Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

Podsumowanie

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Streszczenie

In this protocol, we demonstrate a method for the synthesis of 2'-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Wprowadzenie

Molecular imaging represents a fundamental technique for understanding biological processes within living cells.^1-3 The development of fluorescent nucleic acid based probes for such chemical-biological applications has become an expanding research field. These fluorescent probes need to meet a few requirements to become suitable tools for cell imaging. Firstly, the applied dyes should exhibit fluorescence with high quantum yields, large Stokes' shifts and, most importantly, high photostabilities to allow long-term in vivo imaging. And secondly, they should show a reliable fluorescence readout. Conventional chromophore-quencher-systems are based on the readout of a single fluorescence color by simple changes in fluorescence intensities.⁴ This approach bears the risk of false positive or false negative results due to autofluorescence of intracellular components or low signal-to-noise ratios due to undesired quenching by other components.⁴

We recently reported on the concept of "DNA traffic lights" that show dual fluorescence color readouts by using two different chromophores.^5-6 The concept is based on the energy transfer (ET) from the donor dye to the acceptor dye which changes the fluorescence color (see Figure 1). This allows a more reliable readout and thereby provides a powerful tool for fluorescent imaging probes. Labelling of oligonucleotides with fluorescent dyes can be achieved by two different approaches. Dyes can be incorporated during the chemical DNA synthesis on a solid phase by using correspondingly modified phosphoramidite building blocks.⁷ This method is limited to dyes that are stable under standard phosphoramidite and deprotection conditions. As an alternative, post-synthetic modification methodologies were established in oligonucleotide chemistry. Here, we demonstrate the synthesis of one of our new photostable energy transfer pairs^8,9 and the post-synthetic labelling of DNA by using copper-catalyzed 1,3-cycloaddition between azides and alkynes (CuAAC).¹⁰

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Protokół

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are toxic and carcinogenic. Please use all appropriate safety practices that are typically required in organic chemistry laboratories, such as wearing a laboratory coat, safety glasses and gloves.

1. Synthesis of the Dyes

Note: Both dyes can be synthesized by the same types of reaction. Figure 2 shows an overview of these reactions.

1. Synthesis of 1-(3-azidopropyl)-4-(2-(1-methyl-1H-indol-3-yl)vinyl)pyridin-1-ium iodide (dye 1)
   1. Synthesis of 1-(3-iodopropyl)-4-methylpyridin-1-ium iodide (Figure 2A, step a)
      1. Dissolve 466 mg 4-picoline and 5.91 g 1,3-diiodopropane in 10 ml of acetonitrile in a 20 ml headspace vial and seal tightly by a septum cap.
      2. Heat to 85 °C for 16 hr.
      3. Allow to cool to RT, then remove the solvent under reduced pressure using a rotatory evaporator.
      4. Add 20 ml of ethyl acetate to the residual oil and treat the mixture in an 120 W ultrasonic bath for 3 min.
      5. Collect the formed precipitate by filtration and wash five times with ethyl acetate. Dry the solid product under vacuum O/N.
   2. Synthesis of 1-(3-azidopropyl)-4-methylpyridin-1-ium (Figure 2A, step b)
      1. Dissolve 900 mg 1-(3-iodopropyl)-4-methylpyridin-1-ium iodide in 12 ml of acetonitrile in a 20 ml headspace vial, add 376 mg of sodium azide, and seal tightly by a septum cap.
      2. Heat to 85 °C for 16 hr.
      3. Allow to cool to RT, then remove the solvent under reduced pressure using a rotary evaporator.
      4. Add 15 ml of dichloromethane to the residue.
      5. Filter off and discard the resulting precipitate.
      6. Remove the solvent under reduced pressure using a rotary evaporator to obtain the product as brown oil.
   3. Synthesis of 1-methyl-1H-indole-3-carbaldehyde (Figure 2A, step c)
      1. Under inert gas (argon), dissolve 1.45 g indole-3-carbaldehyde, 1.52 g potassium carbonate and 2.70 g dimethyl carbonate in 10 ml absolute dimethylformamide in a 50 ml round-bottomed flask equipped with a reflux condenser.
      2. Stir the mixture at 130 °C for 19 hr.
      3. Allow to cool to RT, then pour the mixture on ice.
      4. Extract the aqueous layer three times with 150 ml ethyl acetate.
      5. Combine the organic layers, wash them with water, dry them over sodium sulfate and remove the solvent at 50 °C under reduced pressure using a rotary evaporator.
   4. Coupling to 1-(3-azidopropyl)-4-(2-(1-methyl-1H-indol-3-yl)vinyl)pyridin-1-ium iodide (dye 1) (Figure 2A, step d)
      1. Work under argon and under exclusion of moisture. Dissolve 90 mg 1-(3-azidopropyl)-4-methylpyridin-1-ium and 48 mg 1-methyl-1H-indole-3-carbaldehyde in 4 ml ethanol in a 20 ml round-bottomed flask equipped with a reflux condenser.
      2. Add 0.07 ml piperidine and heat to 80 °C for 4 hr.
      3. Allow to cool to RT.
      4. Collect the resulting precipitate by filtration and wash three times with diethylether.
      5. Add diethylether to the supernatant and collect the resulting precipitate by filtration. Wash three times with diethylether.
      6. Combine the precipitates.
2. Synthesis of 1-(3-azidopropyl)-4-(2-(1-methyl-2-phenyl-1H-indol-3-yl)vinyl)quinolin-1-ium iodide (dye 2)
   1. Synthesis of 1-(3-iodopropyl)-4-methylquinolin-1-ium iodide (Figure 2B, step a)
      1. Dissolve 715 mg 4-methylquinoline and 5.91 g 1,3-diiodopropane in 10 ml of acetonitrile in a 20 ml headspace vial and seal tightly by a septum cap.
      2. Heat to 85 °C for 16 hr.
      3. Allow to cool to RT, then remove the solvent under reduced pressure using a rotary evaporator.
      4. Add 20 ml of ethyl acetate to the remaining oil and treat the mixture in an 120 W ultrasonic bath for 3 min.
      5. Collect the formed precipitate by filtration and wash five times with ethyl acetate. Dry the solid product under vacuum O/N.
   2. Synthesis of 1-(3-azidopropyl)-4-methylquinolin-1-ium (Figure 2B, step b)
      1. Dissolve 900 mg 1-(3-iodopropyl)-4-methylquinolin-1-ium iodide in 12 ml of acetonitrile in a 20 ml headspace vial, add 333 mg of sodium azide, and seal tightly by a septum cap.
      2. Heat to 85 °C for 16 hr.
      3. Allow to cool to RT, then remove the solvent under reduced pressure using a rotary evaporator.
      4. Add 15 ml of dichloromethane to the residue.
      5. Filter off and discard the resulting precipitate.
      6. Remove the solvent under reduced pressure using a rotary evaporator to obtain the product as brown oil.
   3. Synthesis of 1-methyl-2-phenyl-1H-indole-3-carbaldehyde (Figure 2B, step c)
      1. Under inert gas (argon), dissolve 1.45 g 2-phenyl-1H-indole-3-carbaldehyde, 0.996 g potassium carbonate and 1.77 g dimethyl carbonate in 10 ml absolute dimethylformamide in a 50 ml round-bottomed flask equipped with a reflux condenser.
      2. Stir the mixture at 130 °C for 19 hr.
      3. Allow to cool to RT, then pour the mixture on ice.
      4. Extract the aqueous layer three times with 150 ml ethyl acetate.
      5. Combine the organic layers, wash them with water, dry them over sodium sulfate and remove the solvent under reduced pressure using rotary evaporator.
   4. Coupling to 1-(3-azidopropyl)-4-(2-(1-methyl-2-phe nyl-1H-indol-3-yl)vinyl)quinolin-1-ium iodide (Figure 2B, step d)
      1. Work under argon and under exclusion of moisture. Dissolve 90 mg 1-(3-azidopropyl)-4-methylquinolin-1-ium and 59.7 mg 1-methyl-2-phenyl-1H-indole-3-carbaldehyde in 4 ml ethanol in a 20 ml round-bottomed flask equipped with reflux condenser.
      2. Add 0.06 ml piperidine and heat to 80 °C for 4 hr.
      3. Allow to cool to RT.
      4. Collect the resulting precipitate by filtration and wash with diethylether three times.
      5. Add diethylether to the supernatant and collect the resulting precipitate by filtration. Wash three times with diethylether.
      6. Combine the precipitates.

2. Synthesis of the DNA Strands

Note: The synthesis of the DNA strands is carried out using the phosphoramidite method on a solid phase, as described by M. Caruthers¹¹ on a DNA synthesizer. The functioning of the synthesizer is tested before the synthetic procedure, and reagents are renewed if necessary.

1. Prearrangements
   1. Dissolve the commercially available 2'-O-propargyl-deoxyuridinephosphoramidite (cU) in 1.2 ml of amidite diluent (extra dry acetonitrile). Move solution in the vial to a synthesizer vial and screw into synthesizer.
   2. Perform a "leak test" (according to manufacturer's instructions) to make sure that no argon gas leak exists. If the "leak test" fails, screw in the vial more tightly.
   3. Prime the cU solution by filling the tube that connects to the solid phase synthesis chamber.
   4. Wash all lines with acetonitrile.
2. Synthesis of DNA strands
   1. Use the connected computer to enter the DNA sequence and coupling method, following the prompts from the manufacturer's protocol. The coupling step of the cU building block takes 168 sec with 7 pulses (each pulse are 16 µl) compared to 40 sec with 7 pulses for a conventional phosphoramidite as building blocks.
   2. Mount the column containing the CPG (controlled pore glass) as solid phase that is modified with 1 µmol of the first base (DNA synthesis is performed from 3' to 5') into the synthesizer.
   3. Start the synthesis on the DNA synthesizer¹¹.
3. Workup of synthesized DNA strands
   1. Dry the columns with the synthesized DNA strands under vacuum O/N.
   2. Use a pincer to open the column and release the CPG into a reaction vial. Add 700 µl of concentrated aqueous ammonia solution to deprotect the oligonucleotide and release the DNA strand from the CPG.
   3. Close the vial and apply a security lid onto the reaction vessel to prevent it from bursting, and heat to 50 °C for 18 hr.
   4. Remove ammonia by centrifugation under reduced pressure (100 mbar) at 30 °C for 30 min.
   5. Start filtration from CPG: Centrifuge vessel at 11,000 x g for 3 min. Take supernatant and transfer it into a centrifugal device with a pore size of 0.45 µm. Centrifuge at 1,000 x g for 4 min.
   6. Meanwhile add 300 µl of double distilled water to the CPG, vortex for 20 sec and centrifuge at 11,000 x g for 3 min. Transfer the supernatant into the centrifugal device and centrifuge at 1,000 x g for 4 min. Repeat this washing procedure twice.
   7. Remove the water from the combined aqueous solutions by centrifuging at 0.1 mbar and 25 °C O/N.

3. "Clicking" Procedure

1. Add 50 µl doubly distilled water, 25 µl of a sodium ascorbate solution (0.4 M in water), 34 µl tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (0.1 M in DMSO/tBuOH 3:1), 114 µl of the azide (0.01 M in DMSO/tBuOH 3:1) and 17 µl of a tetrakis(acetonitrile) copper(I)hexafluorophosphate solution (0.1 M in DMSO/tBuOH 3:1) to the lyophilized alkyne-modified DNA sample.
2. Incubate the sample at 60 °C for 1.5 hr.
3. Cool to RT.
4. Add 150 µl Na₂EDTA (0.05 M in water) and 450 µl sodium acetate (0.3 M in water) to the DNA sample and transfer to a 10 ml tube.
5. Add 10 ml ethanol (100%) and keep at -32 °C for 16 hr.
6. Centrifuge vessel for 15 min at 1,000 x g and remove supernatant.
7. Wash DNA-pellet with 2 ml cold ethanol (80%).
8. Dry pellet under reduced pressure.

4. HPLC Purification

Note: Before separating the DNA strands make sure that the HPLC is working properly, enough solvent is available and the column is clean. Rinse column with the starting concentration of buffer/acetonitrile.

1. Dissolve the crude DNA-pellet in 250 µl of doubly distilled water and transfer to an HPLC vial.
2. Start HPLC run of 45 min with a gradient of 0% acetonitrile to 15% of acetonitrile for dye 1 and a gradient of 0% acetonitrile to 17% of acetonitrile for dye 2. Monitor the run by checking 260 nm (DNA absorption) and 459 nm (dye 1) or 542 nm (dye 2). Collect those fractions that show absorption in both wavelength channels.
3. Check the collected fractions for the right mass by matrix assisted laser desorption ionization (MALDI) mass spectrometry using a matrix consisting of 3-hydroxypicolinic acid (3HPA) and diammoniumhydrogencitrate.
   1. Prepare the matrix by mixing 900 µl of a saturated 3-HPA solution in 1:1-mixture of acetonitrile and water with 100 µl of a diammoniumhydrogencitrate solution (100 g/L) in water.
   2. Pipette 1-2 µl of the DNA probe on the target and let it dry on air.
   3. Add 0.2 µl of the 3-HPA/diammoniumhydrogencitrate matrix to the sample and mix until it crystallizes.
   4. Perform MALDI mass spectrometry.
   5. Combine those HPLC fractions that exhibit the right mass.

5. Determination of Concentration

Note: The concentration is determined by measuring the absorption at 260 nm using a UV/Vis spectrophotometer, based on the extinction coefficients (ε₂₆₀) of the DNA bases and the dye.

1. Dissolve the DNA in 100 - 200 µl doubly distilled water.
2. Apply 1 µl of the DNA solution on the UV/Vis spectrophotometer.
3. Measure the absorption at 260 nm. Repeat three times.
4. Take the average value to calculate the concentration of the solution. For the calculation of the extinction coefficients, use ε_260nm of the four natural bases¹² and the ε_260nm of the purchased building block cU (considered as natural uridine)¹² to obtain the ε_260nm of the whole DNA strand. For dye 1, use ε_260nm = 10,200 L*mol^-1*cm^-1 and for dye 2, use ε_260nm = 13,100 L*mol^-1*cm^-1. Calculate the concentration of the solution following Lambert-Beers' law based on the extinction coefficients and measured absorbances.¹³

6. Sample Preparation and Spectroscopy

1. Preparation of single strand samples
   1. Prepare separately 1 ml of 2.5 µM solutions of each of the modified DNA1 and DNA2 in 200 mM NaCl, 50 mM NaP_i buffer, pH = 7.
2. Preparation of double strand samples
   1. Prepare 1 ml of a solution containing 2.5 µM of DNA1 and 2.5 µM of DNA2 in 200 mM NaCl, 50 mM NaP_i buffer, pH = 7 in a reaction vessel.
   2. Secure the lid with a safety cap and heat to 90 °C for 10 min. Then turn off heating and allow to cool down to RT O/N.
3. Absorption spectroscopy
   Note: To determine the excitation wavelength for the fluorescence spectroscopy absorption spectra are recorded.
   1. Single strand measurements
      1. Record a blank measurement containing only 200 mM NaCl, 50 mM NaP_i buffer, pH = 7.
      2. Transfer the prepared solution of DNA1 into a 1 cm quartz glass cuvette and record the absorption. Correct the measurement against the blank data. Determine the maximum value of dye absorption.
      3. Repeat for DNA2.
4. Fluorescence spectroscopy
   1. Single strand measurements
      1. Record a blank measurement containing only 200 mM NaCl, 50 mM NaP_i buffer, pH = 7; using the excitation wavelength of dye 1.
      2. Transfer the DNA1 solution into a 1 cm quartz glass cuvette.
      3. Record the fluorescence spectrum.
   2. Double strand measurement
      1. Transfer the double strand solution into a quartz glass cuvette.
      2. Record the fluorescence spectrum using the excitation wavelength of dye 1.
5. Visualization experiments
   Caution: Appropriate eye protection should be worn to avoid UV damage to the eyes!
   1. To gain a better understanding of what the recorded spectra are showing, irradiate the cuvettes with handheld UV-lamps (Figure 5). Observe the change in fluorescence color from the single to the double stranded DNA.

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Wyniki

Absorption and fluorescence spectra of the single and double stranded DNA are recorded as shown in Figure 4.

The recorded absorption spectra (Figure 4 right) show absorption maxima λ_max at 465 nm for single-stranded DNA1 (dye 1) and 546 nm for single-stranded DNA2 (dye 2). The annealed DNA1_2 (dye 1 & dye 2) shows maxima at both 469 nm and 567 nm. Both absorption maxima show ...

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Dyskusje

This protocol shows the complete procedure to label DNA post-synthetically via CuAAC by azide-modified fluorescent dyes. This includes the synthesis of the dyes and the alkyne-modified DNA as well as the labeling procedure.

The synthesis of the dyes follows four steps. All products can be obtained by a rather simple precipitation due to their positive charge and no time consuming column chromatography is needed. The introduction of the azide functionalities before the central coupling steps sh...

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Materiały

Name	Company	Catalog Number	Comments
synthesis
4-Picoline	Sigma Aldrich	239615
1,3-Diiodopropane	Sigma Aldrich	238414
Acetonitrile	Fisher Scientific	10660131	HPLC grade
Ethyl acetate	Fisher Scientific	10456870	technical grade
Sodium azide	Sigma Aldrich	71290	p.a. grade
Dichloromethane	Fisher Scientific	10626642	technical grade
Indole-3-carboxaldehyde; 98%	ABCR	AB112969
Potassium carbonate, 99+%	Acros	424081000
dimethylcarbonate	Sigma Aldrich	517127
N,N-Dimethylformamide, 99.8%, Extra Dry over Molecular Sieve	Acros	348435000
Sodium sulfate	Bernd Kraft	12623.46
Ethanol, 99.5%	Acros	397690010
Piperidine, 99%	Acros	147181000
Diethylether	Fisher Scientific	10407830	technical grade
2-Phenylindole-3-carboxaldehyde; 97%	ABCR	AB125050
4-Methylquinoline	ABCR	AB117222
DNA synthesis
Expedite 8909 Nucleic Acid Synthesizer	Applied Biosystems	-
DMT-dA(bz) Phosphoramidite	Sigma Aldrich	A111081
DMT-dT Phosphoramidite	Sigma Aldrich	T111081
DMT-dG(dmf) Phosphoramidite	Sigma Aldrich	G11508
DMT-dC(bz) Phosphoramidite	Sigma Aldrich	C11108
Amidite Diluent for DNA synthesis	Sigma Aldrich	L010010
Ultrapure Acetonitrile for DNA synthesis	Sigma Aldrich	L010400
Cap A	Sigma Aldrich	L840000
Cap B	Sigma Aldrich	L850000
CPG dT Column 1.0 µmole	Proligo Reagents	T461010
CPG dA(bz) Column 1.0 µmole	Proligo Reagents	A461010
CPG dG(ib) Column 1.0 µmole	Proligo Reagents	G461010
CPG dC(bz) Column 1.0 µmole	Proligo Reagents	C461010
ammonia (aqueous solution)	Fluka Analytical	318612
centrifugal devices nanosep 0.45 µm	Pall	ODGHPC34
5-(Benzylthio)-1H-tetrazole (Activator)	Sigma Aldrich	75666
2'-O-propargyl deoxyuridinephosphoramidite	Chem Genes	ANP-7754
workup
vacuum concentrator	Christ
clicking procedure
Tetrakis(acetonitrile)copper(I) hexafluorophosphate	Sigma Aldrich	346276
Sodium acetate	Sigma Aldrich	S2889
(+)-Sodium L-ascorbate	Sigma Aldrich	A7631
EDTA disodium salt	Sigma Aldrich	E5134
TBTA-ligand	-	-	synthesized according to a literature procedure1
HPLC
HPLC-system	Shimadzu
MALDI-Biflex-IV spectrometer	Bruker Daltonics
LC-318 C18 column	Supelcosil via Sigma Aldrich	58368
determination of concentration
ND 1000 Spectrophotometer	nanodrop
sample preparation and spectroscopy
Cary 100 Bio	Varian
Fluoromax-3 fluorimeter	Jobin-Yvon
1 R. Chan Timothy, R. Hilgraf, K. B. Sharpless, V. Fokin Valery, Org Lett 2004, 6, 2853-2855.