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

Published: July 6th, 2016



1Institute of Organic Chemistry, Karlsruhe Institute of Technology
* These authors contributed equally

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....

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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.......

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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 .......

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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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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.


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Name Company Catalog Number Comments
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
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 procedure [1]
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.

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