Site-Specific Fluorescent Labeling of DNA Using Staudinger Ligation

Bioconjugate Chem. 2003, 14, 697−701

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Site-Specific Fluorescent Labeling of DNA Using Staudinger Ligation Charles C.-Y. Wang,†,§ Tae Seok Seo,† Zengmin Li, Hameer Ruparel, and Jingyue Ju* Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, and Department of Chemical Engineering, Columbia University, New York, New York 10027. Received November 13, 2002; Revised Manuscript Received February 26, 2003

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5′ end by an azido group was selectively reacted with 5-[(N-(3′-diphenylphosphinyl-4′-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (∼90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.

INTRODUCTION

Selective chemical coupling methods are widely used to synthesize modified oligonucleotides for use in fundamental and applied biological research. These oligonucleotides can be used as primers for DNA sequencing (1) and polymerase chain reaction (PCR) (2), antisense agents for therapeutic applications (3), molecular beacons for detecting genetic mutations (4), and probes for measuring gene expression in DNA microarrays and gene chips (5). Several methods have been developed to couple a variety of functional groups to oligonucleotides (6). The most common method is to first synthesize modified phosphoramidites (7-10) or phosphodiester residues (11) labeled with the desired functionality and then incorporate them into DNA sequences at precise sites during automated solid-phase DNA synthesis (12). Another approach involves postsynthetic DNA modification (13). In this method, a specific chemical functional group is inserted in the DNA molecule during solid-phase synthesis, and then the group is selectively reacted with a reporter of choice in solution coupling conditions. Fluorescent oligonucleotides are often synthesized using coupling chemistry between the succinimidyl ester of a fluorophore and a primary alkylamine-modified oligonucleotide (14). However, a limitation of this technique is the requirement of aqueous conditions for the coupling reaction which can hydrolyze the succinimidyl ester. A method to address this limitation involves the direct coupling of phosphoramidite derivatives of the fluoro* To whom correspondence should be addressed. J. Ju, Room 405A Russ Berrie Medical Science Pavilion, Columbia University, College of Physicians and Surgeons, 1150 St. Nicholas Ave., New York, NY 10032. Email: [email protected]. Phone: 212851-5172. Fax: 212-851-5215. † Both contributed equally to this work. § Current address: Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China.

phore to oligonucleotides during the solid-phase oligonucleotide synthesis (15). However, this approach is limited to functional groups that are stable to the basic deprotection condition used in the solid-phase DNA synthesis. Therefore, there is still a need to develop a very specific coupling chemistry to modify DNA which overcomes the above limitations. Recently, we have demonstrated the use of the "Click chemistry” 1,3-dipolar cycloaddition between an alkynyl 6-carboxyfluorescein (Fam) and an azido-labeled single-stranded (ss) DNA to synthesize fluorescent DNA with a high selectivity, stability, and yield (16). We also investigated an alternative coupling chemistry for DNA modification based on Staudinger ligation that was developed by Bertozzi et al. (17, 18). This reaction allows the chemoseletive formation of amide-linked products from azides and triarylphosphines. The classical Staudinger reaction (19, 20) between a phosphine and an azide produces an unstable intermediate, aza-ylide, which hydrolyzes spontaneously to form a primary amine and the corresponding phosphine oxide. To produce a stable amide-linked product, Bertozzi et al. designed a specifically engineered triarylphosphine that allows rearrangement of the unstable aza-ylide to a stable covalent adduct by intramolecular cyclization (17). This chemical reaction is highly selective and efficient and proceeds rapidly at room temperature under physiological conditions. These characteristics enabled the Staudinger ligation to chemoselectively modify cell surface (17) and proteins (21). Here, we report the synthesis of fluorescent ssDNA using the Staudinger ligation and its application as a primer in a Sanger dideoxy chain termination reaction (22) to produce DNA sequencing fragments. EXPERIMENTAL SECTION

Materials and General Procedures. 5-(Aminoacetamido)fluorescein was purchased from Molecular Probe, Inc. Amino-linker-modified oligonucleotide (5′-amino-GTT

10.1021/bc0256392 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/02/2003

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TTC CCA GTC ACG ACG-3′; M13-40 universal forward sequencing primer) and an internal standard oligonucleotide for mass measurement of DNA were obtained from Midland, Inc. PD-10 column and DNA sequencing reagents were obtained from Amersham Biosciences, and an oligonucleotide purification cartridge (OPC) was obtained from Applied Biosystems. All reagents for polymerase chain reaction (PCR) and chemicals for organic synthesis were purchased from Sigma-Aldrich. UV-vis spectra of DNA samples in acetonitrile/water (1:1, v/v) were recorded on a Perkin-Elmer Lambda 40 spectrophotometer. The 1H, 13C, and 31P NMR spectra were recorded on Bruker 300, 400, and 500 MHz NMR spectrometers. 31P NMR spectra were measured using 85% H3PO4 as an external standard. High-resolution mass spectrometry (HRMS) data were obtained using a JEOL JMS HX 110A mass spectrometer. Mass measurement of oligonucleotides was performed using an Applied Biosystems Voyager DE MALDI-TOF mass spectrometer. Succinimidyl 5-Azidovalerate, 1. This compound was prepared by coupling 5-azidovaleric acid (23) with N-hydroxysuccinimide mediated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as previously described (16). Synthesis of an Azido-Labeled DNA, 2. To incorporate the azido group at the 5′-end of the oligonucleotide, 10 nmol of the amino-modified oligonucleotide in 40 µL of 0.25 M Na2CO3/NaHCO3 buffer (pH 9.0) was incubated for 12 h at room temperature with 10 µmol of succinimidyl 5-azidovalerate 1 in 12 µL of dimethyl sulfoxide. The azido-labeled DNA was purified as described previously (16) with an isolated yield of 96%. Succinimidyl 3-Diphenylphosphino-4-methoxycarbonylbenzoate, 3. 3-Diphenylphosphino-4-methoxycarbonylbenzoic acid (216 mg, 0.593 mmol) (17), dicyclohexylcarbodiimide (DCC) (129 mg, 0.623 mmol), and N-hydroxysuccinimide (NHS) (72 mg, 0.623 mmol) were dissolved in diethyl ether (20 mL) under Ar and stirred overnight at room temperature. After the diethyl ether solvent was evaporated, the yellow crystalline product was isolated by filtration. Recrystallization from 2-propanol (20 mL) yielded 164 mg (60%) of 3 as yellow crystals. 1H NMR (CDCl3, 400 MHz) δ 8.15 (s, 2H), 7.67 (d, 1H, J ) 3.2 Hz), 7.39-7.22 (m, 10H), 3.75 (s, 3H), 2.82 (s, 4H, COCH2CH2CO); 13C NMR (CDCl3, 75 MHz) δ 169.4 (COCH2CH2CO), 166.7, 161.5, 143.0, 142.6, 140.1, 139.9, 137.0, 136.8, 136.6, 134.5, 134.2, 131.3, 130.2, 129.6, 129.2, 129.1, 128.1, 53.0, 26.0 (COCH2CH2CO); 31P NMR (CDCl3, 121 MHz) δ -3.05; HRMS (FAB+) Calcd for C25H21O6NP, 462.1107 (M + H)+; found, 462.1093. 5-[(N-(3′-Diphenylphosphinyl-4′-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein, 4. The FAM-tethered triphenylphosphine 4 was prepared by mixing a 1:0.8 ratio of 5-(aminoacetamido)fluorescein (5 mg, 12.3 µmol) and 3 (4.5 mg, 10 µmol) in 1 mL of DMF under argon at room temperature. After reacting for 12 h, the solvent was evaporated under vacuum. The residue was chromatographed on a silica gel plate in CHCl3/MeOH (9:1), yielding 4 quantitatively. 1H NMR (CD3OD, 500 MHz) δ 8.21 (d, 1H, J ) 1.6 Hz), 8.05 (dd, 1H, J ) 3.4, 4.6 Hz), 7.85 (dd, 1H, J ) 1.4, 6.5 Hz), 7.70 (dd, 1H, J ) 1.9, 6.4 Hz), 7.48 (dd, 1H, J ) 1.5, 2.1 Hz), 7.25-7.30 (m, 6H), 7.19-7.24 (m, 4H), 7.07 (d, 1H, J ) 8.3 Hz), 6.58-6.62 (m, 4H), 6.49 (d, 1H, J ) 2.4 Hz), 6.47 (d, 1H, J ) 2.4 Hz), 4.2 (s, 2H), 3.62 (s, 3H); 13C NMR (CD3OD, 100 MHz) δ 175.0, 171.4, 169.9, 169.6, 168.4, 168.3, 154.5, 142.6, 142.3, 141.5, 138.7, 138.6, 138.5, 138.1, 135.2, 134.9, 134.7, 131.6, 131.5, 130.4, 130.1, 129.7, 129.5, 127.9, 127.6, 126.2, 116.6, 114.3, 111.8,

Wang et al.

103.5, 52.6, 44.6, 26.7; 31P NMR (CD3OD, 121 MHz) δ -3.00; HRMS (FAB+) Calcd for C43H32O9N2P, 751.7088 (M + H)+ ; found, 751.7115. Fam-Labeled DNA, 5. A DMF solution (30 µL) of 4 (0.4 mg, 0.5 µmol) was added to a solution of 2.4 nmol of the azido-labeled DNA 2 in 100 µL of 0.25 M Na2CO3/ NaHCO3 buffer (pH 9.0) solution in a 500 µL Eppendorf tube. The tube was shaken for 12 h at room temperature on a shaker, and then the sample was purified by passing through a PD10 size-exclusion column followed by desalting with an OPC column. Calculation of concentration based on the absorbance at 260 nm indicated that DNA was collected with ∼90% yield. The sample was characterized by MALDI-TOF mass spectrometry, which showed that a majority of the sample gave a single peak at 6463 Da (calcd value for 5: 6464 Da). Mass Spectrum of DNA. 30 pmol of the DNA product was mixed with 10 pmol of the internal mass standard and the mixture was suspended in 2 µL of 3-hydroxypicolinic acid matrix solution. 0.5 µL of this mixture was spotted on a stainless steel sample plate, air-dried and analyzed. The measurement was taken using a positive ion mode with 25 kV accelerating voltage, 94% grid voltage, and a 350 ns delay time. PCR Amplification of DNA Template. A PCR DNA product amplified from a pBluescript II SK(+) phagemid vector was used as a sequencing template, as it has a binding site for M13-40 universal primer. Amplification was carried out using the M13-40 universal forward and reverse primers in a 20 µL reaction, which contained 1X Accutaq LA Reaction Buffer, 250 pmol of each dNTP, 40 pmol of each primer, 0.5 units of Jumpstart Red Accutaq LA DNA Polymerase, and 100 ng of the phagemid template. The reaction was performed in a DNA thermal cycler using an initial activation step of 96 °C for 1 min. This was followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min. At the end of the PCR reaction, 20 µL of an enzymatic mixture containing 5 units of shrimp alkaline phosphatase (SAP), 4 µL of 10X SAP buffer, 6 units of E. coli exonuclease I, and 10 µL of water was added to the PCR reaction to degrade the excess primers and dNTPs. The reaction mixture was incubated at 37 °C for 90 min and then heated at 72 °C for 30 min to inactivate the enzymes. Generation and Detection of Sanger DNA Sequencing Fragments. A Sanger DNA sequencing reaction was performed using the FAM-labeled DNA 5 as a primer and the above PCR product as a template. A 30 µL reaction mixture was made, consisting of 2.22 nmol of each of the dNTPs (A, C, G, T), 37 pmol of Biotin-11ddATP (Perkin-Elmer), 20 pmol of primer, 9 units of Thermo Sequenase DNA polymerase, 1X Thermo Sequenase Reaction Buffer, and 20 µL of PCR product. The reaction consisted of 30 cycles of 94 °C for 20 s, 50 °C for 20 s, and 60 °C for 90 s. DNA fragments correctly terminated by Biotin-11-ddATP were purified from other reaction components using solid-phase capture according to the published method (24). The fluorescent DNA fragments in 8 µL of formamide were electrokinetically injected at 3 kV into a capillary filled with linear polyacrylamide (LPA) gel in a capillary array fluorescent DNA sequencer (Amersham), and then separated at 8kV in LPA buffer to produce a fluorescence electropherogram. RESULTS AND DISCUSSION

Succinimidyl 5-azidovalerate 1 was reacted with the amino-linker modified oligonucleotide (5′-amino-GTT

Technical Notes

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Scheme 1a

a Reagents: (a) NaN , DMSO; (b) NaOH, MeOH, H O; (c) HCl; 3 2 (d) N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, CH2Cl2; (e) Na2CO3/NaHCO3 buffer (pH 9.0), DMSO.

TTC CCA GTC ACG ACG-3′) to produce the azido-labeled DNA 2 with a 96% yield (Scheme 1) (16). The MALDITOF MS spectrum of 2 shows a single major peak at 5757 Da (calcd value for 2: 5758 Da). 3-Diphenylphosphino4-methoxycarbonylbenzoic acid was prepared according to the procedure described by Bertozzi et al. (17). Treatment of 3-diphenylphosphino-4-methoxycarbonylbenzoic acid with N-hydroxysuccinimide forms the corresponding NHS ester 3. The reaction of 3 with 5-(aminoacetamido)-

Figure 1. MALDI-TOF mass spectrum of 5.

fluorescein produced FAM-tethered triphenylphosphine 4 which was then coupled with the azido-modified oligonucleotide 2 to form Fam-labeled DNA 5 (Scheme 2). During the Staudinger ligation between 2 and 4, an azaylide intermediate was first formed by the nucleophilic attack of the phosphine on the azide accompanied by the release of N2. A methoxycarbonyl group situated on one

Scheme 2a

a Reagents: (a) NaNO , HCl, H O; (b) KI, H O; (c) Ph PH, Pd(AcO) , triethylamine, MeOH; (d) N-hydroxysuccinimide, DCC; (e) 2 2 2 2 2 5-(aminoacetamido)fluorescein, DMF; (f) Na2CO3/NaHCO3 buffer (pH 9.0), DMF.

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Figure 2. Electropherogram of the DNA sequencing fragments generated with 5.

of the aryl rings of the phosphine traps the aza-ylide in a five-membered ring intermediate, resulting in an amide-linked phosphine oxide 5 after hydrolysis (18). The Fam-labeled DNA 5 was purified by size-exclusion chromatography and then desalted with an OPC column. The collected DNA was examined by UV/Vis spectrophotometry, and the yield of the DNA 5 was found to be 90% based on the absorbance at 260 nm. In the UV/vis spectrum of 5, in addition to the peak at 260 nm, there is a strong peak centered at 496 nm, indicating that the Fam moiety 4 was successfully coupled to the DNA by the Staudinger ligation. The product 5 was characterized by MALDI-TOF mass spectrometry, and the result is shown in Figure 1. In the presence of an internal mass standard (7221 Da), the mass spectrum of the modified DNA product revealed that a majority of the Staudinger ligation product had a mass value of 6463 Da, that matched well with the theoretical value of 6464 Da for 5. To demonstrate the utility of 5 for genetic analysis, we used the Fam-labeled oligonucleotide 5 as a primer in a Sanger dideoxy DNA sequencing reaction to produce DNA sequencing fragments terminated by biotinylated dideoxyadenine triphosphate (ddATP-biotin). The reaction mixture consisted of all four dNTPs (A, C, G, T) along with a biotinylated ddATP. Consequently, the resulting DNA sequencing fragments were all terminated with an ‘A’ at the 3′ end and labeled with a fluorophore (Fam) at the 5′ end. Solid-phase capture using streptavidin-coated magnetic beads allows the isolation of pure DNA extension fragments free from false terminations and other components of the sequencing reaction (24). The purified DNA fragments were separated in a capillary electrophoresis fluorescent DNA sequencer and resolved to produce an electropherogram as shown in Figure 2. The peaks represent the Fam fluorescence emission from each DNA fragment that was extended from 5 and terminated by ddATP. This “A” sequencing ladder shown in Figure 2 matched exactly with the sequence of the DNA template. In conclusion, we have synthesized a fluorescent oligonucleotide by the Staudinger ligation with a high yield and selectivity. Without the requirement of conventional purification methods, such as gel electrophoresis or HPLC, simple purification by size-exclusion chromatography and desalting of the product is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which were subsequently analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and sitespecifically to prepare fluorescent oligonucleotide to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection. Since the amide bond formed by the Staudinger

ligation that couples a fluorophore to the oligonucleotide is very stable, the relatively high temperature of 94 °C used in DNA cycle sequencing caused no degradation of the DNA. It is expected that the Staudinger ligation can also be used to site-specifically introduce other functional groups to DNA for PCR-based genetic analysis as well as for selective covalent attachment of DNA on a solid surface. ACKNOWLEDGMENT

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