KR20070072268A - Novel fret system consisting of a qd donor and a dye accepotor attached to dna - Google Patents

Abstract

A fluorescence resonance energy transfer(FRET) system which is very sensitive and highly efficient compared to conventional systems is provided to be used for manufacturing a sensitive FRET-based sensor and used as a useful manufacturing material for assembling a novel and functional nano-structure by low accepter to donor ratio in the mono-molecule level. The FRET system comprises a quantum dot such as CdSe/ZnS core shell QD as a donor, and a DNA which is labeled with Alexa 594 fluorophore as an accepter. The method for preparing a QD-DNA conjugate comprises the steps of: (a) capping the QD using MPA to prepare an MPA-QD; (b) removing excessive and unbound free MPA from the MPA-QD solution; and (c) conjugating the MPA-QD with DNA with or without removing the MPA therefrom.

Description

Novel FRET system consisting of a QD donor and a dye accepotor attached to DNA}

1 is a schematic of a QD-dye-labeled DNA FRET system.

FIG. 2 shows absorption spectra (black lines, left scale) and fluorescence spectra (red lines, right scale) of Alexa 594 labeled DNA (1 μM), and fluorescence spectra of MPA-capped CdSe / ZnS core / shell QDs (blue lines) , Right scale).

FIG. 3A shows the fluorescence spectra (excited at 450 nm) of DNA-QD conjugates at different DNA / QD ratios after removal of free MPA.

Figure 3b is a bulk (black squares), and single-shows the apparent FRET efficiency from molecular studies (red dots) _(A I / (I _A + I _D)) for DNA / QD ratio.

3C shows representative fluorescence emission trajectories of QD-DNA conjugates at 1: 1. Green eruptions are eruptions from the donor (QD) and red eruptions represent eruptions from the receptor (Alexa 594).

3D-3G depict FRET histograms of QD-DNA conjugates at different DNA to QD ratios: DNA to QD ratio = 0.25 (FIG. 3D), 0.50 (FIG. 3E), 1.0 (FIG. 3F) and 2.0 ( 3g).

4 shows tapping mode AFM phase images of QD-DNA at DNA: QD ratios of 0.25 (A, left scale bar) and 2 (B, right scale bar).

5A depicts the fluorescence spectra of QD-DNA conjugates prepared without removing free MPA at different ratios of dye-labeled DNA to QD. Blue arrows indicate a gradual change in fluorescence as the DNA: QD ratio increases from 1: 1 to 1:10.

5B shows a plot of FRET efficiency obtained from bulk measurement to DNA: QD ratio.

5C depicts a representative single-molecule FRET histogram obtained from QD-DNA conjugate at a DNA to QD ratio of 1: 1.

6 shows the fluorescence spectra of MPA-QD (red line, right scale) and QD-DNA (blue line, left scale) at a DNA: QD ratio of 4. FIG.

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Field of technology

The present invention relates to a novel fluorescence resonance energy transfer (FRET) system. Specifically, the present invention relates to a FRET system using quantum dots (QDs) as donors and dye-labeled DNA as receptors.

Prior art

Quantum dots (QDs) are currently of great interest due to their stable dependence of size-dependentness, symmetry, and narrow angle, which allow for extended observation and multiplexing (see above: 1). QDs are excellent donors for FRET because they can be widely applied due to their narrow emission and broad excitation spectrum, which is a broad donor for effective separation of donor and acceptor fluorescence and to reduce background It allows the selection of excitation wavelengths in the range (see supra: 2-4). Studies on FRET between two different colored pigmented QDs (see above: 2), ie QD and dye-labeled biomolecules (see above 3-5), QD and gold nanoparticles (see above: 6) , And QD and dye-labeled polymers (see above) 7 have been reported. Most of the previous studies were performed in bulk solutions (see Documents 2-4 above), providing only total averaged FRET information. In addition, these studies were mostly based on QD and dye-labeled proteins, resulting in low FRET efficiency due to the large size of the QDs and proteins (see Document 3-5). Recently, single-molecular FRET between protein-coated QD and dye-labeled DNA has been reported, but the FRET efficiency was low (ie 1-2%) (see supra: 5). To achieve high FRET, a number of receptors (> 10) are required for each QD in the QD-protein design (see above, 3, 4). Since numerous binding events for the conjugated protein are needed to produce detectable changes in the FRET signal, this significantly reduces the sensitivity of these conjugates as FRET-based sensors. In this context, we coupled the dye-labeled DNA receptor directly to the QD donor via a thiol linker. This coupling reduced the donor-receptor distance, significantly increasing FRET efficiency. FRET between QD and dye-labeled DNA was studied at both bulk solution and single-molecule levels.

It is an object of the present invention to provide a novel FRET system. The FRET system of the present invention consists of QD as donor and dye-labeled DNA as acceptor, the donor and acceptor being coupled with a thiol linker.

The design of the QD-DNA conjugate according to the present invention can be depicted as shown in FIG. The receptor Alexa 594 fluorophore labeled double stranded (ds) DNA is coupled directly via a C ₆ -thiol linker to the donor CdSe / ZnS core-shell QD (see above: 8). The FRET system of the present invention as shown in FIG. 1 reduces the donor-receptor distance, thus significantly increasing the FRET efficiency. Trioctyl-phosphine oxide (TOPO) capped CdSe / ZnS core-shell QDs (peak emission ˜533 nm, Evident Technologies, New York) according to the literature method were obtained using MPA in a mixed solvent of chloroform / methanol at pH ˜10. Treatment with (3-mercaptopropionic acid) yielded water-soluble MPA-capped QD (MPA-QD) (see supra: 9). MPA-QD showed narrow angle emission (FWHM 30nm, quantum yield ˜15%) equivalent to a slightly red-shifted peak at 558 nm (see FIG. 2). QD emission overlaps with the absorption spectrum of the dye, suggesting that efficient FRET can occur between the QD donor and the dye receptor. Based on the spectral overlap, bulk quantum yield of QD and absorption coefficient of dye, we estimated Forster distance R ₀ (distance showing 50% FRET efficiency) of ˜4.2 nm in the FRET system of the present invention (see above: 4). There was little overlap between the donor and the receptor fluorescence (see FIG. 2), which resulted in an efficient separation of the donor fluorescence from the fluorescence of the receptor. All measurements in FIG. 2 were performed in Tris buffer (10 mM Tris.HCl, 100 mM NaCl, pH 7.6) at room temperature. Alexa 594-labeled dsDNA (at different DNA to QD ratios) was conjugated to MPA-QD according to literature methods (see above: 8).

To achieve high conjugation (FRET) efficiency between QD and dye-labeled DNA, it is necessary to remove the excess unbound MPA that was used to make QD water soluble. Removal of excess MPA is accomplished by precipitation with ethanol and then centrifugation for 20 minutes at 14,000 rpm. Unbound MPA is soluble in ethanol but MPA-QD is not under centrifugation conditions. Removal of excess MPA eliminated competition for QD binding from MPA and greatly increased the conjugation efficiency of DNA to QD. Fluorescence spectra of QD-DNA conjugates at different ratios of DNA to QD are shown in FIG. 3A. Conjugation of dye-labeled DNA to QD dissipated donor (QD) fluorescence at 560 nm, while enhancing receptor (dye) fluorescence at 618 nm via FRET at the same time. The estimated FRET efficiency, defined as I _A / ( I _A + I _D ), where I _A and I _D are receptor and donor intensities, increased with the ratio of initial DNA to QD, but at a DNA: QD ratio of 0.5. It stabilized quickly, where ˜50% FRET efficiency was obtained (see FIG. 3B). FRET efficiency was higher than expected from the equation E = nR ₀ ⁶ / ( nR ₀ ⁶ + r ⁶ ) for FRET with n identical receptors interacting with a single donor (see supra: 4). By using the FRET efficiency and the above equation, we estimated the average donor-receptor distance r for DNA: QD ratios of 0.25, 0.50, 1.0 and 2.0 to 4.10, 3.80, 4.33 and 4.60 nm, respectively. The estimated distance from the dye to the center of the QD was 4.60 nm, taking into account the size of the QD and assuming the DNA was extended. Therefore, the experimental values were in agreement with the prediction only at the DNA: QD ratio of 2, and at other ratios the distance obtained was much smaller than the prediction.

Through a single-molecular study that simultaneously records QD (donor) and dye (receptor) fluorescence emission as each DNA-QD conjugate diffuses through the laser focal point (probe volume), low DNA versus Very bright receptor fluorescence eruptions were found at the QD ratio (up to several hundred counts per ms) (see FIG. 3C). They were much brighter than typical ejection (˜10 counts per ms) from a single fluoropor under the same conditions. Receptor ejection at the DNA: QD ratio ≦ 1 was stronger than the ejection at the higher ratio (ie 2), which contradicts the notion that a higher DNA: QD ratio should lead to stronger receptor ejection. This also means that the solution needs to be agitated using a pipette during the collection of fluorescence eruptions at the DNA: QD ratio ≦ 1, but is unnecessary at higher ratios (ie 2.0). This suggests that the QD-DNA conjugates aggregate at low ratios (ie ≦ 1), but not at higher ratios (ie 2.0) or only slightly aggregated. To demonstrate this, we present a planar TSG (template) modified with 6-mercapto-N-hexylpyridinium bromide, a self-assembled monolayer of positively charged thiols. The QD-DNA conjugate was immobilized on a stripped gold, typical roughness <0.2 nm surface (see supra: 11). Tapping mode AFM topological images (see above: 12) showed that the QD-DNA conjugates aggregated a lot at a DNA: QD ratio of 0.25 (see left A in FIG. 4), but they were at a DNA: QD ratio of 2: Most were isolated (see right B in FIG. 4), suggesting that the single-molecule FRET measured at the DNA: QD ratio of 2 actually originated from individual single QD-DNA conjugates. In FIG. 4 the image size of left A is 5 × 5 μm ² and B is 2 × 2 μm ² . In addition, samples were deposited on a newly fabricated ultra-planar TSG surface modified with self-assembled monolayer 6-mercaptohexyl- N -pyridinium.

3C-3G depict FRET histograms at different DNA: QD ratios. All histograms show a single distribution of FRET efficiency with mean FRET increasing from 0.40 to 0.88 as the DNA: QD ratio increased from 0.25 to 2.0 (FIG. 3B). FRET efficiencies obtained from single-molecular studies were higher than bulk efficiencies, which means that the single molecule method only detects bright QD-DNA conjugates (threshold of 50 counts per ms for the sum of donor and acceptor fluorescence signals). Was used to differentiate single molecule ejections from the background), the bulk method is presumably because it measures signals from all QD-DNA conjugates, ie bright and faint signals. If we had estimated a quantum yield of 0.9 for bright QDs we would have obtained a Forster distance of about 5.6 nm and therefore the FRET efficiency would be 0.77 for 1: 1 DNA / QD conjugates, which is shown in FIG. 3F. Very consistent with our single-molecule results. Thus we demonstrated that it is possible to achieve high FRET efficiency between QD and dye-labeled DNA with little agitation at single-molecule (QD) level at a DNA: QD ratio of 2.0.

Aggregation of QD-DNA conjugates observed at low DNA: QD ratios may be due to interactions between the QD and DNA backbones, and may be a common feature of the QD-biomolecule conjugates used in FRET studies (see Literature: 2-4). However, without independent analysis by the single-molecular method, this information will be eliminated by the total-average provided by the bulk method. Thus, the single-molecule method provides an easy way to examine the agitation of the sample and to guide the optimization of sample preparation.

It should be understood that excess MPA removal and control of the DNA: QD ratio are key to achieving high FRET without significant aggregation. Conjugation of DNA to MPA-QD prior to removal of excess unbound MPA yields a low FRET efficiency of only about 2.6% per receptor, where the apparent overall FRET increases linearly with the DNA: QD ratio (FIG. 5a to 5c), presumably because not all DNA is conjugated with QD due to competition for QD binding from unbound MPA. Single-molecule studies have revealed a broad distribution of FRET efficiencies (see FIG. 5C) and reflect the heterogeneity of the sample because it can attach to variable labeled DNA single QDs (see above). After removal of excess unbound MPA, the use of higher DNA: QD ratios reduced QD-DNA conjugate aggregation, but this was found to be limited. If the DNA: QD ratio (unlabeled, identical sequence) is further increased to 4, significant surface defect emission is accompanied by a dramatic decrease in QD fluorescence (FIG. 6), which indicates that QD fluorescence is dependent upon the environment and its surface ligand coating. Presumably because it is very sensitive (see above). In FIG. 6 the same sequence as labeled but non-labeled DNA used in FRET studies was used and both spectra were recorded under the same experimental conditions at a QD concentration of 1 μM excited at 450 nm. 6 clearly shows that QD fluorescence is significantly reduced when conjugated with four DNA molecules, and surface defect emission is also clearly observed.

Example

Example 1: Coupling of MPA-QD and DNA

3-mercaptopropionic acid (99%, MPA), tetramethylalmonium hydroxide, 1M TrisHCl buffer (pH 7.6), high purity NaCl (without DNAnase and RNAnase), methanol, chloroform and other chemicals and reagents Purchased from Sigma-Aldrich, Dorset, UK and used in the present invention unless otherwise noted. Toluene solution of trioctylphosphine oxide (TOPO) capped CdSe / ZnS core / shell QD (TOPO-QD, peak emission ˜553 nm) is purchased from Evident Technologies, New York, USA It was soluble in nonpolar solvents and made water soluble by ligand exchange with MPA according to literature methods (see supra: 15-16). In summary, 2 mL of chloroform is added to 0.5 mL of toluene solution of TOPO-QD (5 mg / mL), which is MPA in methanol (30 mM, pH adjusted to ˜10 by tetramethylalmonium hydroxide). 2 mL was added dropwise. The mixture was stirred at room temperature for 30 minutes and then 5 mL of water was added. After shaking and mixing, most of the QD is transferred to the aqueous layer, which indicates that MPA capped QD (MPA-QD) has been successfully formed. The aqueous layer was separated from the organic layer. The obtained MPA-QD was directly coupled to thiolated DNA or coupled to DNA after removal of the free MPA ligand.

Example 2: Removal of Free MPA from MPA-QD

The volume of the aqueous phase obtained in Example 1 was reduced to -0.2 mL under reduced pressure in a rotary evaporator, and then 5 mL ethanol was added to precipitate MPA-QD. The resulting solution was centrifuged at 14,000 rpm for 20 minutes. The clear solution on top of the centrifuge tube (non-fluorescent upon UV irradiation) was discarded and the red pellet at the bottom of the tube was collected. The pellet was washed, sonicated and centrifuged twice at 14,000 rpm for 20 minutes to remove excess unbound MPA (MPA is soluble in ethanol). The obtained MPA-QD pellets were finally dissolved in MilliQ water, sonicated and stored at 4 ° C. MPA-QD is very stable under these storage conditions and no change in fluorescence properties was observed for 3 months.

Dual HPLC purified single-helix (ss) DNA was purchased from IBA GmbH (Gottingen, Germany). Hybridized double-helix (ds) DNA by heating a mixed solution of two complementary strands at 90 ° in a 1: 1 molar ratio in Tris buffer (10 mM Tris.HCl, 100 mM NaCl, pH 7.6) Was prepared and then slowly cooled to room temperature over 2 hours (see supra: 17). The sequence of duplex DNA is as follows:

HSC ₆ H ₁₂ -5'-CAT AAA AGA GCT CCA TAT CCA ACC TGC ACG-3 '

3'-GTA TTT T CT CGA GGT ATA GGT TGG ACG TGC-5 '

Wherein the base T is labeled with Alexa 594 fluorophore.

Example 3: Preparation of QD-DNA Conjugate

DNA was conjugated to QD according to literature methods (see above: 18). In summary, an amount of duplex DNA was added to the MPA-capped QD solution (50 μl, 6.8 μM) to obtain a pre-controlled DNA to QD molar ratio. After mixing, the solution was stored for 2 days in a 4 ° C. refrigerator. A calculated amount of 10 × Tris buffer (0.1 M Tris.HCl, 1 M NaCl, pH 7.6) was then added to give a final buffer concentration of 1 × Tris buffer (10 mM Tris.HCl, 0.1 M NaCl, pH 7.6). . The solution was mixed and stored at 4 ° C. for an additional 2 days to allow efficient conjugation of DNA and QD. 1 mL ethanol was then added to each sample to precipitate the QD-DNA conjugate. The resulting solution was centrifuged at 14,000 rpm for 20 minutes and the clear solution was colorless and non-fluorescent upon UV lamp irradiation. This means that not only DNA but also QDs were not removed from the sample, and the conjugation efficiency of DNA and QDs was nearly single. Then 50 μl 1 × Tris buffer was added to each sample to dissolve the pellets. After sonication, 1 mL of ethanol was added and the solution was further centrifuged for 20 minutes at 14,000 rpm. The resulting pellet was dissolved in 1 × Tris buffer to bring the desired concentration for measurement.

Example 4: UV-Vis Spectrum

Absorption spectra of the QD-DNA conjugates were measured on a Cary 300 Bio UV-vis spectrophotometer (Varian Inc., CA, USA). The spectral range of 360-800 nm was recorded with a scan speed of 600 nm / min with a slit width of 2 nm. The spectral background was corrected using blank lx Tris buffer using the same cuvette.

Example 5: Fluorescence Spectrum

All fluorescence spectra were recorded on an Aminco-Bowman Series 2 Luminescence spectrometer (Sim-Aminco Spectronic Instruments Inc, Rochester, NY). Emission spectra (range 500-800 nm) were recorded under a fixed excitation wavelength of 450 nm at a scan rate of 2 nm / s. Excitation and emission bandwidths of 4 nm were used. For QD-DNA conjugates in which DNA was labeled with Alexa 594 fluoropores, the fluorescence spectra were corrected from direct excitation of the dye by using dye-labeled DNA as a reference. Quantum yield of QD was determined by using Rhodamine-6G (95% under 480 nm excitation) in ethanol as reference. The optical density of the QD and rhodamine-6G solutions used was 0.05 at 480 nm. Since the fluorescence quantum yield of QD in the QD-DNA conjugate is dependent on the DNA to QD ratio, the approximate FRET efficiency was estimated using E = I _A / ( I _A + I _D ) rather than using the donor extinction method. Where I _D and I _A are donor and receptor fluorescence intensities, respectively.

Example 6: Atomic force microscopy

Freshly prepared ultra-planar template stripped gold (TSG) surfaces were used as substrates (19 supra). First, a positively charged self-assembled monolayer of 6-mercaptohexyl- N -pyridinium bromide (MHP.Br) was coated on the TSG surface to enable effective immobilization of negatively charged QD-DNA conjugates. 5 μl of QD-DNA conjugate was then applied to the TSG surface and left for 1 hour. The surface was rinsed entirely with MilliQ water and air dried using N ₂ before the image was taken.

All AFM experiments were performed on a digital instrument, Dimension 3100 AFM (Veeco, Santa Babara, CA) with Nanoscope IV controller in tapping mode at 24 ± 1 ° C. with a scan rate of 0.8-1 Hz. Ultra-Sharp MikroMasch silicon cantilever (NSC15 series, 125 μm length, tip radius <10 nm, spring constant ˜40 N / m, resonant frequency ˜300 KHz) was used. Topographic images were taken under light tapping sufficient to obtain a stable image to minimize the effect of tip compression of the shape. All images were captured at 512 x 512 pixels per image and analyzed using Nanoscope image analysis software (version 5.12r3) using first-class flattening (see above, 19).

Example 7: Single-molecule FRET Measurement

Home-built dual-channel confocal fluorescence microscopy was used to detect single-diffusing molecules (20, 21, supra). QD donors were excited by argon ion laser (Model 35LAP321-230, Melles Griot) using 20 Hz at 488 nm. Donor and receptor fluorescence was collected simultaneously as the QD-DNA conjugate diffused through the laser focal point. Here, green and red fluorescence eruptions occurred simultaneously, using an immersion objective lens (Apochromat x60, NA1.45, Nikon), separated by a dichroic mirror (585DRLP, Omega Optical), long pass filter and band pass filter (510ALP and 545AF75 for donor channels and 560ALP and 645AF75 for acceptor channels, all filters purchased from Omega Optical) and individually detected by two photon-counting modules (SPCM-AQR14, Perkin Elmer) It was. The results of the two detectors were recorded by two computer- performed multi-channel scalar cards (MCS-PCI, EG & G). Single-molecule detection was achieved using a sample solution of 1 nM QDs in the DNA / QD conjugate. This concentration is typically much higher than the required concentration because most of the QDs in the DNA / QD conjugate are dark (quantum yield of MPA-QD is ˜15%). All samples were diluted in Tris buffer (10 mM Tris, 100 mM NaCl, pH 7.6) containing 0.01% Tween 20 to reduce surface uptake. Thresholds of 50 counts per ms for the sum of donor and acceptor fluorescent signals were used to differentiate single molecule ejections from the background. Background, direct excitation from the buffer solution and signal crosstalk from the donor to the receptor were measured using appropriate corresponding control samples and subtracted from the fluorescence emission. The FRET efficiency, E , of each jet was calculated according to E = n _A / ( n _A + γ n _D ). Where n _A and n _D are the acceptor and donor counts, respectively. γ = (φ _A η _A ) / (φ _D η _D ) is a factor indicating the quantum yield of donor and acceptor, φ _A and φ _D , and the detection efficiency for acceptor and donor, difference in η _A and η _D , respectively to be. In the bulk measurement it was found that the ratio η _A / η _D = 1 of the detection efficiency and the quantum yield of the quantum dot φ _D change with the DNA / QD ratio. Thus, we estimated that γ = 1 and only approximate FRET efficiency could be obtained.

In summary, the inventors have demonstrated that direct coupling of dye-labeled thiolated DNA (receptor) to QD (donor) reduces donor-receptor distance, leading to highly improved FRET efficiency. Unlike QD-protein conjugates, which require numerous receptors for each QD to achieve adequate FRET, we found that highly efficient FRET (~ 88%) had low receptor-to-donor ratios at the single-molecule level with little aggregation. That is, it can be obtained in 2). This can be particularly useful for making sensitive FRET-based sensors, where a single interaction with the receptor can produce large changes in the FRET signal. These QD-DNA conjugates can also be useful fabrication materials for the assembly of novel functional nanostructures (14, supra), and research in this respect is currently underway.

As described above, the novel FRET system according to the present invention provides a very sensitive and highly efficient FRET system, unlike the prior art which provides only the total averaged FRET information. The FRET system according to the present invention can be used as a useful fabrication material for the fabrication of novel functional nanostructures as well as for the fabrication of sensitive FRET-based sensors because of its high efficiency at low receptor-to-donor ratios at the single-molecule level.

Claims (5)

A fluorescence resonance energy transfer (FRET) system consisting of QD (quantum dot) as donor and dye-labeled DNA as receptor. The FRET system of claim 1, wherein the QD is a CdSe / ZnS core-shell QD. The FRET system of claim 1, wherein the dye is an Alexa 594 fluorophore. The method of claim 1, wherein the donor and the acceptor is C ₆ - thiol linker (C ₆ -thiol linker) the FRET system characterized in that direct coupling (coupling) via. Capping the QDs with MPAs to produce MPA-QDs; Removing excess unbound free MPA from the MPA-QD solution; A method of making a QD-DNA conjugate, comprising conjugating with DNA with or without removing MPA from MPA-QD.

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