Published: July 6th, 2016
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'-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.
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....
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 thes.......
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 & dye 2) shows maxima at both 469 nm and 567 nm. Both absorption maxima show .......
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.......
|Potassium carbonate, 99+%
|N,N-Dimethylformamide, 99.8%, Extra Dry over Molecular Sieve
|Expedite 8909 Nucleic Acid Synthesizer
|Amidite Diluent for DNA synthesis
|Ultrapure Acetonitrile for DNA synthesis
|CPG dT Column 1.0 µmole
|CPG dA(bz) Column 1.0 µmole
|CPG dG(ib) Column 1.0 µmole
|CPG dC(bz) Column 1.0 µmole
|ammonia (aqueous solution)
|centrifugal devices nanosep 0.45 µm
|EDTA disodium salt
|synthesized according to a literature procedure 
|LC-318 C18 column
|Supelcosil via Sigma Aldrich
|determination of concentration
|ND 1000 Spectrophotometer
|sample preparation and spectroscopy
|Cary 100 Bio
| R. Chan Timothy, R. Hilgraf, K. B. Sharpless, V. Fokin Valery, Org Lett 2004, 6, 2853-2855.
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