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. 2003 Jan 15;31(2):708–715. doi: 10.1093/nar/gkg130

Fluoride-cleavable biotinylation phosphoramidite for 5′-end-labeling and affinity purification of synthetic oligonucleotides

Shiyue Fang 1,2, Donald E Bergstrom 1,2,a
PMCID: PMC140496  PMID: 12527780

Abstract

A fluoride-cleavable phosphoramidite for biotinylation was designed, synthesized and coupled efficiently to the 5′-end of DNA on an automatic synthesizer. The diisopropylsilyl acetal functionality was used to link the biotin moiety through a tertiary hydroxide group to the 5′-end of DNA. This linkage proved to be completely stable under certain post-synthetic DNA cleavage/deprotection conditions [0.05 M K2CO3 in MeOH, room temperature, 24 h and MeNH2 (∼40%)/NH4OH (∼29%), 1:1 v/v, 65°C, 30 min] while it can be readily broken by fluoride ion, releasing unmodified DNA. To demonstrate the use of this DNA biotinylation method, we applied this method in affinity purification of synthetic DNA. As revealed by HPLC analysis, biotinylated full-length DNA can be efficiently attached to NeutrAvidin™ coated microspheres, and failure sequences can be readily removed. Subsequent treatment of the microspheres with pyridine/HF released high quality full-length unmodified DNA in good yield.

INTRODUCTION

The strong affinity between biotin and streptavidin or avidin (association constant 1015/M) is widely used as a means to label DNA and RNA (1). Biotinylation of the target oligonucleotides can be achieved either enzymatically (2) or chemically. Site specific chemical biotinylation can be performed after an oligonucleotide has been synthesized and cleaved from a solid support and fully deprotected (3–6). A more convenient way is to synthesize a biotinylated phosphoramidite, and incorporate this into the oligonucleotide during automated solid phase synthesis (7–17). Phosphoramidite reagents are available that allow one to label either the 3′- or 5′-end or, by way of a modified base, any site in between. Most linkers between the biotin and the phosphoramidite have been designed for permanent attachment to the oligonucleotide (9–18). Alternatively, cleavable linkers provide a means for reversible biotinylation. By using a cleavable linker, the harsh conditions for breaking the biotin–streptavidin binding can be avoided when it is necessary. An example is incorporation of a disulfide bond into the linker between biotin and the oligonucleotide, which can be broken by a reducing reagent such as dithiothreitol (19–22). The drawback of this method is that part of the linker is left on the oligonucleotide, and the oligonucleotide is thus modified, which may not be ideal for many applications. Consequently, development of biotinylation phosphoramidites that can be cleaved and leave unmodified oligonucleotide have received considerable attention. Gildea et al. reported an acid-labile linker for biotinylation at the 5′-end of DNA. Although they successfully applied their linker for affinity purification of synthetic DNA by streptavidin media, the synthesis of the biotinylation reagent required many steps; moreover, this reagent has to be attached to the 5′-end of DNA in a separate step (3). Rothschild and co-workers reported a photocleavable biotinylation phosphoramidite for 5′-end-labeling. Affinity purification, followed by photocleavage proved effective for the synthesis of oligonucleotide 5′-phosphates (7,8). The formation of thymine–thymine photodimers under UV irradiation is a concern. Although they minimized the possible irradiation-induced damage to DNA by cutting off wavelengths below 300 nm and using short irradiation times, damaged DNA was detectable. This type of damage may become more significant when long sequences containing more thymidines are involved (23–25). In this paper, we report the synthesis of a fluoride-cleavable biotinylation phosphoramidite and its application in affinity purification of synthetic DNA by avidin coated microspheres. Our biotinylation method can be considered to be complementary to Rothschild’s method because the free 5′-OH rather than 5′-phosphate of DNA is recovered after cleavage.

MATERIALS AND METHODS

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. Methylene chloride and pyridine were distilled over CaH2. Acetone was dried over anhydrous Na2SO4, and the supernatant 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 p.p.m. for 1H and 77.23 p.p.m. for 13C) or CD3C(O)CD2H (δ = 2.05 p.p.m. for 1H and 29.92 p.p.m. for 13C) or CD3OH (δ = 4.87 p.p.m. for 1H and 49.15 p.p.m. for 13C) or triphenylphosphate (δ = 0.00 p.p.m. for 31P). Infrared spectra were recorded on a Nicolet FTIR spectrophotometer. High-resolution mass spectra were obtained on a Finnigan Mat 95XL spectrometer. Melting points were determined using a Mel-Temp melting point apparatus and were uncorrected. Sep-Pak® Cartridges were purchased from Waters Corporation (Milford). UltraLink™ Immobilized NeutrAvidin™ (slurry in water, ∼50% v/v, containing 0.02% sodium azide; pore size, 1000 Å; particle size, 50–80 µm; biotin-binding capacity, ∼0.08 µmol biotin/ml gel) was purchased from Pierce. Biotin was purchased from AnaSpec Inc. Aqueous CH3NH2 (∼40%) and NH4OH (∼29%), pyridine/HF (pyridine, ∼30%; HF, ∼70%) and Me3SiOMe were purchased from Aldrich Inc. THF/pyridine/Pac2O, succinic ester linked DMTr-dT-lcaa-CPG (pore size 1000 Å) and 5′-DMTr, 2-cyanoethyl phosphoramidites benzoyl-dA, isobutyryl-dG, acetyl-dC, Pac-dA, 4-isopropyl-Pac-dG and dT were purchased from Glen Research Inc. TTL buffer: 100 mM Tris–HCl pH 8.0, 0.1% Tween 20, 1.0 M LiCl. TES buffer: 10 mM Tris–HCl, 1 mM EDTA, 1 M NaCl, pH 8.2. 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 Analytical Instruments); solvent A: 0.1 M triethylammonium acetate, 5% acetonitrile; solvent B: 90% acetonitrile; profiles were generated using a linear gradient of solvent B (0–45%) in solvent A over 60 min at flow rates of 1 ml/min (analysis) and 3 ml/min (preparation) by detecting the absorbance of DNA at 260 nm.

Lactone 2

Methyl magnesium bromide in ether (3.0 M, 100 ml, 300 mmol) was added to a solution of levulinic acid (14 ml, 136 mmol) in THF (300 ml) via a syringe at –78°C with stirring. The reaction mixture was stirred for 3 h while warming to room temperature (r.t.) gradually, and then heated to 50°C overnight forming a light yellow solution. After cooling to r.t., AcOH was added until pH 4, and stirring was continued for 12 h. Water (40 ml) was added, and THF and ether were removed under reduced pressure. The red residue was extracted with CH2Cl2 (100 ml × 3), the organic phase was dried over anhydrous MgSO4, and the solvents removed under reduced pressure. Distillation under vacuum gave the product as a colorless oil (9.5 g, 61%): 1H NMR [CDCl3, 250 MHz, consistent with reported data (26)] δ 1.44 (s, 6H), 2.06 (t, 2H, J = 8.23 Hz), 2.63 (t, 2H, J = 8.3 Hz).

Amino alcohol 3

2,2′-(Ethylenedioxy)bis(ethylamine) (24.3 ml, 166 mmol) and 2 (9.5 g, 83.2 mmol) in water (10 ml) were heated to 90°C overnight forming a yellow solution. Water and excess diamine were removed under vacuum. The light yellow oily residue was purified by flash chromatography (4.5 × 30 cm, SiO2, 5:2:2:1 Et2O/MeOH/MeCN/Et3N), giving 3 as a light yellow oil (16.3 g, 75%): Rf = 0.8 (5:2:2:1 Et2O/MeOH/MeCN/Et3N); IR (thin film, cm–1) ν 3289, 2963, 2788, 1650, 1130; 1H NMR (CD3OD, 500 MHz) δ 1.20 (s, 6H), 1.78 (t, 2H, J = 8.1 Hz), 2.35 (t, 2H, J = 7.5 Hz), 2.88 (t, 2H, J = 4.5 Hz), 3.89–3.42 (m, 2H), 3.54–3.57 (m, 4H), 3.62 (s, 4H); 13C NMR (CD3OD, 500 MHz) δ 28.8, 31.0, 38.5, 38.8, 40.8, 69.0, 69.3, 69.6, 69.7, 72.0, 174.0. HRMS (ESI, M + H+) calculated for C12H27N2O4 263.1971, found 263.1961.

N-t-butylbenzoylbiotin (4)

To a suspension of biotin (2.44 g, 10.0 mmol) and DMAP (0.61 g, 5.0 mmol) in dry pyridine (15 ml) was added t-butylchlorodiphenylsilane (6.5 ml, 25 mmol). The reaction mixture was stirred at r.t. overnight. Addition of 4-t-butylbenzoylchloride (3.0 ml, 15 mmol) to the white suspension gave a light yellow solution, which was stirred for 3 h, and then quenched with MeOH (2 ml). Volatile components were removed under reduced pressure. The light yellow residue was dissolved in a mixture of THF (12 ml), MeOH (6 ml), H2O (6 ml) and K2CO3 (6.9 g, 50 mmol), and stirred at r.t. for 0.5 h. The reaction was quenched with citric acid (pH 4, 50 ml) and extracted with EtOAc (20 ml × 6) (27). The organic layer was dried over anhydrous MgSO4, and solvents were removed. The residue was purified by flash chromatography (4.5 × 10 cm, Si2O, first CHCl3, then 19:1 CHCl3/MeOH), giving the product as a light yellow foam (3.07 g, 76%): Rf = 0.5 (9:1:10 CHCl3/MeOH/Et2O); IR (thin film, cm–1) ν 3245, 2960, 2860, 1727, 1653, 1332; 1H NMR (CD3OD, 500 MHz) δ 1.15 (s, 9H), 1.27–1.63 (m, 6H), 2.14 (t, 2H, J = 7.4 Hz), 2.79–2.91 (m, 2H), 3.11–3.15 (m, 1H), 4.07 (dd, 1H, J = 4.6, 7.8 Hz), 4.98–5.00 (m, 1H), 7.25 (d, 2H, J = 8.5 Hz), 7.31 (d, 2H, J = 8.6 Hz); 13C NMR (CD3OD, 500 MHz) δ 26.1, 29.6, 29.9, 31.7, 34.8, 35.9, 38.9, 56.7, 59.3, 64.1, 125.7, 129.8, 133.8, 156.1, 158.2, 171.9, 177.6. HRMS (ESI, M + H+) calculated for C21H29N2O4S 405.1848, found 405.1856.

Biotinyl alcohol 5

To a solution of 4 (1.9 g, 4.8 mmol) and diisopropylethylamine (920 µl, 5.28 mmol) in dry DMF (4 ml) was added isobutylchloroformate (684 µl, 5.27 mmol) via a syringe at 0°C. After stirring for 1 h, DMAP (292 mg, 2.39 mmol) and the amino alcohol 3 (656 mg, 2.5 mmol) in DMF (4 ml) was added via a syringe slowly at 0°C and stirred for 10 min. The reaction was quenched with water (2 ml) and partitioned between citric acid (pH 4, 20 ml) and EtOAc (30 ml × 6). The organic phase was dried over anhydrous Na2SO4, purified by flash chromatography (4.5 × 10 cm, SiO2, 19:1 CH2Cl2/MeOH), and the major UV active fraction was collected, giving 5 as a white foam (2.1 g, 66%): Rf = 0.5 (9:1, CH2Cl2/MeOH); IR (thin film, cm–1) ν 3295, 2963, 2860, 1731, 1642; 1H NMR (CD3OD, 500 MHz) δ 1.24 (s, 6H), 1.38 (s, 9H), 1.46–1.55 (m, 2H), 1.65–1.85 (m, 6H), 2.28 (t, 2H, J = 7.3 Hz), 2.34 (t, 2H, J = 7.7 Hz), 3.06–3.13 (m, 2H), 3.36–3.43 (m, 5H), 3.55–3.66 (m, 8H), 4.31 (t, 1H, J = 6.9 Hz), 5.22 (t, 1H, J = 6.6 Hz), 7.48 (d, 2H, J = 8.2 Hz), 7.54 (d, 2H, J = 8.1 Hz); 13C NMR (CD3OD, 500 MHz) δ 26.9, 29.3, 29.5, 29.9, 31.8, 32.4, 35.9, 36.8, 38.9, 40.3, 40.4, 40.4, 56.7, 59.3, 64.0, 70.7, 70.7, 70.9, 71.4, 79.6, 125.6, 129.8, 133.7, 156.1, 158.1, 171.8, 176.1, 176.6. HRMS (ESI, M + H+) calculated for C33H53N4O7S 649.3635, found 649.3628.

Biotinyl thymidine 6

Biotinyl alcohol 5 (1.7 g, 2.62 mmol) and imidazole (211 mg, 3.1 mmol) in a round-bottomed flask was flushed with argon. Dry DMF (5 ml) and diisopropylethylamine (2.7 ml, 15.5 mmol) were added via a syringe, and the mixture was cooled to 0°C. Diisopropyldichlorosilane (839 µl, 4.65 mmol) was added via a syringe in one portion. The resulting colorless solution was stirred at 0°C for 1 h and r.t. for 4 h. Thymidine (1.5 g, 6.2 mmol) and imidazole (422 mg, 6.2 mmol) in dry DMF (5 ml) were added via a syringe at –60°C. The reaction was maintained at –60°C for 1 h and 0°C for 3 h. Cooled EtOAc (0°C, 50 ml) and 5% NaHCO3 (50 ml) were added, and the phases partitioned. The aqueous phase was further extracted by EtOAc (0°C, 50 ml × 4). The organic phase was dried over anhydrous Na2SO4, and solvents removed leaving a light yellow oil. The UV active spot that has a slightly higher Rf value than thymidine on TLC was isolated by flash chromatography (4.5 × 12 cm, SiO2, 9:1, CHCl3/MeOH) giving 6 as a white foam (2.49 g, 92%): Rf = 0.4 (9:1, CH2Cl3/MeOH); IR (thin film, cm–1) ν 3315, 2959, 2869, 1695, 1123; 1H NMR (CD3OD, 500 MHz) δ 0.91–1.05 (m, 14H), 1.19 (s, 6H), 1.28 (s, 9H), 1.28–1.29 (m, 1H), 1.40–1.43 (m, 2H), 1.58–1.83 (m, 5H), 1.81 (s, 3H), 2.12–2.30 (m, 4H), 2.94–3.05 (m, 2H), 3.25–3.33 (m, 5H), 3.46–3.51 (m, 4H), 3.56–3.57 (m, 4H), 3.90–4.00 (m, 3H), 4.23 (dd, 1H, J = 4.5, 7.7 Hz), 4.38–4.40 (m, 1H), 5.13 (dd, 1H, J = 4.8, 7.7 Hz), 7.37 (d, 2H, J = 8.1 Hz), 7.44 (d, 2H, J = 8.4 Hz), 7.47 (s, 1H); 13C NMR (CD3OD, 500 MHz) δ 12.7, 14.5, 14.7, 18.3, 18.3, 18.3, 18.4, 26.8, 29.4, 29.5, 29.8, 30.2, 31.6, 32.1, 32.5, 35.8, 36.7, 38.8, 40.2, 40.3, 41.0, 41.5, 56.0, 56.6, 59.2, 64.0, 64.1, 70.5, 70.6, 71.3, 71.9, 74.8, 86.0, 88.5, 111.5, 125.5, 129.7, 133.6, 137.5, 152.2, 156.0, 158.0, 166.2, 171.7, 176.0, 176.2, 211.4. HRMS (ESI) calculated for C49H78N6O12SSi 1003.5246, found 1003.5250.

Biotinyl phosphoramidite 1

A round-bottomed flask containing 6 (220 mg, 0.22 mmol) was flushed with argon. Dry CH2Cl2 (5 ml) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (76 µl, 0.23 mmol) were added via a syringe, sequentially. 1H-tetrazole (15.4 mg, 0.22 mmol) was added in three portions over a period of 1 h. After a total of 5 h, the reaction mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography (3.0 × 12 cm, SiO2, 1:1, CHCl3/THF), giving 1 as a white foam (225 mg, 85%): Rf = 0.3 (1:1, CHCl3/THF); 1H NMR (CDCl3, 250 MHz) δ 0.99–1.13 (m, 14H), 1.24 (d, 12H, J = 6.8 Hz), 1.28 (s, 6H), 1.36 (s, 9H), 1.36–1.90 (m, 8H), 1.90 (s, 3H), 2.21–2.39 (m, 6H), 2.75 (dt, 2H, J = 2.0, 5.8 Hz), 3.07–3.10 (m, 2H), 3.32–3.42 (m, 5H), 3.55–3.60 (m, 4H), 3.65 (s, 4H), 3.62–3.92 (m, 4H), 4.01–4.09 (m, 3H), 4.28–4.33 (m, 1H), 4.68–4.72 (m, 1H), 5.20–5.24 (m, 1H), 6.30 (t, 1H, J = 7.5 Hz), 7.49 (d, 4H, J = 7.8 Hz), 7.59 (s, 1H); 31P NMR (CDCl3, 250 MHz) δ 149.7, 149.8.

Biotinyl oligonucleotide 7

Protection, cleavage/deprotection strategy A. The oligonucleotide was synthesized on an ABI DNA/RNA synthesizer at the 1 µmol scale, using the following 5′-DMTr, 2-cyanoethyl phosphoramidites: Pac-dA, 4-isopropyl-Pac-dG, acetyl-dC and dT. The manufacturer’s recommended synthetic cycles were used except that THF/pyridine/Pac2O was used as the capping reagent, and 1 was coupled for 15 min. The CPG was dried under a nitrogen flow and then divided into three portions (0.36, 0.30 and 0.33 µmol). To the 0.30 µmol portion in a screw-capped 5 ml vial was added K2CO3 in anhydrous methanol (0.05 M, 1.0 ml), and the resulting suspension was gently shaken at r.t. for 24 h. Supernatant was transferred to Eppendorf tubes by pipette, and the CPG was washed with NH4OAc (0.5 M, 500 µl) and water (100 µl × 3). The supernatant, washing buffer and water were combined, and dried on a SpeedVac. The residue was redissolved in 500 µl water, of which 40 µl was analyzed by HPLC to generate trace a (Fig. 1). Three aliquots each of 100 µl were taken from the remaining solution and all were dried on a SpeedVac in Eppendorf tubes. To the suspension of the first aliquot in THF (200 µl) was added pyridine/HF (30 µl), the mixture was vortexed (10 s) and allowed to stand at r.t. for 1 h with gentle shaking, followed by quenching the excess fluoride ion by Me3SiOMe (500 µl, r.t., 30 min). The mixture was dried on a SpeedVac, dissolved in 500 µl water, of which 200 µl was analyzed by HPLC to give trace b (Fig. 1). The coupling efficiency of 1 was estimated to be 47% by comparing the area of the peak with retention time of near 36 min to that of the major peak near 15 min in trace a (Fig. 1). This unreasonably low yield was later improved to >99% by performing the synthesis with more care to avoid adventitious moisture.

Figure 1.

Figure 1

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HPLC traces. (a) Crude DNA obtained by 0.05 M K2CO3/MeOH cleavage/deprotection. (b) After treating with pyridine/HF. (c) After affinity purification. (d) Unbiotinylated failure sequences left after NeutrAvidin™ treatment. (e) Control DNA and affinity purified DNA. (f) Affinity purified DNA using PBS as buffer. (g) Control DNA. (h) DMTr-selective cartridge purified DNA. (i) Crude DNA obtained by MeNH2 (40%)/NH4OH (29%) (1:1 v/v) cleavage/deprotection. (j) After treating with pyridine/HF.

Protection, cleavage/deprotection strategy B. A second synthesis was carried out on an ABI DNA/RNA synthesizer at the 1 µmol scale, using the following 5′-DMTr, 2-cyanoethyl phosphoramidites: benzoyl-dA, isobutyryl-dG, acetyl-dC and dT. The manufacturer’s recommended synthetic cycles were used except that 1 was coupled for 15 min. The CPG was dried under nitrogen flow and then divided into three portions (0.34, 0.32 and 0.34 µmol). To the 0.32 µmol portion in a screw-capped 5 ml vial was added NH4OH (∼29%, 300 µl) and methylamine (∼40%, 300 µl), and the resulting suspension was heated to 65°C for 30 min. After cooling 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 10.0 ml water, of which 50 µl was analyzed by HPLC to generate trace i (Fig. 1). Two milliliters were removed from the remaining solution, and dried on a SpeedVac in Eppendorf tubes. THF (200 µl) and pyridine/HF (30 µl) were added, vortexed (10 s) and allowed to stand at r.t. for 1 h with gentle shaking. Me3SiOMe (500 µl) was added, and the mixture was gently shaken for 30 min, and dried on a SpeedVac. The residue was redissolved in 500 µl water, of which 25 µl was analyzed by HPLC to give trace j (Fig. 1). The coupling efficiency of 1 was estimated to be 97% by comparing the area of the peak with retention time of near 36 min to that of the latest peak near 15 min in trace i (Fig. 1).

Affinity purification and HPLC analysis

The second aliquot, obtained by protection, cleavage/deprotection strategy A, was dissolved in TTL buffer (300 µl), and added to UltraLink™ Immobilized NeutrAvidin™ gel (1.0 ml slurry, 0.5 ml gel), which was washed with TTL buffer (200 µl × 3). The mixture was incubated at r.t. for 1 h with gentle shaking, and then centrifuged and the supernatant removed. The gel was washed with TES buffer (200 µl × 4) and water (200 µl × 3). The supernatant and washing buffer and water were combined, dried on a SpeedVac, and redissolved in 500 µl water, of which 200 µl was analyzed by HPLC to generate trace d (Fig. 1). The gel in an Eppendorf tube was dried by washing with acetone (200 µl × 2) and THF (200 µl × 2), and then suspended in THF (200 µl). The cleavage reagent pyridine/HF (30 µl) was added by pipette, and the mixture was incubated at r.t. for 1 h. Excess fluoride ion was quenched with Me3SiOMe (500 µl) by incubating at r.t. for 30 min. After centrifuging, the supernatant was removed, and pure DNA was washed down from the gel by water (200 µl × 7), dried on a SpeedVac and redissolved in 500 µl water, of which 200 µl was analyzed by HPLC to generate trace c (Fig. 1). The recovery yield of full-length DNA 9 was estimated to be 90% by comparing the area of the peak in trace c to that in trace a with a retention time of near 36 min. The identity of 9 (Scheme 2) was confirmed by co-injection of 100 µl of this solution with authentic sample (∼0.005 µmol, synthesized as described below) on HPLC to yield a single peak (Fig. 1, trace e). The third aliquot was treated exactly (including volumes of buffers and water) as described for the second aliquot except that PBS was used as the binding and washing buffer. The purified DNA was dissolved in 500 µl water, and 200 µl was analyzed by HPLC to give trace f (Fig. 1). The recovery yield of full-length DNA 9 was estimated to be 81% by comparing the area of the peak in trace f to that in trace a with a retention time of near 36 min.

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Scheme 2.

Synthesis and purification of control DNA

Oligonucleotide 9 was synthesized independently on an ABI DNA/RNA synthesizer at the 1 µmol scale, using the following 5′-DMTr, 2-cyanoethyl phosphoramidites: benzoyl-dA, isobutyryl-dG, acetyl-dC and dT. The manufacturer’s recommended synthetic cycles were used. In the last cycle, the trityl group was left on. Cleavage/deprotection was performed with NH4OH (∼29%) and methylamine (∼40%) (1:1 v/v) at 65°C for 15 min. The DNA was divided into two equal portions. One portion was purified by preparative reverse phase HPLC and then detritylated following a standard procedure (28). The purified DNA was used as a control. HPLC analysis of the control (∼0.005 µmol) generated trace g (Fig. 1). The other portion was purified by DMTr-selective cartridge purification following a standard procedure (29); HPLC (∼0.005 µmol) analysis gave trace h (Fig. 1).

RESULTS AND DISCUSSION

In a separate project in our laboratory, we developed a new method for highly specific biotinylation of unprotected nucleoside analogs at the 5′ hydroxyl group (results will be published elsewhere). It was soon realized that it might be possible to adapt the method for reversible biotinylation of 5′-OH of DNA during automated solid phase synthesis. The biotinylation phosphoramidite 1 (Scheme 1) designed for this purpose contains a number of important structural features. The 4-t-butylbenzoyl protection group at position N-1 of the biotin moiety aids phosphoramidite solubility in acetonitrile, the commonly used solvent for automatic DNA synthesis, and, at the same time, prevents phosphorylation at the urea moiety of biotin (9–12). The long hydrophilic linker between the diisopropylsilyl group and biotin favors the interaction between biotin and streptavidin or avidin (9,11). Thymidine was chosen as the nucleoside for proof of principle studies, but the chemistry would be expected to be compatible with other nucleosides. The key part of compound 1 is the diisopropylsilyl acetal linkage. This type of linkage was used by Sproat for linking a hydrophobic group to the 5′-end of DNA/RNA to assist reverse phase chromatographic purification (30,31). The Sproat patent also proposes use of the diisopropylsilyl linkage for biotinylation of DNA, but the strategy was not developed and provided no specifications or guidance concerning linker structure. Based on several publications (30–37), we anticipated that this linkage might be compatible with DNA synthesis and certain cleavage/deprotection conditions, while it could be readily broken by treatment with fluoride ion, releasing the free 5′-OH group. In principle, the tertiary hydroxide connected to the silicon atom can be a primary hydroxide, which would render the molecule simpler and easier to synthesize. However, in the project mentioned above, we found that this linkage is not very stable even in water, which is consistent with documented data (36). Alternatively, one could use the t-butylphenylsilyl group to link two primary hydroxide functionalities to form this linkage. This would meet the stability requirement (36), but a chiral silicon center would be created, and diastereoisomers would be formed. In the other project, we found these materials difficult to purify even without any attempt to separate the diastereoisomers, which may react at different rates during DNA synthesis. By using the tertiary hydroxide group, the problems associated with stability and purification are both solved. As discussed later, another advantage was revealed when this linkage was formed by addition of diisopropyldichlorosilane to the tertiary alcohol followed by coupling to the nucleoside. Undoubtedly, because of steric hindrance, no unwanted symmetric dimer of the tertiary alcohol was formed. Coupling to a primary or secondary alcohol would have led to this side product, resulting in loss of material and difficulty in purification (34,35).

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Scheme 1.

Synthesis of phosphoramidite 1 is illustrated in Scheme 1. The lactone 2 was prepared from levulinic acid and methyl magnesium bromide followed by acetic acid treatment. This compound was prepared by Engel et al. (26) and Piva (38) from ethyl 4-methyl 3-pentenoate in the presence of Me3SiI, by Martin et al. (39) from 4-methylpentane-1,4-diol under oxidation conditions and by Ali and Alper (40) through palladium catalyzed carboxylation of 2-methyl-3-buten-2-ol. Heating compound 2 with 2,2′-(ethylenedioxy)bis(ethylamine) in the presence of water at 90°C gave amino alcohol 3 (75%). Without water, the symmetric diacetylation product was exclusively formed. Biotin was protected at N-1 by the base-labile 4-t-butylbenzoyl group to give derivative 4 (76%) (9,12). This early protection strategy makes the following materials easier to manipulate because of their increased lipophilicity. Coupling of compounds 3 and 4 with isobutyl chloroformate gave compound 5 (66%) (41). The key silyl acetal linkage was formed by addition of diisopropyldichlorosilane to a solution of the tertiary alcohol 5 in DMF in the presence of diisopropylethylamine and imidazole at 0°C. Subsequent addition of thymidine in DMF gave product 6 in 92% yield after flash chromatography. No dimeric 5 was detected by TLC, which has been reported to be a problem for such a reaction when primary or secondary alcohols were added to diisopropyldichlorosilane (34,35). Phosphinylation of 6 to give phosphoramidite 1 was performed using Beaucage’s method (85%) (42).

The 5′-end biotinylated oligonucleotide 7 (Scheme 2) was synthesized in an automated DNA/RNA synthesizer, using two common base protection and post-synthetic cleavage/deprotection strategies (A and B). In strategy A, exo-cyclic amino groups on A, G and C were protected by phenoxyacetyl (Pac), 4-isopropyl-Pac and acetyl groups, respectively. Normal synthetic cycles were used, except that the biotinylated phosphoramidite 1 was coupled for 15 min. Cleavage/deprotection was performed under UltraMild conditions (0.05 M K2CO3 in MeOH, r.t., 24 h; Glen Research Inc., 2002 catalog, pp. 22–23). The resulting solution was neutralized by NH4OAc buffer (0.5 M), and dried on a SpeedVac. The crude product was redissolved in water, and divided into five portions (see Materials and Methods for details). One portion was analyzed by HPLC to give trace a (Fig. 1). The second portion was treated with pyridine/HF, followed by quenching excess fluoride ion by methoxytrimethylsilane (43), dried and redissolved in water and analyzed by HPLC to give trace b (Fig. 1). The peaks with a retention time near 15 min are the fully deprotected DNA (Scheme 2, 9) or the failure sequences generated during DNA synthesis. Those with retention times near 36 min are fully deprotected DNA (7), 5′-end biotinylated through the silyl acetal linker. Comparison of traces a and b indicates that the silyl acetal linker is completely stable under these cleavage/deprotection conditions. In strategy B, A, G and C were protected by benzoyl, isobutyryl and acetyl groups, respectively. Normal synthetic cycles were also used, and 1 was also coupled for 15 min. Protection of C by the acetyl group allowed fast cleavage/deprotection by heating the CPG in MeNH2 (∼40%)/NH4OH (∼29%) (1:1 v/v, 65°C, 30 min). These conditions have been used for 2′-OH alkylsilyl protected RNA deprotection to minimize unwanted early desilylation (44,45). The deblocking solution was separated from CPG, which was washed with water. Deblocking solution and water washes were combined and dried on a SpeedVac. The resulting residue was redissolved in water and divided into three portions (see Materials and Methods for details). Two portions were treated exactly as described in strategy A, and generated HPLC traces i and j, which correspond to traces a and b, respectively (Fig. 1). Comparison of traces i and j also undoubtedly indicated that the silyl acetal linkage is stable under these cleavage/deprotection conditions. In both strategies, the 4-t-butylbenzoyl protecting group was removed during the cleavage/deprotection process; this is supported by the observation that the biotinylated DNA binds to NeutrAvidin™ beads, which allows selective removal of unbiotinylated failure sequences as discussed later. The coupling efficiency of the modified phosphoramidite 1 may also be deduced from the HPLC traces a and i together with information provided by trityl assay. In strategy A, the coupling efficiency of phosphoramidite 1 was ∼47%. This unreasonably low yield was later improved to >99% by performing the synthesis with more care to avoid adventitious moisture. In strategy B, it was 97%.

To test the binding efficiency of biotinylated DNA to streptavidin or avidin media, attempts were made to separate 7 from the failure sequences, generated during synthesis, by NeutrAvidin™ mediated affinity purification. Because in every synthetic cycle, failure sequences are capped, only the full-length sequence is biotinylated, and affinity purification followed by fluoride cleavage should give high quality full-length DNA 9 (Scheme 2, avidin bead not shown). Since the DNA obtained by strategy A with unoptimized yield contains more failure sequences, it was chosen as our substrate for this study. One portion was dried on a SpeedVac, dissolved in TTL buffer, and incubated with NeutrAvidin™ coated microspheres for 1 h at room temperature with gentle shaking (it is important to handle these beads gently, otherwise bead material may leak, and recovery yield of full-length DNA may be low). The non-biotinylated failure sequences were washed away by TES buffer and water, which were combined, dried, dissolved in water and analyzed by HPLC to give trace d (Fig. 1). As can be seen, the biotinylated DNA peak with a retention time near 36 min in trace a (Fig. 1) completely disappeared, leaving only the non-biotinylated failure sequences with retention times near 15 min (trace d). The beads were dried by washing with anhydrous acetone and THF sequentially, and suspended in dry THF. The cleaving reagent, pyridine/HF, was then added and incubated at room temperature for 1 h with gentle shaking. The excess fluoride was quenched by methoxytrimethylsilane at room temperature for 30 min (Scheme 2). After centrifuging, the supernatant was removed, dried, dissolved in water and analyzed by HPLC to ensure that no DNA was in this portion (HPLC trace not shown). There is no need for further washing of the beads with THF, because all side products not bound to the beads are volatile with the exception of DNA 9 (Scheme 2, 8 binds to beads, which are not shown), which is an important advantage of this reagent combination. DNA 9 is then washed down with water, dried, redissolved in water and analyzed by HPLC to give trace c (Fig. 1). As shown in trace c, the fully deprotected DNA is very pure. The identity of the DNA was confirmed by comparing HPLC traces c with g (authentic sample), and co-injection of the affinity purified DNA with authentic sample (trace e). We also tested other binding and washing buffers besides TTL and TES, which may be very important in other biological applications. The HPLC trace f was obtained from the sample prepared by the same procedure as described above but using PBS as binding and washing buffers. As expected, similar results were obtained. We also evaluated the recovery yield of this affinity purification method by comparing the peak area in traces c and f with that of the peak with a retention time of near 36 min in trace a. Their ratios should be equal to the recovery yields when specific amounts of materials are injected and assuming DNA 7 and 9 have the same molar extinction coefficient at 260 nm. In both cases, the recovery yields were >80%.

Currently, synthetic DNA is purified by reverse phase HPLC (trityl-on or trityl-off), anion-exchange HPLC or gel electrophoresis. These methods are time consuming and have other drawbacks, which were discussed in detail by Sproat (30). DMTr-selective cartridge purification is convenient, but the quality of DNA thus obtained is less satisfactory, especially when long sequences are purified. For comparison, we also purified DNA obtained in cleavage/deprotection strategy A by this method (29), and evaluated the results by HPLC. As shown in Figure 1, the DNA purified by our method (traces c and f) is purer than that purified by the cartridge method (trace h) even though the sequence is short. We believe that the advantage of this method will be more obvious compared to all of the above methods when longer DNA sequences are involved. The most important aspect of this method for purification of DNA is its potential ability to efficiently isolate target sequence from even very complex mixtures. We anticipate that this will find use in applications that require pure oligonucleotide, such as PCR mediated gene synthesis (46,47) and DNA sequencing (48,49). Importantly, the method is amenable to automated high throughput strategies for oligonucleotide synthesis and purification. The current method may also be readily adapted to biotinylation and purification of synthetic RNA when the 2′-OHs are protected by alkylsilyl groups considering that the 2′-OH protecting groups can be removed under the same conditions for breaking the linker by fluoride ion.

In conclusion, we designed and synthesized a fluoride-cleavable DNA biotinylation phosphoramidite. We demonstrated that the phosphoramidite can be coupled to the 5′-end of DNA efficiently on an automatic synthesizer, and that the linkage is stable under common post-synthetic DNA cleavage/deprotection conditions, while it can be readily cleaved by fluoride ion under mild conditions releasing unmodified DNA. We applied this biotinylation method to synthetic DNA purification, and demonstrated that failure sequences can be readily removed and the full-length unmodified DNA can be recovered in high yield. We anticipate that this biotinylation method will find application where purification of chemically synthesized DNA is otherwise difficult.

Acknowledgments

ACKNOWLEDGEMENTS

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.

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