral sequences, inhibition of RNA translation, and control of DNA replication. Recently, Xu et al. (8)described a strategy for postsynthetic modification of DNA a t the 4-position of thymine. There is a variety of modified monomer phosphoramidites commercially available for automated synthesis of DNA. On the other hand, chemical synthesis of RNA is a much more challenging task due to the presence of a 2’-hydroxyl group. During chemical synthesis of RNA, the 2’-hydroxyl group has to be protected until all other protecting groups have been removed. Due to the synthetic difficulties, modified monomers for
* To whom correspondence should be addressed. Phone: (908) 235-4590. Fax: (908) 235-4073. E. Mail:
[email protected]. Abstract published in Advance A C S Abstracts, October 1, 1994. @
1043-1802/94/2905-0508$04.50/0
chemical synthesis of RNA are not common for structural studies. Photochemical cross-linking has been widely used to study RNA-RNA and RNA-protein interactions (9-13). Recently, Ebright and co-workers have exploited photocrosslinking reactions mediated by site-specificallyplaced bromouracil to identify an amino acid-base contact in GCN4-DNA and Myc-DNA complexes (14,15). Photochemical crosslinking can trap transient association of macromolecules, which might not be possible by available physical methods. In the case of dynamic structures such as ribozyme-substrate interactions, these methods are extremely valuable. However, this technique faces the challenge of how to site-specifically incorporate photoprobes into the internal sequences of RNA. To meet this challenge, we have synthesized phosphoramidites of 5-bromouridine, 5-iodouridine, and 04-triazolouridine. These new phosphoramidites were used to incorporate photo probes a t chosen sites in the RNA sequence during chemical syntheses. The following four sequences were synthesized on a n automated DNA synthesizer: (1)5’AUU AAU UBrAG-3’;(2) 5’-CGC UGU’ CA-3’; (3)5’-AUU CUThiOU G-3’; and (4) 5’-GCC GUU UUU UC-3’ (unmodified control sequence). RNAs containing bromouridine and iodouridine were cleaved from the support, deprotected, and desalted according to standard procedures. Modified RNA phosphoramidites were incorporated into RNA oligomers with more than 98% coupling efficiencies. After chemical synthesis, 04-triazolouridine was converted to 4-thiouridine by the addition of thiolacetic acid during deprotection methods. To analyze the stability of RNA, crude modified oligoribonucleotides were 5‘ labeled with 32Pand run on 20% polyacrylamide-8 M urea gels. Results of this analysis are shown in Figure 1. Three crude sequences containing modified monomers gave single bands on the gel, indicating that modification or deprotection conditions had no effect on the stability of RNA (lanes 2-4, Figure 1). One unmodified control sequence was synthesized and deprotected under similar conditions. Stability of control sequence is shown in lane 0 1994 American Chemical Society
Bioconjugate Chem., Vol. 5, No. 6, 1994 509
Letters
1
2
3
4
Figure 1. Analysis of the chemically synthesized crude RNA sequences by 8 M urea-20% polyacrylamide gel electrophoresis. All RNA sequences were labeled at the 5’ end with 32P.Labeling reactions were carried out a t rt for 30 min in a mixture P ] (6000 containing 10 pM RNA, 10 mM MgC12,0.5 pM [ Y - ~ ~ATP Cilmmol), and 4 units of T4 polynucleotide kinase in 50 mM Tris-HC1 (pH 7.4). Autoradiogram of a typical gel is shown: unmodified control sequence, 5’-GCC GUU ULTU UC-3’ (lane 1);bromouridine containing sequence, 5’-AUU AAU UBrAG-3’ (lane 2); iodouridine containing sequence, 5’-CGC UGUI CA-3’ (lane 3); and RNA sequence modified with 4-thiouridine, 5’-AUU CuThiOUG-3’ (lane 4).
-I
12.5
0
5
10
15
20
25
Time (min)
Figure 2. HPLC profile of crude RNA modified with 04triazolouridine. RNA sequence, 5’-AUU C P O U G-3’, was deprotected as described in the Experimental Procedures and chromatographed on a c8 reversed phase column (Zorbax 300 SB, 4.6 mm x 25 cm). A 25 min linear gradient, from 0.1 M triethylammonium acetate in 5% acetonitrile, pH 6.4, to 12.5% acetonitrile, was used with a flow rate of 1.0 mumin. The shape of the solvent gradient is shown by the dashed line. Retention time of the major peak at 15.58 min is also indicated.
1, Figure 1. These results clearly indicate that there were no significant degradation products during deprotection procedures of modified RNA sequences. During the development of postsynthetic RNA modification methods, the major concern was the stability of RNA toward nonconventional deprotection conditions. To address this question, we synthesized sequence 3 and modified the 4-position of uridine by postsynthetic substitution. After deprotection, the RNA oligomer was analyzed by HPLC. The presence of 4-thiouridine in deprotected sequence 3 was confirmed by monitoring at 335 nm. As shown in Figure 2, crude sequence 3 RNA gave only one major peak with retention time of 15.58 min. The extent of thiation was calculated by measuring the absorbance of sequence 3 at 330 and 260 nm, which showed 97% transformation of 04-triazolouridine into 4-thiouridine. Incorporation of 5-bromouridine and 5-iodouridine in oligomers was assessed by nuclease digest analyses. Quantification of HPLC data showed
1.0 nucleoside of 5-bromouridine and 0.92 nuceoside of 5-iodouridine in sequences 1 and 2, respectively. The synthetic methodology presented in this report is the first example of chemical synthesis of RNA containing bromouridine, iodouridine, and 4-thiouridine at a predetermined site and postsynthetic substitution of RNA. An important application of single site RNA modification is the use of these photoprobes for structural studies of ribozymes. Synthesis of RNA with longer sequences can be accomplished by using T7 RNA polymerase in vitro with oligonucleotide DNA templates (16). Site-specifically modified RNA can be prepared by synthesizing short sequences of modified RNA on automated synthesizer and ligating it into longer pieces of RNA with the use of bacteriophage T4 DNA ligase (17).By the use of this method, RNA can be labeled with photoprobes at predetermined sites in the ribozyme and substrate sequences. EXPERIMENTAL PROCEDURES
(-)-5-Bromouridine, (-)-5-iodouridine, uridine, dimethoxytrityl chloride (DMTCl), tert-butyldimethylsilyl chloride (t-BDMSCl), 2-cyanoethyl NJV-diisopropylchlorophosphoramidite, 1,2,4-triazole, phosphorus oxychloride, triethylamine, tetrabutylammonium fluoride (1M solution in THF), 2,4,6-collidine, thiolacetic acid, DBU, anhydrous N-methylimidazole, dry THF, and pyridine were purchased from Aldrich. 2,4,6-Collidine was dried over 4 A molecular sieves. The CPG linked monomers and the chemicals for synthesizer were obtained from Glen Research (VA). Sep Pak Clg plus cartridges were purchased from Waters (Millipore). Snake venom phosphodiesterase 1(1mg/0.5 mL) and alkaline phosphatase (1000 U/mL) were obtained from Boehringer Mannheim (Indianapolis, IN). Solvents were HPLC grade and were degassed immediately before use. lH NMR spectra were recorded at 200 MHz on a Gemini 200 spectrometer (Varian). All spectra were taken in CDC13. Mass spectra were recorded using fast atom bombardment (FAB) ionization. TLC were performed with precoated 0.2 mm silica gel 60 F-254 TLC plates (EM Reagents, Darmstadt, FRG). Plates were visualized under short wave U V light and with iodine vapors. Dimethoxytrityl-containing compounds were visualized by exposing the TLC plate to concentrated HCl vapors. Column chromatography was performed with silica gel (70-230 mesh, 60 A) purchased from Aldrich. Reversed-phase HPLC analysis of oligomers was carried out on Beckman 344 with a variable wavelength detector (Beckman Model 165). Reagent grade chemicals were used without purification unless otherwise stated. All the reactions involving (-)-5-bromouridine and (-)-5iodouridine were performed in the dark. Synthesis of (-)-5-Bromouridine Phosphoramidite (4). The synthesis of phosphoramidite 4 is delineated in Scheme 1. The 5’ OH of bromouridine 1 was protected as DMT ether 2 using DMTCl and pyridine, followed by selective 2’ OH protection as the corresponding t-BDMS ether 3 using Ogilvie’s method (18).Finally, nucleoside 3 was converted to the phosphoramidite 4 using 2cyanoethyl N,N-diisopropylchlorophosphoramidite(19). 5‘-0-[(4,4’-Dimethoxyphenyl)methyl]-(-)-5-Bromouridine (2). To a solution of (-)-5-bromouridine (323 mg, 1mmol) in dry pyridine (4 mL) was added DMTCl(350 mg, 1.03 mmol) and the reaction mixture was stirred overnight under NZ atmosphere. Methanol (1mL) was added, and after 15 min the solution was concentrated to dryness under reduced pressure. A 5% NaHC03 solution (10 mL) was added, the resulting solution was extracted with ethyl acetate (2 x 15 mL), and the organic
510 Bioconjugate Chem., Vol. 5, No. 6,1994
Shah et al.
Scheme 1
-
H N k " DMTCl
selecl~ve2-rilyaion
Pyrldine
I.BDMSCUAgN0,
85%). 'H NMR (CDC13) 6 (ppm): 8.15 ( l H , s, HG),7.397.24 (9H, m, aromatic), 6.83 (4H, d, aromatic, J = 8.83 Hz), 5.99 (lH, d, H1,), 4.61-4.44 (2H, m, Hs. and HT), 4.29-4.00 (3H, m, Hg and OCHZ),3.77 (6H, s, OCH3), 3.61-3.29 (4H, m, Hs, HE,, and 2CH), 2.75 (2H, t, CH2CN J = 6.22 Hz), 1.27 (12 H, m, isopropyl), 0.88 (9H, s, tert -butyl), 0.10 (6H, s, CH3). MS (NaI 2-hydroxyethyl disulfide): mle 963 (M Na)+. TLC (ethyl acetate: dichloromethane 1:4): Rf 0.73,0.58 (two diastereomers).
+
+
Synthesis of (- )-5-Iodouridinephosphoramidite (8). The synthesis of phosphoramidite 8 is outlined in Scheme 1. The 5' OH of iodouridine 5 was protected as DMT ether 6 using DMTCl and pyridine, followed by selective 2' OH protection as the corresponding t-BDMS ether 7. Finally, nucleoside 3 was converted to the phosphoramidite 8 using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. 5' - 0-[(4,4'Dime t hoxy p he ny 1)met h y I/- (--)- 5 - iodo uridine (6). A solution of (')-5-iodouridine (370 mg, 1 mmol) and DMTCl (340 mg, 1 mmol) in dry pyridine (4 mL) was stirred for 6 h a t rt. Methanol (1 mL) was added, and after 15 min the solution was concentrated to dryness under reduced pressure. A 5% NaHC03 solution (10 mL) was added, the resulting solution was layer was dried over molecular sieves and reconcentrated extracted with ethyl acetate (2 x 15 mL), and the organic to yield a gum, which was purified by column chromalayer was dried over molecular sieves and reconcentrated tography (ethyl acetate). Nucleoside 2 was obtained a s to yield a gum, which was purified by column chromaa white solid (413 mg, 0.66 mmol; 66%). lH NMR (CDCl3) tography (ethyl acetate:benzene 1:l)to yield 5'-DMT 6 (ppm): 8.09 ( l H , s, H6), 7.41-7.17 (9H, m, aromatic), ether 6 as a white solid (417 mg, 0.62 mmol, 62%). 'H 6.82 (4H, d, aromatic, J = 8.7 Hz), 5.90 (lH, d, HI,, J = NMR (CDCl3) 6 (ppm): 8.10 ( l H , s, &), 7.44-7.18 (9H, 4.04 Hz), 4.46 ( l H , dd, Hy, J = 4.2 Hz), 4.41 (lH, t, H3,), m, aromatic), 6.83 (4H, d, aromatic, J = 8.8 Hz), 5.94 4.23 (lH, d, Hc, J = 3.08 Hz), 3.75 (6H, S, OCHs), 3.40 ( l H , d, His, J 4.18 Hz), 4.50 (lH, t, Hz,, J = 4.46 Hz), (2H, br s, Hg, and Hs,). MS (NaI MNBA): mle 647 (M 4.42 (lH, t, H3,, J = 4.46 Hz), 4.22 ( l H , br s, H ~ J )3.73 , -H Na)+. TLC (ethyl acetate): Rf 0.33. (6H, s, OCH3), 3.40 (2H, br s, Hg, and Hv). MS (NaI MNBA): mle 695 (M Na)+. TLC (ethyl acetate): Rf 5'-0-[(4,4'-Dimethoxyphenyl)methyl]-2'-O-(tertbutyl0.42. dimethylsilyl)-(-)-5-bromouridine (3). To a solution of nucleoside 2 (312 mg, 0.5 mmol) in dry THF (5 mL) was 5'-0-[(4,4'-Dimethoxyphenyl)methyl]-2'-0-(tertbutyladded dry pyridine (0.4 mL, 5 mmol) and silver nitrate dimethylsilyl)-(-)-5-iodouridine (?). To a solution of (102 mg, 0.6 mmol). The reaction mixture was stirred nucleoside 6 (269 mg, 0.4 mmol) in dry THF (5 mL) was for 15 min, followed by the addition of tert-butyldimethadded dry pyridine (0.16 mL, 2.0 mmol) and silver nitrate ylsilyl chloride (98 mg, 0.65 mmol). Stirring was con(82 mg, 0.48 mmol). The reaction mixture was stirred tinued for an additional 2 h, followed by filtration into for 15 min, followed by the addition of tert-butyldimeth5% NaHC03 solution (10 mL). The aqueous layer was ylsilyl chloride (79 mg, 0.52 mmol). Stirring was conextracted with ethyl acetate (3 x 10 mL), dried over tinued for an additional 2 h, followed by filtration into molecular sieves, and evaporated under reduced pres5% NaHC03 solution (10 mL). The aqueous layer was sure. Column purification (ethyl acetate:benzene 1:9) extracted with ethyl acetate (3 x 10 mL), dried over provided 2'-silylnucleoside as a white solid (249 mg, 0.33 molecular sieves, and evaporated under reduced presmmol, 67.5%). lH NMR (CDCl3) 6 (ppm): 8.17 ( l H , s, sure. The 2'-t-BDMS ether 7 was obtained as a white HC),7.45-7.23 (9H, m, aromatic), 6.85 (4H, d, aromatic, solid (270 mg, 0.34 mmol, 85.5%)after column purificaJ = 8 . 7 8 H ~ ) , 6 . 0 1 ( 1 H , d , H 1 , , J = 5 . 1 6 H ~ ) , 4 . 5 1 ( 1 H , t , tion (ethyl acetate:benzene 1:9). IH NMR (CDC13) 6 Hy, J = 5.14 Hz), 4.32 (lH, q, Hy, J = 4.04 Hz), 4.19 (ppm): 8.23 (lH, s, HG),7.47-7.25 (9H, m, aromatic), 6.87 (lH, d, Hq, J = 3.14Hz), 3.80 (6H, S, OCH3), 3.44 (2H, d, (4 H, d, aromatic, J = 8.82 Hz), 6.05 (lH, d, HI,,J = 5.4 Hg' and HE,, , J = 2.12 Hz), 0.93 (9H, s, t- butyl), 0.17 H~),4.52(1H,t,Hz.,J=5.12H~),4.30(1H,q,H3,,J= 3.94 Hz), 4.21 (lH, br s, Hc), 3.81 (6H, s, OCHs), 3.50 (6H, s, CH3). MS (NaI MNBA): mle 761 (M - H Na)+. TLC (ethyl acetate:benzene 1:l):Rf 0.83. (2H, d, Hy, J = 9.36 Hz), 3.40 (2H, d, HE", J = 9.78 Hz) 0.94 (9H, s, tert-butyl), 0.16 (6H, s, CH3). MS (NaI 5'-0-[(4,4'-Dimethoxyphenyl)methyl]-2'-O-(tertbutylMNBA): mle 809 (M + Na)+. TLC (benzene:ethyl acetate dimethylsilyl)-(-)-5-bromouridine3'-0-[2-~yanoethylN,N1:l):Rf 0.76. (diisopropylamino)phosphoramidite] (4). To a stirred solution of nucleoside 3 (200 mg, 0.27 mmol) in dry THF 5'-0-[(4,4'-Dimethoxyphenyl)methyl]-2'-O-(tertbutyl(5 mL) was added dry 2,4,6-collidine (0.26 mL, 2.02 dimethylsilyl)-(-)-5-iodouridine3'-0-[2-Cyanoethyl (N,Nmmol), followed by N-methylimidazole (0.01 mL, 0.135 diisopropylamino)phosphoramidite] (8). To a stirred mmol). 2-Cyanoethyl N,N-diisopropylchlorophosphorsolution of nucleoside 7 (78 mg, 0.1 mmol) in dry THF (2 amidite (45 pL, 0.20 mmol) in dry THF (1mL) was added mL) was added dry 2,4,6-collidine (0.1 mL, 0.75 mmol), dropwise over a period of 5 min a t rt. Stirring was followed by N-methylimidazole (4 pL, 0.05 mmol). 2continued for 1h a t rt. The reaction mixture was worked Cyanoethyl N,N-diisopropylchlorophosphoramidite (45 up by diluting it with ethyl acetate (25 mL) and washing pL, 0.20 mmol) in dry THF (1mL) was added dropwise the organic phase with 5%NaHC03 solution (5 mL) and over a period of 5 min a t rt. Stirring was continued for brine (5 mL). Evaporation followed by column purifica1 h a t rt. The reaction mixture was worked up by tion (ethyl acetate:benzene 1:9, 0.05% Et3N) yielded diluting it with ethyl acetate (25 mL) and washing the phosphoramidite 4 as a white solid (215 mg, 0.22 mmol, organic phase with 5%NaHC03 solution (5 mL) and brine
+
+
+
+
+
+
+
Bioconjugafe Chem., Vol. 5, No. 6,1994 511
Letters
Scheme 2 0
0 OMTO
'.., Chemical synthesis of RNA
OAN'
~
thioiacelic acidIDeprololection O
%-Af+
I
i
Y
P
I
OH
+Pr2N*P'OCH2CH2CN
Modified RNA
(lQ)
(5 mL). Evaporation followed by column purification (ethyl acetate:benzene 2:8, 0.05% EtsN) yielded phosphoramidite 8 as a white solid (78 mg, 0.8 mmol, 80%). lH NMR (CDC13) 6 (ppm): 8.18 (lH, s, H6), 7.35-7.26 (9H, m, aromatic), 6.83 (4H, d, aromatic, J = 7.7 Hz), 5.99 (lH, m, HI,), 4.61-4.35 (2H, m, HS. and Hy), 4.214.03 (3H, m, Hc and OCHZ), 3.77 (6H, s, OCHs), 3.503.15 (4H, m, HE', HV and 2CH), 2.80-2.55 (2H, m, CHzCN), 1.27 (12 H, m, isopropyl), 0.88 (9H, s, tert -butyl), 0.05 (6H, s, CH3). MS (NaI MNBA): mle 1010 (M Na)+. TLC (ethyl acetate:dichloromethane 3:7) Rf 0.62, 0.50 (two diastereomers). Synthesis of 04-Triazolouridine Phosphoramidite (10). The synthesis of triazolouridine phosphoramidite 10 is outlined in Scheme 2. Triazolouridine phosphoramidite (10) was obtained from commercially available uridine phosphoramidite 9 by using triazole, POC13, and triethylamine (20).
+
+
5'-O-[(Dimethoxyphenyl)methyl]-2'-O-(tert-butyldimethylsily1)triazolouridine 3'-0-[2-Cyanoethyl N,N-(diisopropylamino)phosphoramidite] (10). To a n ice-cooled stirred suspension of 1,2,4-triazole (209 mg, 3.03 mmol) in dry acetonitrile (4 mL) was added POC13 (0.06 mL), followed by dry Et3N (0.45 mL). After 30 min a solution of nucleoside 9 (43 mg, 0.05 mmol) in dry acetonitrile (1 mL) was added over a period of 15 min and stirring continued for 2 h. The reaction was stopped with saturated NaHC03 solution (5 mL), the aqueous layer extracted with ethyl acetate (3 x 10 mL), the organic layer washed with saturated NaHC03 (5 mL) and brine (5 mL) and dried over molecular sieves, and solvent removed under reduced pressure. The crude compound thus obtained was purified by column chromatography (ethyl acetate:benzene 8:2). Yield: 28 mg, 0.03 mmol, 62%. 'H NMR (CDCl3) 6 (ppm): 8.82-8.52 (lH, m, H6), 8.22 (2H, s, aromatic), 7.36-7.19 (9H, m, aromatic), 6.86-6.80 (4H, m, aromatic), 6.68-6.38 (lH, m, Hv), 6.05-5.80 ( l H , m, H5), 4.70-4.06 (5H, m, Hz,,HY,OCHz and Hc), 3.90-3.30 (4H, m, 2CH, Hg, and Hv), 3.79 (6H, s, OCH3), 2.80-2.50 (2H, m, CHzCN), 1.30-1.20 (12 H, m, isopropyl), 0.91-0.74 (9H, m, tert-butyl), 0.22-0.03 (6H, m, CH3). MS (NaI 2-hydroxyethyl disulfide): mle 937 (M + Na Hz)+. TLC (ethyl acetate:dichloromethane 2:3): Rf 0.46, 0.35 (two diastereomers). RNA Synthesis and Deprotection. All RNA syntheses were performed on AI31 synthesizer Model 392
+
+
using standard protocols. All the monomers of (2cyanoethy1)phosphoramidites were obtained from Glen Research. The fully protected (with 5'-DMT removed) sequence 3, 5'-AUU C P a z UG-3' attached to the CPG support was treated with 10%thiolacetic acidacetonitrile (1mL) overnight a t rt. Resin was filtered, washed with excess acetonitrile, and dried. Cleavage from the support and deprotection was carried out with 10% DBUl methanol (1mL) for 16 h at rt. Product was filtered and purified by Sep Pak cl8 cartridge by standard methods. Purified and 2'-protected RNA was dried and further treated with 0.5 mL of TBAF' (1.0 M solution in THF) for 24 h a t rt. The reaction was quenched with 1.0 mL of 0.1 M TEAA, pH 7.0, and dried to a total volume of 1.0 mL. Desalting was achieved by Sep Pak cartridge purification. The other three sequences of RNA were deprotected using 10% DBU/methanol (1 mL) followed by TBAF treatment and purification as described above. Enzymatic Digestion of Oligonucleotides. Enzymatic digestion was carried out by incubating 0.M260unit of purified oligonucleotide a t 37 "C overnight with 6 pL (12 pg) of snake venom phosphodiesterase and 2 pL (2 units) of alkaline phosphatase in a total volume of 78.2 pL of 32 mM Tris, pH 7.5, and 15 mM MgClz (21). Nucleosides were recovered by adding 10 pL of 3 M NaOAc, pH 5.2, and 234 pL of 95% ethanol, mixing and chilling to -80 "C for 30 min. After centrifugation a t 13 000 rpm for 20 min a t 4 "C, the supernatent was taken and dried in Speed-Vac (Savant) and redissolved in 200 pL of water for HPLC analysis. HPLC Analysis of Oligonucleotides. Nucleosides were chromatographed on a cl8 reversed phase column (Beckman 5 pm 4.6 mm x 25 cm, Ultrasphere). Solvent A: 97.5% 0.01 M KHzPO4 (pH 5), 2.5% methanol; Solvent B: 80% 0.01 M KHzPO4 (pH 5.1),20%methanol. Gradient: a t 0 min, 0% B; a t 7 min, start ramp to 10% B over 5 min; a t 12 min, start ramp to 25% B over 3 min; a t 15 min, start ramp to 60% B over 5 min; a t 20 min, start ramp to 62% B in 2.5 min; a t 22.5 min, start ramp to 100% B over 6 min; at 28.5 min, continued 100% B for 10 min. UV absorbance of nucleosides was monitored a t 254 nm. Retention times for ribonucleosides (in min) were as follows: C, 7.19; U, 10.44; G, 22.47; 5-bromo-U, 24.9; 5-iodo-U, 28.14; A, 31.25. Quantitation of HPLC data was performed by comparison to authentic nucleoside standards and integration of the absorbance as described by Eadie et al. (21). ACKNOWLEDGMENT
We wish to thank Dr. Edward Browning for his help in HPLC analysis of oligonucleotides and Dr. Narayan C. Chaudhuri for helpful discussions. This research was supported by a Research Grant AI 34785 from the National Institutes of Health. LITERATURE CITED (1) Cech, T. R. (1991) Self splicing of RNA. Curr. Opin. Cell. Biol. 59, 543-568. (2) Dock-Bregeon, A. C., Chevrier, B., Podjarny, A., Johnson, J., De Bear, J. S., Gough, G. R., Gilham, P. T., and Moras, D. (1989) Crystallographic structure of an RNA helix: [U(UA)6Al2. J . Mol. Biol. 209, 459-474. (3) Holbrook, S. R., Cheong, C., Tinoco, I., Jr., and Kim, S-H. (1991) Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs. Nature 353, 579581. (4) Varani, G., and Tinoco, I., Jr. (1991) RNA structure and NMR spectroscopy. Q. Rev.Biophys. 24, 479-532.
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