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Bioconjugate Chem. 2003, 14, 80−85
Reversible Biotinylation Phosphoramidite for 5′-End-Labeling, Phosphorylation, and Affinity Purification of Synthetic Oligonucleotides Shiyue Fang†,§ and Donald E. Bergstrom*,†,§ Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907 and Walther Cancer Institute, Indianapolis, Indiana 46208. Received October 14, 2002
A fluoride/amine-cleavable phosphoramidite designed for biotinylation, phosphorylation, and affinity purification of synthetic oligonucleotides was synthesized and coupled efficiently to the 5′-end of DNA on a solid-phase automatic synthesizer. The two hydroxyl groups of diethyl bis(hydroxymethyl)malonate were used to link biotin and the 5′-end of DNA together through a diisopropylsilyl acetal functionality and a phosphate ester group, respectively. The DNA was cleaved from solid support and fully deprotected by treating with a mixture of MeNH2 (∼40%) and NH4OH (∼29%) (1:1, v/v, 65 °C, 30 min), and the linkage between biotin and DNA was found completely stable under these conditions. The biotinylated full-length DNA was efficiently attached to NeutrAvidin coated microspheres and failure sequences and other impurities were simply removed by washing with buffer and water. The microspheres were then treated with HF/pyridine/THF (rt, 1 h) and MeNH2 (∼40%, rt, 15 min) sequentially to yield high quality full-length 5′-end phosphorylated unmodified DNA as revealed by HPLC analysis. It is anticipated that this method will find applications in areas that require efficient isolation of 5′-end phosphorylated DNA from a complex mixture.
INTRODUCTION
MATERIALS AND METHODS
The high affinity between biotin and streptavidin or avidin (association constant 1015/M) has been widely used as a means to label DNA and RNA (1), and as a result, chemical biotinylation of these biopolymers has received considerable attention (2-9). For some applications, reversible biotinylation may be required in order to regenerate unmodified DNA/RNA that can be biochemically or biologically processed. For 5′-end reversibly biotinylated DNA/RNA, after cleavage, both 5′-end nonphosphorylated and 5′-end phosphorylated DNA/RNA are required to meet different applications. For the former purpose, Gildea and co-workers reported an acid-labile biotinylation phosphoramidite (8). For the latter purpose, Rothschild and co-workers reported a photocleavable biotinylation phosphoramidite (9). Although they successfully applied their methods for affinity purification of synthetic oligonucleotides, Gildea’s method requires a challenging and inconvenient reagent synthesis, and in Rothschild’s method, formation of thymine-thymine photodimer under UV irradiation is a concern (10-12). We recently reported a fluoride-cleavable biotinylation phosphoramidite for 5′-end-labeling, affinity purification of synthetic oligonucleotides (13). In this method, nonphosphorylated unmodified DNA was obtained after removal of the biotin tag. Complementary to this method, we describe here a reversible biotinylation phosphoramidite that on removal of the biotin tag affords 5′-end phosphorylated unmodified DNA. * To whom correspondence should be addressed. . Telephone: 765-494-6275 Fax: 765-494-9193 email: bergstrom@ purdue.edu. † Purdue University. § Walther Cancer Institute.
General. All reactions were performed under a blanket of dry argon. Reagents and solvents available from commercial sources were used as received unless otherwise noted. Tetrahydrofuran was distilled from a Na/ benzophenone ketyl. Acetonitrile was distilled over CaH2. Acetone was dried over anhydrous Na2SO4, and the supernatant was used directly. Thin-layer chromatography (TLC) was performed using Sigma-Aldrich TLC plates, silica gel on aluminum 60F-254, 200 µm thickness. Flash column chromatography was performed using ‘Baker’ silica gel (40 µm). NMR spectra were obtained using a 250 or 500 MHz Bruker spectrometer. Chemical shifts (δ) are reported relative to CHCl3 (δ ) 7.27 ppm for 1H and 77.23 ppm for 13C) or triphenyl phosphate (δ ) 0.00 ppm for 31P). Infrared spectra were recorded on a Nicolet FTIR spectrophotometer. High-resolution mass spectra were obtained on a Finnigan Mat 95XL spectrometer. UltraLink Immobilized NeutrAvidin (slurry in water, approximately 50% v/v, containing 0.02% sodium azide; pore size, 1000 Å; particle size, 50-80 µm; biotinbinding capacity, ∼0.08 µmol biotin/mL gel) was purchased from Pierce. Aqueous MeNH2 (∼40%) and NH4OH (∼29%), HF/pyridine (HF, ∼70%; pyridine, ∼30%), diethyl bis(hydroxymethyl)malonate and Me3SiOMe were purchased from Aldrich Inc. Diisopropyldichlorosilane was purchased from Gelest Inc. Succinic ester linked DMTr-dT-lcaa-CPG (pore size 1000 Å), (2-cyanoethyl)[2,2-bis(ethoxycarbonyl)-3-(4,4′-dimethoxytrityloxy)propyl1]-N,N-diisopropylphosphoramidite and 5′-DMTr, 2-cyanoethyl phosphoramidites: benzoyl-dA, isobutyryl-dG, acetyl-dC, and dT were purchased from Glen Research Inc. PBS buffer: 136.9 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, adjusted to pH 7.0 with HCl. HPLC: C-18 reverse phase column (100 Å, 250 × 4.6 mm for analysis, 250 × 10.0 mm for preparation, Varian
10.1021/bc025626o CCC: $25.00 © 2003 American Chemical Society Published on Web 12/19/2002
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Scheme 1
Analytical Instruments); solvent A: 0.1 M triethylammonium acetate, 5% acetonitrile; solvent B: 90% acetonitrile; profiles were generated by detection of the absorbance of DNA at 260 nm using two linear gradient solvent systems: Gradient A: solvent B (0-45%) in solvent A over 60 min at flow rates of 1 mL/min (analysis) and 3 mL/min (preparation); Gradient B: solvent B (020%) in solvent A over 60 min at a flow rate of 1 mL/min (analysis). In all cases, gradient A was used unless otherwise noted. Biotin Diethyl Bis(hydroxymethyl)malonate Conjugate 2. A one-necked round-bottomed flask was charged with biotinyl alcohol 1 (605 mg, 0.93 mmol), synthesized following procedures described previously (13), and imidazole (63 mg, 0.93 mmol) and flushed with Ar. DMF (2 mL) and diisopropylethylamine (498 µL, 2.79 mmol) were added via syringe. After the mixture was cooled to 0 °C, diisopropyldichlorosilane (252 µL, 1.40 mmol) was added via syringe in one portion. The light yellow solution was stirred at 0 °C for 1 h and at rt for 4 h and then added to a solution of diethyl bis(hydroxymethyl)malonate (500 mg, 2.20 mmol) and imidazole (126 mg, 1.86 mmol) in DMF (2 mL) via syringe very slowly (over 45 min). After being stirred at 0 °C for 5 h, the reaction mixture was partitioned between CH2Cl2 (30 mL × 5) and 5% citric acid (50 mL). The organic phase was washed with brine (30 mL) and dried over anhydrous Na2SO4. Removal of volatile components gave a light yellow residue, which was purified by flash column chromatography (3.0 × 12 cm, SiO2, 19:1, CH3Cl/CH3OH). The highest UV active spot (the major one) on TLC (Rf ) 0.5; 9:1, CH3Cl/CH3OH) was easily isolated, giving 2 (Scheme 1)as a white foam (839 mg, 92%): IR (thin film, cm-1) ν 3318, 2954, 2864, 1739, 1652, 1555, 1040; 1H NMR (CDCl3, 500 MHz) δ 0.88-1.01 (m, 14H), 1.24-1.27 (m, 12H), 1.32 (s, 9H), 1.47-1.53 (m, 2H), 1.65-1.85 (m, 6H), 2.18-2.31 (m, 4H), 3.03-3.10 (m, 2H), 3.25 (dt, 1H, J ) 7.4, 4.7 Hz), 3.383.50 (m, 4H), 3.54-3.56 (m, 4H), 3.60 (s, 4H), 4.14-4.27 (m, 9H), 5.22-5.24 (m, 1H), 6.37 (t, 1H, J ) 5.5 Hz), 6.58 (t, 1H, J ) 5.3 Hz), 7.39 (d, 2H, J ) 8.5 Hz), 7.57 (d, 2H, J ) 8.5 Hz); 13C NMR (CDCl3, 500 MHz) δ 13.4, 14.2, 17.7, 27.8, 29.8, 30.5, 31.3, 32.0, 35.1, 35.6, 38.4, 39.2, 39.5, 40.0, 55.2, 57.5, 61.6, 61.7, 61.8, 62.7, 70.0, 70.1 (X 2), 70.3, 73.8, 124.7, 129.1, 131.9, 155.1, 156.3, 169.2, 170.2, 173.3, 174.2. HRMS (ESI, M + H+) Calcd for C48H81N4O13SSi 981.5290, found 981.5295.
Biotin Diethyl Bis(hydroxymethyl)malonate Conjugate Phosphoramidite 3. To a solution of 2 (715 mg, 0.72 mmol) in dry acetonitrile (2 mL) were added 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (264 µL, 0.80 mmol) and 1H-tetrazole (0.45 M in acetonitrile, 1.68 mL, 0.76 mmol) sequentially. After stirring at rt for 3 h, some white precipitate formed. The reaction was quenched with NaHCO3 (5%, 50 mL), and the mixture was extracted by CH2Cl2 (30 mL × 5). The organic phase was dried over anhydrous Na2SO4, solvents were removed under reduced pressure, and the residue was purified by flash column chromatography (3.0 × 10 cm, SiO2, 7:3: 0.5, CHCl3/THF/Et3N), giving 3 as a white foam (520 mg, 61%): Rf ) 0.5 (7:3:0.5, CHCl3/THF/Et3N); 1H NMR (CDCl3, 250 MHz) δ 0.91-1.07 (m, 14H), 1.14-1.18 (m, 12H), 1.22-1.29 (m, 12H), 1.32 (s, 9H), 1.41-1.51 (m, 2H), 1.65-1.82 (m, 4H), 2.16-2.34 (m, 4H), 2.60 (t, 2H, J ) 6.8 Hz), 3.04-3.06 (m, 2H), 3.21-3.26 (m, 1H), 3.403.48 (m, 4H), 3.52-3.61 (m, 8H), 3.76-3.82 (m, 2H), 4.05-4.25 (m, 11H), 5.19-5.22 (m, 1H), 6.28 (t, 1H, J ) 5.5 Hz), 6.39 (t, 1H, J ) 5.3 Hz), 7.39 (d, 2H, J ) 8.0 Hz), 7.57 (d, 2H, J ) 8.3 Hz); 31P NMR (CDCl3, 250 MHz) δ 166.36. Biotinyl Oligonucleotide 4, Synthesis, Cleavage, Deprotection, and HPLC Analysis. The oligonucleotide was synthesized on an ABI DNA/RNA synthesizer at 1 µmol scale, using the following 5′-DMTr, 2-cyanoethyl phosphoramidites: benzoyl-dA, isobutyryl-dG, acetyldC and dT. The manufacture recommended synthetic cycles were used except that 3 (0.1 M in acetonitrile) was coupled for 15 min. The CPG was dried under nitrogen flow and then divided into two portions (0.52 µmol, 0.48 µmol). To the 0.52 µmol portion in a screw-capped 5-mL vial was added NH4OH (∼29%, 200 µL) and methylamine (∼40%, 200 µL), and the resulting suspension was heated to 65 °C for 30 min. After being cooled to -20 °C, the supernatant was taken out, and the CPG was washed with water (200 µL × 5). The supernatant and water washes were combined and dried on a SpeedVac. The residue was redissolved in 1.0 mL water, of which 10 µL was diluted to 100 µL, and 50 µL was analyzed by HPLC to generate trace a (Figure 1). From the 1.0 mL solution was taken out a 100 µL portion and dried on a SpeedVac. The residue was suspended in dry THF (200 µL) in an Eppendorf tube, 30 µL of HF/pyridine was added via pipet. The mixture was vortexed for 1 min and then
82 Bioconjugate Chem., Vol. 14, No. 1, 2003
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Figure 1. HPLC traces of (a) crude oligonucleotide 4, containing failure sequences. (b) HF/pyridine-treated crude 4. (c) HF/pyridineand aqueous methylamine-treated crude 4. (d) Unbiotinylated failure sequences left after treating crude 4 with NeutrAvidin gel. (e) Affinity-purified oligonucleotide 7. (f) Authentic 6. (g) Authentic 6 and HF/pyridine-treated crude 4. (h) Authentic 7 and affinitypurified 7. (i) Authentic 7. (j) Authentic 7 and affinity-purified 7 eluted with solvent gradient B. (k) Authentic 8 and affinity-purified 7 eluted with solvent gradient B.
allowed to stand at rt for 1 h with occasional shaking. The excess fluoride ion was quenched with Me3SiOMe (500 µL) by gentle shaking at rt for 30 min. Volatile components were removed on a SpeedVac, the residue was dissolved in 500 µL water, and 50 µL was analyzed by HPLC to generate trace b (Figure 1). From the 1.0 mL solution was taken out another 100 µL portion, which was treated with HF/pyridine and Me3SiOMe exactly as described above. To the resulting residue was added MeNH2 (∼40%, 200 µL), the mixture was gently shaken at rt for 15 min and then dried on a SpeedVac. The residue was dissolved in 500 µL water, and 50 µL was analyzed by HPLC to generate trace c (Figure 1). The coupling efficiency of 3 was estimated to be more than 81% by comparing the area of the peak with retention time near 43 min to that of the major peak near 15 min in trace a (Figure 1). Affinity Purification and HPLC Analysis. Another 100 µL was taken out from the above-described 1.0 mL solution of crude oligonucleotide 4, dried on a SpeedVac, redissolved in 300 µL of PBS buffer, and incubated with UltraLink Immobilized NeutrAvidin gel (0.5 mL gel, 1.0 mL gel slurry), which was washed with PBS buffer (300 µL × 3), for 1 h at rt with gentle shaking (it is important to handle the gel gently, otherwise, gel material may leak, and recovery yield of full-length DNA may be low). The mixture was centrifuged, the supernatant removed, and the gel washed with PBS buffer (300 µL × 2). The supernatant and PBS buffer washes were combined, dried on a SpeedVac, redissolved in 500 µL water, and 100 µL was analyzed by HPLC to generate trace d (Figure 1). The gel was further washed with PBS buffer (500 µL × 3) and water (500 µL × 3), dried by washing with dry acetone (500 µL × 3) and THF (500 µL × 3), and then suspended in THF (300 µL). To the suspension was added HF/pyridine (30 µL), and the mixture was incubated at rt for 1 h with gentle shaking. The excess fluoride ion was quenched with Me3SiOMe (500 µL, rt, 30 min), the mixture was centrifuged, and the supernatant was removed. The gel was washed with THF (300 µL × 2) and then incubated in MeNH2 (∼40%, 500 µL) at rt for 15 min. The mixture was centrifuged, the supernatant obtained, and the gel washed with water (300 µL × 8).
The supernatant and water washes were combined and dried on a SpeedVac. The residue was redissolved in water (1.5 mL), and 100 µL was analyzed by HPLC to give trace e (Figure 1). The recovery yield of full-length 5′-end phosphorylated DNA 7 (see Scheme 2 for structure) was estimated to be 72% by comparing the area of the peak in trace e to that in trace a with a retention time of near 43 min. The presence of the 5′-phosphate group on 7 was supported by HPLC analysis through coinjection of authentic 6 (authentic samples were synthesized as described below) with the sample used to generate trace b (trace g, Figure 1). Coinjection of the affinity purified 7 with authentic 7 gave trace h (Figure 1). Coinjection of affinity purified 7 with the nonphosphorylated authentic sample HOACAGTGACTOH (8) eluting with solvent gradient A, however, gave a single peak (HPLC trace not shown). To resolve affinity purified 7 and authentic 8, solvent gradient B was used, and HPLC trace k was generated. Coinjection of affinity purified 7 with authentic 7 under these conditions gave trace j. The identity of affinity purified 7 was also confirmed by MALDI mass spectrum analysis of the failure sequences and full-length 7 (14). The samples, which were used to generate HPLC traces d and e, were desalted by Sep-Pac cartridge independently according to the reported procedure and analyzed separately (14). The failure sequences gave the following peaks: 2718.4 for (HO-TCAGTGACA-OH - H)- calcd 2721.9, 2405.7 for (HO-TCAGTGAC-OH - H)- calcd 2408.7, 2117.7 for (HO-TCAGTGA-OH - H)- calcd 2119.5, 1804.3 for (HOTCAGTG-OH - H)- calcd 1806.3, 1476.5 for (HO-TCAGTOH - H)- calcd 1477.1, 1173.2 for (HO-TCAG-OH - H)calcd 1172.8, 844.9 for (HO-TCA-OH - H)- calcd 843.6, 531.0 for (HO-TC-OH - H)- calcd 530.4. The affinity purified 7 gave a peak of 2804.8 for (HO-TCAGTGACAOPO3H2 - H)- calcd 2801.9. Synthesis and Purification of Authentic Oligonucleotides 6, 7, and 8. Oligonucleotide 6 and 7 were synthesized independently on an ABI DNA/RNA synthesizer at 1 µmol scale, using the following 5′-DMTr, 2-cyanoethyl phosphoramidites: benzoyl-dA, isobutyryldG, acetyl-dC and dT; and (2-cyanoethyl)[2,2-bis(ethoxycarbonyl)-3-(4,4′-dimethoxytrityloxy)propyl-1]-N,N-diiso-
Reversible Biotinylation Phosphoramidite
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Scheme 2
propylphosphoramidite. Manufacture recommended synthetic cycles were used. Cleavage/deprotection was performed by NH4OH (∼29%) and MeNH2 (∼40%) (1:1, v/v) at 65 °C for 15 min. Following the procedure described by Guzaev (15), trityl-on reverse phase preparative HPLC purification, detritylation gave 6 (HPLC trace f, Figure 1); removal of the 5′-end diethyl bis(hydroxymethyl)malonate moiety gave 7 (HPLC trace i, Figure 1). Authentic 8 was synthesized following a procedure described previously (13). RESULTS AND DISCUSSION
We recently reported the design and synthesis of a fluoride-cleavable phosphoramidite for 5′-end biotinylation and affinity purification of synthetic oligonucleotides (13). The biotinylated structure obtained by such a method after cleavage of the oligonucleotide from solid support and ammonia deprotection is illustrated in Figure 2 (9). The diisopropylsilyl acetal linkage, which connects the biotin moiety and the 5′-OH of the synthetic oligonucleotide through a tertiary hydroxide group, was found completely stable under certain postsynthetic DNA cleavage/deprotection conditions. However, this linkage can be readily broken by treating with HF/pyridine under mild conditions to yield unmodified DNA carrying a free 5′-OH. In 1995, Guzaev and co-workers described an approach that used the dimethoxytrityl protected diethyl bis(hydroxymethyl)malonate phosphoramidite for chemical phosphorylation of oligonucleotides at the 5′-terminus (15). The structure of DNA obtained by their method is also shown in Figure 2 (10). The DMTr-protected diethyl bis(hydroxymethyl)malonate moiety that is linked to the 5′-OH of DNA through a phosphodiester group is stable under common amino cleavage/deprotection conditions.
Figure 2.
This property is useful for purification of synthetic DNA by reverse phase HPLC. After purification and removal of the DMTr group, the 5′-end phosphate group is released by briefly treating with aqueous amine under mild conditions. On the basis of these two reports, we reasoned that substitution of the DMTr group in 10 by the diisopropylsilyl acetal functionality linked to biotin as in 9 would provide a simple method for reversible biotinylation and phosphorylation of DNA. This led to design of the reversible biotinylation phosphoramidite 3 (Scheme 1). The new linkage between DNA and biotin, like 9 and 10, should be stable under certain postsynthetic DNA cleavage/deprotection conditions. Removal of the silyl acetal group by treating with fluoride ion should generate the same species as treating 10 with acid, and 5′-end phosphorylated DNA should be produced in the presence of aqueous amine under mild conditions. Synthesis of the phosphoramidite 3 is shown in Scheme 1. The biotinyl tertiary alcohol 1 was prepared according to procedures described previously (13). The tertiary hydroxyl group of 1 was first silylated by diisopropyldichlorosilane in DMF in the presence of diisopropylethylamine (2 equiv) and imidazole. This solution was added to a solution of diethyl bis(hydroxymethyl)malonate and imidazole in DMF via syringe very slowly to avoid possible deformylation of the hydroxymethylmalonate under basic conditions, and 2 was obtained as a white foam in excellent yield. The biotinyl alcohol 2 was then phosphinylated under a slightly acidic conditions following Guzaev’s procedure to give 3 (61%) (15). The biotinyl oligonucleotide 4 (Scheme 2) was synthesized on an automatic DNA/RNA synthesizer according to standard synthetic cycles. The following 5′-DMTr, 2-cyanoethyl phosphoramidites were used: benzoyl-dA,
84 Bioconjugate Chem., Vol. 14, No. 1, 2003
isobutyryl-dG, acetyl-dC and dT, and in the last cycle, 3 (0.1 M in acetonitrile) was coupled for 15 min. Protection of C with an acetyl group allowed fast cleavage/deprotection of the DNA by treating with MeNH2 (∼40%)/NH4OH (∼29%) (1:1, v/v, 65 °C, 30 min) (16, 17). The 4-tertiary butyl benzoyl protecting group on biotin was also removed under these conditions, giving the diisopropylsilyl acetal linked biotinyl oligonucleotide 4 as the product (13). Analysis by HPLC generated trace a (Figure 1); the major peak with a retention time near 43 min should be the biotinylated oligonucleotide 4 (Scheme 2). A portion was treated with HF/pyridine, followed by quenching the excess fluoride ion by methoxytrimethylsilane (18), dried, redissolved in water, and analyzed by HPLC to give trace b (Figure 1); the major peak with a retention time near 20 min should be 6 (Scheme 2). The peaks with retention time around 15 min in traces a and b should be failure sequences and other impurities. Another portion was treated similarly with HF/pyridine, followed by methoxytrimethylsilane, but was then further incubated in aqueous MeNH2 (∼40%) at room temperature to give the 5′-end phosphorylated unmodified oligonucleotide 7 (Scheme 2) (15). As expected, HPLC analysis (trace c, Figure 1) indicated that the major peaks in traces a (retention time ∼43 min) and b (retention time ∼20 min) was replaced by a peak near 15 min. Comparison of traces a with b and c indicates that the linkage between biotin and the 5′-end of DNA is completely stable under these postsynthetic DNA cleavage/deprotection conditions. The coupling efficiency of phosphoramidite 3 was estimated to be 81% by comparing the area of the peak in trace a with a retention time of near 43 min to that of the largest peak with retention time near 15 min. To demonstrate application of this biotinylation strategy, the crude oligonucleotide 4, contaminated by failure sequences and other impurities (see trace a, Figure 1), was then purified by NeutrAvidin-coated microspheremediated affinity purification. Because the failure sequences and other impurities do not contain biotin, incubation of the crude product with the microsphere followed by washing with buffer should leave only the biotinylated full-length 4 on the solid phase. In practice, crude 4 was incubated with the microspheres in PBS buffer at room temperature for 1 h. After being centrifuged, the supernatant was removed and analyzed by HPLC (trace d, Figure 1). The major peak in trace a (retention time ∼43 min) disappeared in trace d, showing the high efficiency of binding 4 to the NeutrAvidin-coated microspheres. The microspheres were then further washed with buffer and water, dried by washing with anhydrous acetone and THF, and then suspended in dry THF. The cleavage reagent HF/pyridine was added, incubated at room temperature, followed by quenching excess fluoride ion by methoxytrimethylsilane to give 6 (Scheme 2). The mixture was centrifuged and the supernatant removed. The gel was washed with THF and incubated at room temperature in aqueous MeNH2 (∼40%) to remove the diethyl bis(hydroxymethyl)malonate moiety and generate 5′-end phosphorylated 7 (15). After centrifugation, the supernatant was obtained, and the gel was washed with water. The supernatant and water washes were combined, dried on a SpeedVac, redissolved in water, and analyzed by HPLC to give trace e (Figure 1). As can be seen, the full-length, 5′-end phosphorylated DNA 7 is very pure. The recovery yield of 7 by this purification method was estimated by comparing the area of the peak in trace e (Figure 1) with that in trace a with a retention time of near 43 min, and it was 72%. To confirm the identity of 7, authentic samples of 6 and 7 were prepared
Fang and Bergstrom
following the published procedure (15). We first confirmed the structure of 6 by coinjection of the sample used to generate HPLC trace b with authentic 6 to give trace g. The identity of 7 was confirmed by coinjection of affinity purified sample with authentic 7, whereupon only one peak was observed (trace h, Figure 1). However, when we coinjected affinity purified 7 with nonphosphorylated authentic sample HOACAGTGACTOH (8), prepared according to procedures described previously (13), a single peak was also observed (HPLC trace not shown). To resolve affinity purified 7 and authentic 8, the solvent gradient B was used, and HPLC trace k was generated, which had two peaks. To make certain the affinity purified 7 and authentic 7 were also identical under these conditions, they were also coinjected for HPLC analysis, eluting with solvent gradient B; as expected, a single peak was observed (trace j). The identity of 7 was also conveniently confirmed by MALDI mass spectrum analysis of the failure sequences and full-length 7 (samples used to generate HPLC traces d and e, respectively), because the failure sequences were easily enriched after efficient separation from the full-length biotinylated 4 (14). In conclusion, we designed and synthesized a novel reversible biotinylation, phosphorylation phosphoramidite, and successfully used it to biotinylate and phosphorylate the 5′-end of DNA on an automatic synthesizer. We demonstrated the use of this method by application in affinity purification of a synthetic oligonucleotide. The full-length biotinylated DNA was efficiently attached to NeutrAvidin-coated microspheres by brief incubation, and failure sequences and other impurities were removed by simple washing with buffer and water. High quality full-length 5′-end phosphorylated DNA was recovered by brief treatments with fluoride ion and aqueous amine, sequentially. The procedure is simple, and the recovery yield is high. We anticipate that this method will find applications in areas that require efficient isolation of 5′-end phosphorylated DNA from complex mixtures. ACKNOWLEDGMENT
Grant support from the National Institutes of Health (GM53155) and the Showalter Trust Fund is gratefully acknowledged. Assistance from the National Cancer Institute Grant (P30 CA23168) awarded to Purdue University is also gratefully acknowledged. LITERATURE CITED (1) McInnes, J. L., and Symons, R. H. (1989) Preparation and detection of nonradioactive nucleic acid and oligonucleotide probes. Nucleic Acid Probes (R. H. Symons, Ed.) pp 33-80, CRC Press, Boca Raton, FL. (2) Zhao, Z., and Ackroyd, J. (1999) A biotin phosphoramidite reagent for the automated synthesis of 5′-biotinylated oligonucleotides. Nucleosides Nucleotides 18, 1231-1234. (3) Kumar, P., Bhatia, D., Garg, B. S., and Gupta, K. C. (1994) An improved method for synthesis of biotin phosphoramidites for solid-phase biotinylation of oligonucleotides. Bioorg. Med. Chem. Lett. 4, 1761-1766. (4) Neuner, P. (1996) New non nucleosidic phosphoramidite reagent for solid-phase synthesis of biotinylated oligonucleotides. Bioorg. Med. Chem. Lett. 6, 147-152. (5) Pieles, U., Sproat, B. S., and Lamm, G. M. (1990) A protected biotin containing deoxycytidine building block for solid-phase synthesis of biotinylated oligonucleotides. Nucleic Acids Res. 18, 4355-4360. (6) Shimkus, M., Levy, J., and Herman, T. (1985) A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns. Proc. Natl. Acad. Sci. U.S.A. 82, 2593-2597.
Reversible Biotinylation Phosphoramidite (7) Herman, T. M., and Fenn, B. J. (1990) Chemically cleavable biotin-labeled nucleotide analogs. Methods Enzymol. 184, 584-588. (8) Gildea, B. D., Coull, J. M., and Koster, H. (1990) A versatile acid-labile linker for modification of synthetic biomolecules. Tetrahedron Lett. 31, 7095-7098. (9) Olejnik, J., Krzymanska-Olejnik, E., and Rothschild, K. J. (1996) Photocleavable biotin phosphoramidite for 5′-endlabelling, affinity purification and phosphorylation of synthetic oligonucleotides. Nucleic Acids Res. 24, 361-366. (10) Greenberg, M. M. (1995) Photochemical release of protected oligodeoxyribonucleotides containing 3′-glycolate termini. Tetrahedron 51, 29-38. (11) Greenberg, M. M., and Gilmore, J. L. (1994) Cleavage of oligonucleotides from solid-phase supports using o-nitrobenzyl photochemistry. J. Org. Chem. 59, 746-753. (12) Cadet, J., and Vigny, P. (1990) The photochemistry of nucleic acids. Bioorganic Photochemistry (H. Morrison, Ed.) Vol. 1, pp 170-184, John Wiley & Sons, New York. (13) Fang, S., and Bergstrom, D. E. (2003) Fluoride-cleavable biotinylation phosphoramidite for 5′-end-labeling, affinity purification of synthetic oligonucleotides. Nucleic Acids Res., in press. (14) Alazard, D., Filipowsky, M., Raeside, J., Clarke, M., Majlessi, M., Russell, J., and Weisburg, W. (2002) Sequencing
Bioconjugate Chem., Vol. 14, No. 1, 2003 85 of production-scale synthetic oligonucleotides by enriching for coupling failures using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Anal. Biochem. 301, 57-64. (15) Guzaev, A., Salo, H., Azhayev, A., and Lo¨nnberg, H. (1995) A new approach for chemical phosphorylation of oligonucleotides at the 5′-terminus. Tetrahedron 51, 9375-9384. (16) Reddy, M. P., Hanna, N. B., and Farooqui, F. (1994) Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 35, 4311-4314. (17) Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, Z. (1995) Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 23, 2677-2684. (18) Tallarico, J. A., Depew, K. M., Pelish, H. E., Westwood, N. J., Lindsley, C. W., Shair, M. D., Schreiber, S. L., and Foley, M. A. (2001) An alkylsilyl-tethered, high-capacity solid support amenable to diversity-oriented synthesis for one-bead, one-stock solution chemical genetics. J. Comb. Chem. 3, 312-318.
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