Name: Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes (Video) | JoVE Journal
Uploaded: 2016-07-06T13:00:00.000+00:00
Duration: 7 min 44 s
Description: Learn to synthesize wavelength-shifting DNA hybridization probes using photostable cyanine dyes and copper-catalyzed click chemistry. This method enables real-time visualization of energy transfer for molecular imaging applications.

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5 minutes

Financial support by the Deutsche Forschungsgemeinschaft (DFG, Wa 1386/17-1), the Research Training Group GRK 2039 (funded by DFG) and KIT is gratefully acknowledged.

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.
<ol>
	<li>Synthesis of 1-(3-azidopropyl)-4-(2-(1-methyl-1H-indol-3-yl)vinyl)pyridin-1-ium iodide (dye 1)
	<ol>
		<li>Synthesis of 1-(3-iodopropyl)-4-methylpyridin-1-ium iodide (Figure 2A, step a)
		<ol>
			<li>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.</li>
			<li>Heat to 85 &#176;C for 16 hr.</li>
			<li>Allow to cool to RT, then remove the solvent under reduced pressure using a rotatory evaporator.</li>
			<li>Add 20 ml of ethyl acetate to the residual oil and treat the mixture in an 120 W ultrasonic bath for 3 min.</li>
			<li>Collect the formed precipitate by filtration and wash five times with ethyl acetate. Dry the solid product under vacuum O/N.</li>
		</ol>
		</li>
		<li>Synthesis of 1-(3-azidopropyl)-4-methylpyridin-1-ium (Figure 2A, step b)
		<ol>
			<li>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.</li>
			<li>Heat to 85 &#176;C for 16 hr.</li>
			<li>Allow to cool to RT, then remove the solvent under reduced pressure using a rotary evaporator.</li>
			<li>Add 15 ml of dichloromethane to the residue.</li>
			<li>Filter off and discard the resulting precipitate.</li>
			<li>Remove the solvent under reduced pressure using a rotary evaporator to obtain the product as brown oil.</li>
		</ol>
		</li>
		<li>Synthesis of 1-methyl-1H-indole-3-carbaldehyde (Figure 2A, step c)
		<ol>
			<li>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.</li>
			<li>Stir the mixture at 130 &#176;C for 19 hr.</li>
			<li>Allow to cool to RT, then pour the mixture on ice.</li>
			<li>Extract the aqueous layer three times with 150 ml ethyl acetate.</li>
			<li>Combine the organic layers, wash them with water, dry them over sodium sulfate and remove the solvent at 50 &#176;C under reduced pressure using a rotary evaporator.</li>
		</ol>
		</li>
		<li>Coupling to 1-(3-azidopropyl)-4-(2-(1-methyl-1H-indol-3-yl)vinyl)pyridin-1-ium iodide (dye 1) (Figure 2A, step d)
		<ol>
			<li>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.</li>
			<li>Add 0.07 ml piperidine and heat to 80 &#176;C for 4 hr.</li>
			<li>Allow to cool to RT.</li>
			<li>Collect the resulting precipitate by filtration and wash three times with diethylether.</li>
			<li>Add diethylether to the supernatant and collect the resulting precipitate by filtration. Wash three times with diethylether.</li>
			<li>Combine the precipitates.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Synthesis of 1-(3-azidopropyl)-4-(2-(1-methyl-2-phenyl-1H-indol-3-yl)vinyl)quinolin-1-ium iodide (dye 2)
	<ol>
		<li>Synthesis of 1-(3-iodopropyl)-4-methylquinolin-1-ium iodide (Figure 2B, step a)
		<ol>
			<li>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.</li>
			<li>Heat to 85 &#176;C for 16 hr.</li>
			<li>Allow to cool to RT, then remove the solvent under reduced pressure using a rotary evaporator.</li>
			<li>Add 20 ml of ethyl acetate to the remaining oil and treat the mixture in an 120 W ultrasonic bath for 3 min.</li>
			<li>Collect the formed precipitate by filtration and wash five times with ethyl acetate. Dry the solid product under vacuum O/N.</li>
		</ol>
		</li>
		<li>Synthesis of 1-(3-azidopropyl)-4-methylquinolin-1-ium (Figure 2B, step b)
		<ol>
			<li>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.</li>
			<li>Heat to 85 &#176;C for 16 hr.</li>
			<li>Allow to cool to RT, then remove the solvent under reduced pressure using a rotary evaporator.</li>
			<li>Add 15 ml of dichloromethane to the residue.</li>
			<li>Filter off and discard the resulting precipitate.</li>
			<li>Remove the solvent under reduced pressure using a rotary evaporator to obtain the product as brown oil.</li>
		</ol>
		</li>
		<li>Synthesis of 1-methyl-2-phenyl-1H-indole-3-carbaldehyde (Figure 2B, step c)
		<ol>
			<li>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.</li>
			<li>Stir the mixture at 130 &#176;C for 19 hr.</li>
			<li>Allow to cool to RT, then pour the mixture on ice.</li>
			<li>Extract the aqueous layer three times with 150 ml ethyl acetate.</li>
			<li>Combine the organic layers, wash them with water, dry them over sodium sulfate and remove the solvent under reduced pressure using rotary evaporator.</li>
		</ol>
		</li>
		<li>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)
		<ol>
			<li>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.</li>
			<li>Add 0.06 ml piperidine and heat to 80 &#176;C for 4 hr.</li>
			<li>Allow to cool to RT.</li>
			<li>Collect the resulting precipitate by filtration and wash with diethylether three times.</li>
			<li>Add diethylether to the supernatant and collect the resulting precipitate by filtration. Wash three times with diethylether.</li>
			<li>Combine the precipitates.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Prearrangements
	<ol>
		<li>Dissolve the commercially available 2&#39;-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.</li>
		<li>Perform a &#34;leak test&#34; (according to manufacturer&#39;s instructions) to make sure that no argon gas leak exists. If the &#34;leak test&#34; fails, screw in the vial more tightly.</li>
		<li>Prime the cU solution by filling the tube that connects to the solid phase synthesis chamber.</li>
		<li>Wash all lines with acetonitrile.</li>
	</ol>
	</li>
	<li>Synthesis of DNA strands
	<ol>
		<li>Use the connected computer to enter the DNA sequence and coupling method, following the prompts from the manufacturer&#39;s protocol. The coupling step of the cU building block takes 168 sec with 7 pulses (each pulse are 16 &#181;l) compared to 40 sec with 7 pulses for a conventional phosphoramidite as building blocks.</li>
		<li>Mount the column containing the CPG (controlled pore glass) as solid phase that is modified with 1 &#181;mol of the first base (DNA synthesis is performed from 3&#39; to 5&#39;) into the synthesizer.</li>
		<li>Start the synthesis on the DNA synthesizer11.</li>
	</ol>
	</li>
	<li>Workup of synthesized DNA strands
	<ol>
		<li>Dry the columns with the synthesized DNA strands under vacuum O/N.</li>
		<li>Use a pincer to open the column and release the CPG into a reaction vial. Add 700 &#181;l of concentrated aqueous ammonia solution to deprotect the oligonucleotide and release the DNA strand from the CPG.</li>
		<li>Close the vial and apply a security lid onto the reaction vessel to prevent it from bursting, and heat to 50 &#176;C for 18 hr.</li>
		<li>Remove ammonia by centrifugation under reduced pressure (100 mbar) at 30 &#176;C for 30 min.</li>
		<li>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 &#181;m. Centrifuge at 1,000 x g for 4 min.</li>
		<li>Meanwhile add 300 &#181;l 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.</li>
		<li>Remove the water from the combined aqueous solutions by centrifuging at 0.1 mbar and 25 &#176;C O/N.</li>
	</ol>
	</li>
</ol>
3. &#34;Clicking&#34; Procedure
<ol>
	<li>Add 50 &#181;l doubly distilled water, 25 &#181;l of a sodium ascorbate solution (0.4 M in water), 34 &#181;l tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (0.1 M in DMSO/tBuOH 3:1), 114 &#181;l of the azide (0.01 M in DMSO/tBuOH 3:1) and 17 &#181;l of a tetrakis(acetonitrile) copper(I)hexafluorophosphate solution (0.1 M in DMSO/tBuOH 3:1) to the lyophilized alkyne-modified DNA sample.</li>
	<li>Incubate the sample at 60 &#176;C for 1.5 hr.</li>
	<li>Cool to RT.</li>
	<li>Add 150 &#181;l Na2EDTA (0.05 M in water) and 450 &#181;l sodium acetate (0.3 M in water) to the DNA sample and transfer to a 10 ml tube.</li>
	<li>Add 10 ml ethanol (100%) and keep at -32 &#176;C for 16 hr.</li>
	<li>Centrifuge vessel for 15 min at 1,000 x g and remove supernatant.</li>
	<li>Wash DNA-pellet with 2 ml cold ethanol (80%).</li>
	<li>Dry pellet under reduced pressure.</li>
</ol>
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.
<ol>
	<li>Dissolve the crude DNA-pellet in 250 &#181;l of doubly distilled water and transfer to an HPLC vial.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Prepare the matrix by mixing 900 &#181;l of a saturated 3-HPA solution in 1:1-mixture of acetonitrile and water with 100 &#181;l of a diammoniumhydrogencitrate solution (100 g/L) in water.</li>
		<li>Pipette 1-2 &#181;l of the DNA probe on the target and let it dry on air.</li>
		<li>Add 0.2 &#181;l of the 3-HPA/diammoniumhydrogencitrate matrix to the sample and mix until it crystallizes.</li>
		<li>Perform MALDI mass spectrometry.</li>
		<li>Combine those HPLC fractions that exhibit the right mass.</li>
	</ol>
	</li>
</ol>
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 (&#949;260) of the DNA bases and the dye.
<ol>
	<li>Dissolve the DNA in 100 - 200 &#181;l doubly distilled water.</li>
	<li>Apply 1 &#181;l of the DNA solution on the UV/Vis spectrophotometer.</li>
	<li>Measure the absorption at 260 nm. Repeat three times.</li>
	<li>Take the average value to calculate the concentration of the solution. For the calculation of the extinction coefficients, use &#949;260nm of the four natural bases12 and the &#949;260nm of the purchased building block cU (considered as natural uridine)12 to obtain the &#949;260nm of the whole DNA strand. For dye 1, use &#949;260nm = 10,200 L*mol-1*cm-1 and for dye 2, use &#949;260nm = 13,100 L*mol-1*cm-1. Calculate the concentration of the solution following Lambert-Beers&#39; law based on the extinction coefficients and measured absorbances.13</li>
</ol>
6. Sample Preparation and Spectroscopy
<ol>
	<li>Preparation of single strand samples
	<ol>
		<li>Prepare separately 1 ml of 2.5 &#181;M solutions of each of the modified DNA1 and DNA2 in 200 mM NaCl, 50 mM NaPi buffer, pH = 7.</li>
	</ol>
	</li>
	<li>Preparation of double strand samples
	<ol>
		<li>Prepare 1 ml of a solution containing 2.5 &#181;M of DNA1 and 2.5 &#181;M of DNA2 in 200 mM NaCl, 50 mM NaPi buffer, pH = 7 in a reaction vessel.</li>
		<li>Secure the lid with a safety cap and heat to 90 &#176;C for 10 min. Then turn off heating and allow to cool down to RT O/N.</li>
	</ol>
	</li>
	<li>Absorption spectroscopy 
	Note: To determine the excitation wavelength for the fluorescence spectroscopy absorption spectra are recorded.
	<ol>
		<li>Single strand measurements
		<ol>
			<li>Record a blank measurement containing only 200 mM NaCl, 50 mM NaPi buffer, pH = 7.</li>
			<li>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.</li>
			<li>Repeat for DNA2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fluorescence spectroscopy
	<ol>
		<li>Single strand measurements
		<ol>
			<li>Record a blank measurement containing only 200 mM NaCl, 50 mM NaPi buffer, pH = 7; using the excitation wavelength of dye 1.</li>
			<li>Transfer the DNA1 solution into a 1 cm quartz glass cuvette.</li>
			<li>Record the fluorescence spectrum.</li>
		</ol>
		</li>
		<li>Double strand measurement
		<ol>
			<li>Transfer the double strand solution into a quartz glass cuvette.</li>
			<li>Record the fluorescence spectrum using the excitation wavelength of dye 1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Visualization experiments 
	Caution: Appropriate eye protection should be worn to avoid UV damage to the eyes!
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol><li>Kobayashi, H., Ogawa, M., Alford, R., Choyke, P. L., Urano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+strategies+for+fluorescent+probe+design+in+medical+diagnostic+imaging%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">New strategies for fluorescent probe design in medical diagnostic imaging.</a> Chem Rev. 110 (5), 2620-2640 (2010).</li>
<li>Berezin, M. Y., Achilefu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+Lifetime+Measurements+and+Biological+Imaging%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">Fluorescence Lifetime Measurements and Biological Imaging.</a> Chem. Rev. 110 (5), 2641-2684 (2010).</li>
<li>Lee, J. S., Vendrell, M., Chang, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity-oriented+optical+imaging+probe+development%5BTitle%5D)+AND+%22Curr.+Opin.+Chem.+Biol%22%5BJournal%5D)">Diversity-oriented optical imaging probe development.</a> Curr. Opin. Chem. Biol. 15 (6), 760-767 (2011).</li>
<li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicolor+molecular+beacons+for+allele+discrimination%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Multicolor molecular beacons for allele discrimination.</a> Nat. Biotechnol. 16 (1), 49-53 (1998).</li>
<li>Holzhauser, C., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%2334%3BDNA+Traffic+Lights%26%2334%3B%3A+Concept+of+Wavelength-Shifting+DNA+Probes+and+Application+in+an+Aptasensor%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">#34;DNA Traffic Lights&#34;: Concept of Wavelength-Shifting DNA Probes and Application in an Aptasensor.</a> ChemBioChem. 13 (8), 1136-1138 (2012).</li>
<li>Holzhauser, C., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+and+RNA+%26%2334%3BTraffic+Lights%26%2334%3B%3A+Synthetic+Wavelength-Shifting+Fluorescent+Probes+Based+on+Nucleic+Acid+Base+Substitutes+for+Molecular+Imaging%5BTitle%5D)+AND+%22J.+Org.+Chem%22%5BJournal%5D)">DNA and RNA &#34;Traffic Lights&#34;: Synthetic Wavelength-Shifting Fluorescent Probes Based on Nucleic Acid Base Substitutes for Molecular Imaging.</a> J. Org. Chem. 78 (15), 7373-7379 (2013).</li>
<li>Berndl, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+Color+Readout+of+DNA+Hybridization+with+Thiazole+Orange+as+an+Artificial+DNA+Base%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Fluorescent Color Readout of DNA Hybridization with Thiazole Orange as an Artificial DNA Base.</a> Angew. Chem. Int. Ed. 48 (13), 2418-2421 (2009).</li>
<li>Bohländer, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+of+a+Photostable+Energy-Transfer+Pair+for+%26%2334%3BDNA+Traffic+Lights%26%2334%5BTitle%5D)+AND+%22Eur.+J.+Org.+Chem%22%5BJournal%5D)">Synthesis of a Photostable Energy-Transfer Pair for &#34;DNA Traffic Lights&#34.</a> Eur. J. Org. Chem. 34, 7547-7551 (2014).</li>
<li>Walter, H. K., Bohländer, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+Wavelength-Shifting+Fluorescent+Module+for+the+Adenosine+Aptamer+Using+Photostable+Cyanine+Dyes%5BTitle%5D)+AND+%22ChemistryOpen%22%5BJournal%5D)">Development of a Wavelength-Shifting Fluorescent Module for the Adenosine Aptamer Using Photostable Cyanine Dyes.</a> ChemistryOpen. 4 (2), 92-96 (2015).</li>
<li>Gierlich, J., Burley, G. A., Gramlicj, P. M. E., Hammond, D. M., Carell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Click+chemistry+as+a+reliable+method+for+the+high-density+postsynthetic+functionalization+of+alkyne-modified+DNA%5BTitle%5D)+AND+%22Org.+Lett%22%5BJournal%5D)">Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA.</a> Org. Lett. 8 (17), 3639-3642 (2006).</li>
<li>Matteucci, M. D., Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+of+deoxyoligonucleotides+on+a+polymer+support%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Synthesis of deoxyoligonucleotides on a polymer support.</a> J. Am. Chem. Soc. 103 (11), 3185-3191 (1981).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Handbook+of+Biochemistry+and+Molecular+Biology%2C+Volume+1%3A+Nucleic+Acids%22">Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids</a>. Fasman, G. D. , CRC Press. 589(1975).</li>
<li>Puglisi, J. D., Tinoco, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absorbance+melting+curves+of+RNA%5BTitle%5D)+AND+%22Meth.+Enzymol%22%5BJournal%5D)">Absorbance melting curves of RNA.</a> Meth. Enzymol. 180, 304-325 (1989).</li>
<li>Johansson, M. K., Fidder, H., Dick, D., Cook, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intramolecular+Dimers%3A+A+New+Strategy+to+Fluorescence+Quenching+in+Dual-Labeled+Oligonucleotide+Probes%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes.</a> J. Am. Chem. Soc. 124, 6950-6956 (2002).</li>
<li>Barrois, S., Wörner, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Role+of+Duplex+Stability+for+Wavelength-Shifting+Fluorescent+DNA+Probes%3A+Energy+Transfer+vs+Excition+Interactions+in+DNA+%26%2334%3BTraffic+Lights%26%2334%3B%2C+Photochem%5BTitle%5D)+AND+%22Photobiol.+Sci%22%5BJournal%5D)">The Role of Duplex Stability for Wavelength-Shifting Fluorescent DNA Probes: Energy Transfer vs Excition Interactions in DNA &#34;Traffic Lights&#34;, Photochem.</a> Photobiol. Sci. 13, 1126-1129 (2014).</li>
</ol>

The authors have nothing to disclose.

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...

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&#39; 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 &#34;DNA traffic lights&#34; 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 pairs8,9 and the post-synthetic labelling of DNA by using copper-catalyzed 1,3-cycloaddition between azides and alkynes (CuAAC).10

<table><tbody><tr><td colspan="4">&lt;strong&gt;synthesis&lt;/strong&gt;</td></tr><tr><td>4-Picoline</td><td>Sigma Aldrich</td><td>239615</td><td></td></tr><tr><td>1,3-Diiodopropane</td><td>Sigma Aldrich</td><td>238414</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>10660131</td><td>HPLC grade</td></tr><tr><td>Ethyl acetate</td><td>Fisher Scientific</td><td>10456870</td><td>technical grade</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td>p.a. grade</td></tr><tr><td>Dichloromethane</td><td>Fisher Scientific</td><td>10626642</td><td>technical grade</td></tr><tr><td>Indole-3-carboxaldehyde; 98%</td><td>ABCR</td><td>AB112969</td><td></td></tr><tr><td>Potassium carbonate, 99+%</td><td>Acros</td><td>424081000</td><td></td></tr><tr><td>dimethylcarbonate</td><td>Sigma Aldrich</td><td>517127</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide, 99.8%, Extra Dry over Molecular Sieve</td><td>Acros</td><td>348435000</td><td></td></tr><tr><td>Sodium sulfate</td><td>Bernd Kraft</td><td>12623.46</td><td></td></tr><tr><td>Ethanol, 99.5%</td><td>Acros</td><td>397690010</td><td></td></tr><tr><td>Piperidine, 99%</td><td>Acros</td><td>147181000</td><td></td></tr><tr><td>Diethylether</td><td>Fisher Scientific</td><td>10407830</td><td>technical grade</td></tr><tr><td>2-Phenylindole-3-carboxaldehyde; 97%</td><td>ABCR</td><td>AB125050</td><td></td></tr><tr><td>4-Methylquinoline</td><td>ABCR</td><td>AB117222</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;DNA synthesis&lt;/strong&gt;</td></tr><tr><td>Expedite 8909 Nucleic Acid Synthesizer</td><td>Applied Biosystems</td><td>&amp;nbsp;-</td><td></td></tr><tr><td>DMT-dA(bz) Phosphoramidite</td><td>Sigma Aldrich</td><td>A111081</td><td></td></tr><tr><td>DMT-dT Phosphoramidite</td><td>Sigma Aldrich</td><td>T111081</td><td></td></tr><tr><td>DMT-dG(dmf) Phosphoramidite</td><td>Sigma Aldrich</td><td>G11508</td><td></td></tr><tr><td>DMT-dC(bz) Phosphoramidite</td><td>Sigma Aldrich</td><td>C11108</td><td></td></tr><tr><td>Amidite Diluent for DNA synthesis</td><td>Sigma Aldrich</td><td>L010010</td><td></td></tr><tr><td>Ultrapure Acetonitrile for DNA synthesis</td><td>Sigma Aldrich</td><td>L010400</td><td></td></tr><tr><td>Cap A</td><td>Sigma Aldrich</td><td>L840000</td><td></td></tr><tr><td>Cap B</td><td>Sigma Aldrich</td><td>L850000</td><td></td></tr><tr><td>CPG dT Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>T461010</td><td></td></tr><tr><td>CPG dA(bz) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>A461010</td><td></td></tr><tr><td>CPG dG(ib) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>G461010</td><td></td></tr><tr><td>CPG dC(bz) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>C461010</td><td></td></tr><tr><td>ammonia (aqueous solution)&amp;nbsp;</td><td>Fluka Analytical</td><td>318612</td><td></td></tr><tr><td>centrifugal devices nanosep 0.45&amp;nbsp;&amp;micro;m</td><td>Pall</td><td>ODGHPC34</td><td></td></tr><tr><td>5-(Benzylthio)-1H-tetrazole (Activator)</td><td>Sigma Aldrich</td><td>75666</td><td></td></tr><tr><td>2&#039;-O-propargyl deoxyuridinephosphoramidite</td><td>Chem Genes</td><td>ANP-7754</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;workup&lt;/strong&gt;</td></tr><tr><td>vacuum concentrator</td><td>Christ</td><td></td><td></td></tr><tr><td>&lt;strong&gt;clicking procedure&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tetrakis(acetonitrile)copper(I) hexafluorophosphate</td><td>Sigma Aldrich</td><td>346276</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma Aldrich</td><td>S2889</td><td></td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Sigma Aldrich</td><td>A7631</td><td></td></tr><tr><td>EDTA disodium salt</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>TBTA-ligand</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>synthesized according to a literature procedure&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;HPLC&lt;/strong&gt;</td></tr><tr><td>HPLC-system</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>MALDI-Biflex-IV spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>LC-318 C18 column</td><td>Supelcosil via Sigma Aldrich</td><td>58368</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;determination of concentration&lt;/strong&gt;</td></tr><tr><td>ND 1000 Spectrophotometer</td><td>nanodrop</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;sample preparation and spectroscopy&lt;/strong&gt;</td></tr><tr><td>Cary 100 Bio</td><td>Varian</td><td></td><td></td></tr><tr><td>Fluoromax-3 fluorimeter</td><td>Jobin-Yvon</td><td></td><td></td></tr><tr><td colspan="4">&lt;sup&gt;1&lt;/sup&gt; R. Chan Timothy, R. Hilgraf, K. B. Sharpless, V. Fokin Valery, &lt;em&gt;Org Lett&lt;/em&gt; 2004, &lt;strong&gt;6&lt;/strong&gt;, 2853-2855.</td></tr></tbody></table>

synthesis of wavelength-shifting dna hybridization probes by using photostable cyanine dyes

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

In this protocol, we demonstrate a method for the synthesis of 2&#39;-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.

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 λmax at 465 nm for single-stranded DNA1 (dye 1) and 546 nm for single-stranded DNA2 (dye 2). The annealed DNA1_2 (dye 1 &amp; dye 2) shows maxima at both 469 nm and 567 nm. Both absorption maxima show a bat...

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

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

The overall goal of this experiment is to visualize the energy transfer from one dye modified DNA strand to another. This method can help on the key questions in the field of molecular biology. Such as RNA interference by monitoring siRNA processing and integrity in living cells in real time.The main advantage of this technique, is the ease of monitoring the florescence reach out and the color change, giving a clear result. So false positive results are eliminated. The begin this procedure, add the appropriate reagents as indicated in the text protocol to a 20 milliliter, round bottom flask, equipped with a magnetic stir bar and reflux condenser.Then, add zero point zero six milliliters of piperidine. And heat the reaction mixture to 80 degrees celsius for four hours. After the reaction has cooled to room temperature, collect the resulting precipitate by filtration.And wash with diethyl ether three times. Next, add one hundred milliliters of diethyl ether to the supernatant. And collect the resulting precipitate by filtration.After washing three times with diethyl ether, combine the precipitate with isolated solid from the first filtration. Using the computer, connected to a DNA synthesizer, enter the desired DNA sequence and coupling method. Following the prompts from the manufacturer's protocol.Next, mount the column containing the controlled pour glass, that is modified with one micro mole of the first face into the synthesizer. Now, start the synthesis on the DNA synthesizer. Following workup of the DNA strands, add the appropriate regents as indicated in the text protocol to the diethylized alkyne, modified DNA sample.Incubate the sample at 60 degrees celsius for one point five hours. Then, transfer the sample to a 10 milliliter tube. After cooling to room temperature, add 150 microliters, of a zero point zero five molar EDTA disodium salt solution, and 450 microliters of a zero point three molar sodium acetate solution to the DNA sample.Following this, add 10 milliliters of ethanol to the sample. And keep it at minus 32 degrees celsius for 16 hours. Next, centrifuge the sample for 15 minutes at one thousand times G.When finished, remove the supernatant.Wash the resulting DNA pellet with two milliliters of cold 80%ethanol. Then dry the pellet under reduced pressure using a freeze drier. Purify the crude DNA pellet by HP LC.Monitor the run by checking 260 nanometers, and 459 nanometers for Dye one.Or 542 nanometers for Dye two. Collect the fractions that show absorption in both wave length channels. For single strand absorption spectroscopy measurements, record a blank measurement containing only 200 millimolar sodium chloride and 50 millimolar sodium phosphate buffer.Next, transfer the previously prepared DNA1 solution into a one centimeter, quartz glass cuvette. Record the absorption. Then determine the maximum value of dye absorption.For single strand fluorescent spectroscopy measurements, record a blank measurement containing only 200 millimolar sodium chloride and 50 millimolar sodium phosphate, using the excitation wave length of Dye one. Following this, place the cuvette with the DNA1 solution into the spectrometer. Record the florescence spectrum.To perform the double strand, fluorescent spectroscopy measurement, place the cuvette with the double strand of DNA1 and DNA2 solution into the spectrometer. Then record the florescent spectrum using the excitation wave length of Dye one. To gain a better understanding of what the recorded spectra are showing, irradiate the cuvettes with hand held UV lamps.Observe the change in florescence color from the single to the double stranded DNA. The recorded absorption spectra show the maxima at 465 nanometers for single stranded DNA1 and 546 nanometers for single stranded DNA2. The annealed double strand of DNA1 and DNA2 shows maxima at both 469 and 567 nanometers.Both absorption maxima show a bathaohromic shift. And the smallest spectroscopic changes are the result of excitonic interactions between the dyes within the double helical DNA architecture. The corresponding fluorescent spectra exhibit a maxima at 537 nanometers for single stranded DNA1.At 607 nanometers for single stranded DNA2. And at 615 nanometers for annealed, double strand of DNA1 and DNA2. Double stranded of DNA1 and DNA2, shows solely the emission maximum of Dye two, although Dye one is selectively excited, which evidences that the energy transfer occurs efficiently from Dye one to Dye two.After watching this video, you should have a good understanding of how to post synthetically modify DNA by a couple catalytic reaction of an alkyne labeled DNA strand with an A side.

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Research

JoVE Journal

Biology

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robust 3D DNA FISH Using Directly Labeled Probes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

This protocol outlines the design of split hybridization probes utilizing fluorescent light-up aptamer DAP-10-42 as a signal reporter and their application for nucleic acid detection and differentiation between targets with single-nucleotide substitutions.

Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

Learn to synthesize wavelength-shifting DNA hybridization probes using photostable cyanine dyes and copper-catalyzed click chemistry. This method enables real-time visualization of energy transfer for molecular imaging applications.

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

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