Fluorescence labeling of DNA based on photochemical ligation

Abstract Fluorescent labeling of oligonucleotides has been attracting interest in connection with the development of methods for distinguishing and detecting nucleic acids sequences. And photochemical ligation has the merit of the avoiding the need for additional reagents. Fujimoto et al. reported template directed DNA photoligation using 5-carboxyvinyl-deoxyuridine (CVU). Here, we describe the synthesis and photocrosslinking ability of fluorescent reporter analogue (Cy5) tethered CVU-containing ODN.


Introduction
Fluorescent labeling of oligonucleotides has been attracting current interest in connection with the development of new methods for distinguishing and detecting nucleic acids sequences for diagnostics and homogeneous hybridization assays. Being dependent on the local environment fluorescence has proved an indispensable tool for the study of molecular interactions and several cellular functions. Fluorescently labeled oligonucleotide probes are nowadays in much regular use for nucleic acid sequencing [1], sequencing by hybridization (SBH) [2], fluorescence in situ hybridization (FISH) [3], fluorescence resonance energy transfer (FRET) [4], molecular beacons [5,6], taqman probes [7]. This has made fluorescent probes an important tool for clinical diagnostics and made possible real-time monitoring of oligonucleotide hybridization. Chemical approaches involving cross-linking reactions have been quiet useful [8,9].
Photochemical ligation has the merit of the avoiding the need for additional reagents. Their actions are controllable within space and time by the choice of proper irradiation methods. Thus, the photoligation methods can be used as 'photopadlocking' of circular DNA, as a tool for DNA engineering and nanotechnology, and as photoregulated diagnostic and therapeutic agents [10][11][12][13]. And branched DNA molecules have various uses in signal amplification technology [14], nanotechnology applications such as DNA computing [15], DNA nanostructures using self-assembled branched units [16], DNA sensors [17], and nanoelectronic devices [18].
Fujimoto et al. reported template directed DNA photoligation using 5-carboxyvinyl-deoxyuridine ( CV U). CV U-containing ODN have high photoreactivity at 366 nm irradiation. And the resulting ligated DNA is quantitatively reverted to the original oligonucleotides by 302 nm irradiation. By using this novel photoligation method, a convergent and versatile synthesis of branched ODN that would be particularly useful in DNA nanotechnology is possible [12].
Here, we describe the synthesis and photocrosslinking ability of fluorescent reporter analogue (Cy5) tethered CV Ucontaining ODN.

(E)-5-(2-carboxyvinyl)-2 0 -deoxyuridine (5)
Compound 4 (1.10 g, 3.54 mmol) was added to 3 M NaOH (water/ethanolZ1/1, 10 ml) and this mixture stirred at room temperature for 3 h. HCl was powered into this solution on cooling ice bath to give white precipitate. The precipitate was filtered washed with hexane, and sucked dry. The product was dried under vacuum to yield white powder. 1 To a solution of 5 (0.79 g, 2.65 mmol) in anhydrous pyridine (12 ml) was added DMAP (0.03 g, 0.03 mmol) at ambient temperature. To a solution of DMTrCl (0.99 g, 2.91 mmol) in anhydrous pyridine (8 ml) was added at 0 8C. The reaction mixture was stirred for 4 h at ambient temperature. TLC analysis showed the absence of starting material. The reaction mixture was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl 3 :MeOHZ97:3, v/v) to afford 6 (0.68 g, 43%) as white solid. 1 To a solution of 6 (0.30 g, 0.50 mmol) in dry CH 3 CN (20 ml) was added N-hydroxysuccinimide (0.075 g, 0.60 mmol) and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride salt (0.114 g, 0.60 mmol) cooling by ice bath and the solution was stirred for 18 h at ambient temperature, then 2 (0.078 g, 0.50 mmol) was added and stirred for 6 h. The precipitate was removed by filtration, and evaporated. The residue was extracted with chloroform (20 ml!3) and water (30 ml), and washed with brine (20 ml!2). The organic layer was collected, dried over anhydrous magnesium sulphate, and evaporated to dryness under reduce pressure. The crude was purified by column chromatography (ethyl acetate) to 7 as a white solid. 1   To a solution of 7 (0.24 g, 0.32 mmol) in dry CH 3 CN (5 ml) in a sealed bottle was added 0.45 M tetrazole in CH 3 CN (0.71 ml, 0.32 mmol) and 2-cyanoethyl N,N,N 0 ,N 0 -tetra-isopropylphosphorodiamidite (0.10 ml, 0.32 mmol) and the reaction mixture was stirred for 2 h at ambient temperature. The reaction mixture was diluted with EtOAc and organic layer was washed with a saturated aqueous solution of NaHCO 3 . The organic layer was collected, dried over anhydrous sodium sulfate, and evaporated to dryness to yield 8, which was directly used in an automated DNA synthesizer without further purification.

Synthesis of amine containing oligonucleotides (ODN 1)
Oligonucleotides were prepared by the b-(cyanoethyl)phosphoroamidite method on a controlled pore glass supports by using Applied Biosystems Model 3400 snthesizer.

Synthesis of Cy5 containing oligonucleotides (ODN 2)
To a solution of ODN 1 (0.25 mmol) in 100 mM tetraborate buffer (pH 8.5) was added Cy5-NHS (0.2 mg, 0.25 mM) and the reaction mixture was incubated for 2 h at ambient temperature. The crude was purified by reverse phase HPLC to afford ODN 2 (0.11 mmol, 42%). The purity and concentration of all nucleotides were determined by digestion with AP, Nuclease P1 to 2 0 -deoxymononucleotide at 37 8C for 3 h. MALDI-TOF MS: calcd for ODN 2 [M C ] 2559.6075, found 2559.6084.

Photoligation of DNA oligomer as monitored by reverse phase HPLC
The reaction mixture (total volume 300 ml) containing ODN 2 (20 mM, strand conc.), ODN 3 (20 mM, strand conc.), ODN 4 (25 mM, strand conc.) in 50 mM sodium cacodylate buffer (pH 7.0) and 100 mM NaCl in a Pyrex tube was irradiated at 0 8C with a 25 W transilluminator (366 nm, 5,700 mW cm K2 ) under otherwise identical conditions. After irradiation, 5 mL of aliquot was taken up and subjected to reverse phase HPLC analysis (elution with a solvent mixture of 50 mM ammonium formate, pH 7.0, linear gradient over 15 min from 6 to 9% acetonitrile then over 15 min from 9 to 40% acetonitrle at a flow rate of 0.8 ml min K1 ). Photoligated product was separated and identified by MALDI-TOF-MS. The purified ligated product was enzymatically digesed with Nuclease P1 and AP at 37 8C for 4 h to 2 0 -deoxymononucleosides and photoligated dimmer (HPLC condition; 0.05 M ammonium formate containing 3-20% acetonitrile, linear gradient (30 min) at a flow rate of 1.0 ml/min).

Synthesis of branched ODN as monitored by reverse phase HPLC
The reaction mixture (total volume 300 ml) containing ODN 2 (20 mM, strand conc.), ODN 6 (20 mM, strand conc.), ODN 4 (25 mM, strand conc.) in 50 mM sodium cacodylate buffer (pH 7.0) and 100 mM NaCl in a Pyrex tube was irradiated at 0 8C with transilluminator (366 nm) under otherwise identical conditions. After irradiation, 5 mL of aliquot was taken up and subjected to reverse phase HPLC analysis (elution was with 0.05 M ammonium formate containing 6-12% acetonitrile, linear gradient (30 min) at a flow rate of 1.0 ml min K1 ). Photoligated product was separated and identified by MALDI-TOF-MS. The purified ligated product was enzymatically digesed with Nuclease P1 and AP at 37 8C for 4 h to 2 0deoxymononucleosides and photoligated dimmer (HPLC condition; 0.05 M ammonium formate containing 3-20% acetonitrile, linear gradient (30 min) at a flow rate of 1.0 ml/min).

Measurement of fluorescent spectrum
Fluorescent spectrum of ODN 7 was obtained using JASCO FP-6500 spectrofluorometer at room temperature using 5 mm path length cell. The excitation bandwidth was 1 nm. The emission bandwidth was 1 nm.

Synthesis
One of the amino groups of ethylenediamine was protected by trifluoroacetate group to yield 2. 5-Iododeoxyuridine 3 was transferred to 4 by the Heck reaction. Methyl ester group of 4 was hydrolyzed then dimethoxytritylated and coupled with 2 by condensation reaction to yield 7. Compound 7 was converted to the corresponding cyanoethyl phosphoramidite using a conventional method. The CV U with amino group-containing ODN (ODN 1) was synthesized on an ABI3400 DNA synthesizer. Succinimidyl ester of Cy5 was coupled with ODN 1 to yield Cy5 and CV U-containing ODN (ODN 2) (Scheme 1). A formation of ODN 2 was confirmed by enzymatic digestion and MALDI-TOF MS.

Photoligation of DNA oligomer
We determined the feasibility of photoligation of the Cy5 tethered CV U-containing ODN (Scheme 2). ODN 2 and 5 0d(TGTGCC)-3 0 (ODN 3) were irradiated at 366 nm for 180 min at 0 8C in the presence of template ODN 4. HPLC analysis of a mixture of ODN 2 and ODN 3 photoirradiated with template ODN 4 indicated a clean and efficient formation of ligated ODN 5 and the concomitant disappearance of ODN 2 and ODN 3 ( Figs. 1 and 2). Enzymatic digestion of isolated ODN 5 showed the formation of dC, dG, and dT in a ratio of 2:5:3, together with a new product. The molecular weight of ODN 5 was equal to the sum of the molecular weight of ODN 2 and ODN 3. It is strongly suggested that the photoligation reaction proceeded via [2C 2] cycloaddition between the double bond of CV U side chain and the C5-C6 double bond of cytosine, giving rise to the formation of a cyclobutane structure as observed for CV U without Cy5 [11,12].

Synthesis of branched ODN
We next tried the synthesis of branched ODN (Scheme 3). ODN 2 and 5 0 -d(TGTGCCAAAAA)-3 0 (ODN 6) were irradiated at 366 nm for 180 min at 0 8C in the presence of template ODN 4. HPLC analysis of a mixture of ODN 2 and ODN 6 photoirradiated with template ODN 4 indicated a clean and efficient formation of ligated ODN 7 and the concomitant disappearance of ODN 2 and ODN 6 ( Figs. 3 and 4). Enzymatic digestion of isolated ODN 7 showed the formation of dC, dG, and dT in a ratio of 2:5:3, together with a new product. The molecular weight of ODN 7 was equal to the sum of the molecular weight of ODN 2 and ODN 6. It is strongly suggested that Synthesis of branched ODN succeeded via [2C 2] cycloaddition.

Fluorescent spectrum
Fluorescent spectrum of ODN 7 was measure (Fig. 5). A strong fluorescence at 663 nm was observed on excitation at 635 nm, characteristic of Cy5. This results shows that Cy5labeled ODN was successfully synthesized.

Conclusion
In conclusion, we synthesized Cy5 tethered CV U-containing ODN. We determined the feasibility of photoligation of the Cy5 tethered CV U-containing ODN. We succeeded the synthesis of branched and Cy5-labeled ODN. These results indicate that method provides the new site-specific labeling method of diagnostic sensing of nucleic acid sequences.