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 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 pairs8,9 and the post-synthetic labelling of DNA by using copper-catalyzed 1,3-cycloaddition between azides and alkynes (CuAAC).10
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.
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.
3. "Clicking" Procedure
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.
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.
6. Sample Preparation and Spectroscopy
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 a bathochromic shift, 4 nm for dye 1 and 21 nm for dye 2. These small spectroscopic changes are the result of excitonic interactions between the dyes within the double-helical DNA architecture.
The corresponding fluorescence spectra (Figure 4 left) exhibit maxima λmax, at 537 nm for single-stranded DNA1 (dye 1, excitation at 435 nm), at 607 nm for single-stranded DNA2 (dye 2, excitation at 535 nm) and at 615 nm for annealed DNA1_2 (dye 1 & dye 2, excitation at 435 nm). DNA1_2 shows solely the emission maximum of dye 2 although dye 1 is selectively excited which evidences that the energy transfer occurs efficiently from dye 1 to dye 2. This gives an emission contrast ratio of 1:57.
The difference between single and double strand is obvious by comparing the emission color in the cuvette during excitation with a standard UV-lamp as shown in Figure 5.
Figure 1. Basic concept of an energy transfer in DNA. The donor modified DNA strand (green) shows fluorescence at 535 nm upon irradiation at 435 nm. If it is hybridized with the acceptor-modified counter strand (red) the resulting double strand shows only the red fluorescence of the acceptor dye at 610 nm. Please click here to view a larger version of this figure.
Figure 2. Synthesis overview of dye 1 and dye 2. The synthesis of the two cyanine dyes 1 (A) and 2 (B) is carried out in three consecutive steps.9 (A) a) 1,3-diiodopropane, CH3CN, 85 °C, 16 hr, 94%; b) NaN3, CH3CN, 85 °C, 16 hr, 87%; c) K2CO3, dimethyl carbonate, DMF, 130 °C, 19 hr, 89%; d) piperidine, EtOH, 80 °C, 4 hr, 80%. (B) a) 1,3-diiodopropane, CH3CN, 85 °C, 16 hr, 49%; b) NaN3, CH3CN, 85 °C, 16 hr, 93%; c) K2CO3, dimethyl carbonate, DMF, 130 °C, 19 hr, 98%; d) piperidine, EtOH, 80 °C, 4 hr, 44%. Please click here to view a larger version of this figure.
Figure 3. Structure of cU-building block, dyes 1 and 2 and sequences of DNA1 and DNA2. The dyes 1 and 2 are linked to the 2' position of the ribose by a triazole linker within the given sequences of DNA1 and DNA2.9 Please click here to view a larger version of this figure.
Figure 4. Fluorescence of single and double stranded DNA strands (left) and absorption of single and double stranded DNA strands (right). Emission spectra of DNA1 (black) excited at 464 nm, DNA2 (red), excited at 535 nm and 435 nm (pink), and DNA1_DNA2 hybrid (blue), excited at 435 nm are shown. The absorption spectra of the single stranded DNA1 (black), DNA2 (red), and the hybridized double strand DNA1_DNA2 (blue) are shown on the right side show. Please click here to view a larger version of this figure.
Figure 5. Emission color of the single stranded DNA with donor dye (left) and double stranded DNA with donor and acceptor dye (right) during excitation by an UV-lamp. The image of the cuvette containing the single stranded DNA1 (left side) shows bright green fluorescence, and the image of double stranded DNA1_DNA2 (right side) shows red fluorescence during excitation by a handheld UV-lamp at 366 nm. Please click here to view a larger version of this figure.
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 shortens the syntheses of the dyes to four instead of five consecutive reaction steps. The application of 1,3-diiodopropane instead of 3-iodopropanol for the alkylation step, the Appel reaction and the following Finkelstein reactions to convert the hydroxyl function into the iodo functionalities were avoided. These changes in comparison to the published procedures allow us to synthesize the dyes in larger scales and shorter periods of time.
The applied dyes exhibit an excellent photostability but not the necessary stability during solid-phase DNA synthesis and workup. Hence, the post-synthetic labelling strategy was chosen. Alternatively, labelling with such precious dyes during the DNA synthesis represents ultra-mild coupling and deprotection conditions (e.g., deprotection with potassium carbonate). This represents a significantly more expensive alternative and requires the synthesis of the corresponding dye-modified phosphoramidites as DNA building blocks or labelling on the solid support (on-bead).
DNA1 is labeled by dye 1, the donor dye, and DNA2 by dye 2, the acceptor dye. When DNA1 and DNA2 are annealed both dyes get into close proximity. An efficient energy transfer is observed when dye 1 is excited. In principal, excitonic interaction between both dyes may interfere with an efficient energy transfer since the latter process requires the uncoupled and excited dye 1 and uncoupled dye 2. Excitation of the coupled dyes would initiate different photophysical pathways.14 Our previous studies9,15 revealed that the applied 3'-5'-diagonal arrangement of dyes provides the structural basis for only little excitonic interactions between the dyes and efficient energy transfer.
The high efficiency of the demonstrated energy transfer enables precise fluorescence readout in in vitro and in vivo experiments. This offers a wide range of applications, including the visualization of DNA hybridization in molecular beacons, binding of target molecules in nucleic acid based aptamers as well as the visualization of the integrity of small interfering RNA (siRNA) inside cells. The major limitation is the requirement to label both DNA strands. In the future we aim to focus our work on the development of doubly labeled oligonucleotide probes bearing both dyes in one strand.
The authors have nothing to disclose.
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.
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 procedure [1] |
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. |