Detection of adenine-rich ssDNA based on thymine-substituted tetraphenylethene with aggregation-induced emission characteristics

Abstract

In this work, tetraphenylethene (TPE) equipped with thymine, TPE–T, was synthesized and used as a fluorescence biosensor for differentiating ssDNA from dsDNA. TPE–T is a “turn-on” fluorescence probe once it forms hydrogen bonds with single-stranded adenine-containing oligonucleotides with high sensitivity and specificity. The working mechanism of TPE–T and its potential in cell imaging are investigated in detail.

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Introduction

Photoluminescence provides a convenient method for analytical research with outstanding sensitivity and specificity.^1–11 Without expensive instrumentation and tedious sample preparation, target analytes can be quantified and qualified by simply using “mix-and-detect” with a high signal-to-noise ratio.^12–17 Facile and reliable fluorescence methods for nucleic acids detection are of great importance in biosensor technology field.^18–20 Some successful intercalating and groove binding dyes including ethidium bromide, SYBR Green I, TOTO-3, Hoechst 33258, DAPI have been reported to detect nucleic acids.^21–24 Take thiazole orange (TO) as an example, its fluorescence will be enhanced dramatically upon dsDNA intercalation.²⁴ The non-radiative decay of photoexcited cyanine dyes is regulated by the rotation rate of the central methine which would be greatly reduced within the constrictive double-stranded DNA (dsDNA) environment. Unluckily, intercalating DNA is suspected to be toxic and accompanied with the aggregation of the dyes which lead to severely signal reduction by aggregation-caused quenching.²⁵

On the other hand, fluorescent probes of detecting single-stranded DNA (ssDNA) are still scarce compared to a huge number of dsDNA probe. We have previously discovered a group of fluorogens which are nonemissive when molecularly dissolved but highly fluorescent when aggregate are potentially used as ssDNA fluorescence probes.²⁶ We coined this phenomenon as aggregation-induced emission (AIE) with the mechanism of the restriction of intramolecular rotations (RIR).²⁷ In the past decade, the AIE fluorogens have been successfully employed in a variety of biological applications²⁸ such as DNA sensing (G-quadruplex, dsDNA), protein sensing (Human serum albumin, insulin, Caspase-3/7, Acetylcholinesterase), small molecule sensing (cysteine, glucose), and long-term cell tracking etc. The fluorescence signals of AIE fluorogens will be turned on upon the binding to biomacromolecules by RIR.²⁹

Results and discussion

In this contribution, an AIE dye can be used for differentiating ssDNA from dsDNA and the hydrogen bonds between complementary base pairs are utilized for promoting the specificity. Based on this principle, a thymine-functionalized TPE derivative, TPE–T, is synthesized for probing adenine-rich ssDNA (Scheme 1).³⁰ The product was obtained as white powder in a yield of 41% and characterized by NMR, high-resolution mass spectroscopy and elemental analysis (see Experimental section in ESI†). The structure of this dye was analyzed using a B3LYP/6-31G hybrid functional for full geometrical optimization (Fig. S1†). The TGA thermogram of TPE–T showed 5% weight loss at 330 °C corresponding to its thermally stable property (Fig. S2†). Fig. S3† shows the UV spectra of TPE–T in the detection medium. The absorption maxima of TPE–T is located at 275 nm with molar absorptivities of 0.26 × 10⁵ M⁻¹ cm⁻¹, owing to the thymine part of TPE–T.

Scheme 1 Schematic representation of ssDNA detection by TPE–T.

TPE–T is soluble and thus non-emissive in ethanol. The fluorescence spectrum exhibits as nearly a flat line parallel to the abscissa when the volume fraction of water (f_w) is lower than 60% (Fig. S4†). When a large amount of water is added, the resulting mixture becomes intensely luminescent with blue emission peaked at 450 nm. The emission intensity of TPE–T in the solution with f_w of 99% is about 400 times higher than that in pure ethanol. This characteristic curve shown in Fig. 1B indicates that TPE–T is a typical AIE active fluorogen with an f_w threshold of 68%, above which the dye molecules aggregate and the emission is turned on. Solution of TPE–T in ethanol–water mixture with f_w of 64% was then used as a detection medium.

Fig. 1 (A) Emission spectra of TPE–T in ethanol–water mixture (36 : 64, v/v; 10 μM) with calf thymus DNA (dsDNA, 30 μg mL⁻¹) or ssDNA (AA, 2 μM). Excitation wavelength: 350 nm. Inset: fluorescent photographs of TPE–T with (left) dsDNA and (right) AA taken under UV illumination. (B) Plot of relative emission intensity (I/I₀) at 450 nm versus the concentration of AA. Inset: linear plot of the relative emission intensity against the AA concentration.

Using this detection system, dsDNA and ssDNA differentiation was investigated, and the sequences of ssDNA are listed in Table 1. In order to check the stability of dsDNA, UV-vis spectra has been conducted. Fig. S5† showed the UV-vis spectra of calf thymus DNA (30 μg mL⁻¹) in ethanol–water mixture (36 : 64, v/v) at different incubation time. It was found that calf thymus DNA was stable in the test system for at least 60 minutes, which is sufficient for our detection. The fluorescence signals of TPE–T to dsDNA and ssDNA in the detection medium were shown in Fig. 1A. The solution of TPE–T in pure ethanol is almost non-emissive. The presence of adenine-containing ssDNA (AA) triggers the fluorescence of the dye whereas dsDNA (calf thymus DNA) cannot turn on the fluorescence of TPE–T in the detection medium. Increasing the concentration of dsDNA gave similar experimental results (Fig. S6†). Formations of hydrogen bond between TPE–T and adenine induce the RIR of the phenyl groups thus lead to the enhancement of the fluorescence. To verify this hypothesis, Hoechst 33342, a conventional nucleic acid dye, was chosen for evaluation by staining DNA through groove binding. Since the groove does not exist in single-stranded DNA, the fluorescence of Hoechst 33342 cannot be turned on. Different from TPE–T, Hoechst 33342 exhibits fluorescence enhancement in the presence of dsDNA rather than ssDNA (Fig. S7†), which implies that TPE–T is not a groove binder of DNA.

Table 1 Abbreviation and sequences of ssDNA used in the present study

Abbreviation	Sequence
AA	5′AAAAAAAAAAAAAAAAAAAAA3′
TT	5′TTTTTTTTTTTTTTTTTTTTT3′
CC	5′CCCCCCCCCCCCCCCCCCCCC3′
GG	5′GGGGGGGGGGGGGGGGGGGGGG3′
AT	5′ATATATATATATATATATATA3′
AC	5′ACACACACACACACACACACA3′
AG	5′AGAGAGAGAGAGAGAGAGAGA3′
TC	5′TCTCTCTCTCTCTCTCTCTCT3′
TG	5′TGTGTGTGTGTGTGTGTGTGT3′
A2T	5′ATTATATTATATTATATTATA3′
A4T	5′ATTTTATTTTATTTTATTTTA3′
A9T	5′ATTTTTTTTTATTTTTTTTTA3′
R1	5′AATCCGTCGAGCAGAGTT3′
R2	5′GGC ATG AAC CGG AGT CCC ATC CTC3′
R3	5′AAT TTC ATA TTG GCT TCA ATC CAA AAT3′

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To investigate the fluorescence response of TPE–T toward ssDNA, titration experiments were performed by adding different sequences of ssDNA into solution of TPE–T in ethanol–water mixture (36 : 64, v/v; 10 μM) (Fig. S8–16†). In the presence of AA, an emission peak with a maximum at 450 nm appears as a result of TPE–T can form hydrogen bond with adenine causing the RIR of the phenyl groups. There were detectable fluorescent signals observed at concentrations of AA as low as 100 nM, and the fluorescence intensity increased with increasing AA concentration (Fig. 1B). In the AA concentration ranged of 1.3–1.9 μM, the plot of fluorescence increment at 450 nm as a function of AA concentration was in a linear regression with a correlation coefficient of 0.9928. Beside sensitivity, the specificity of TPE–T towards adenine-rich ssDNA was also investigated. As shown in Fig. 2, nearly no change was observed when adding the ssDNA without adenine, even at the ssDNA concentrations of 2 μM. At such a high ssDNA concentration, the emission intensity of TPE–T increased nearly 40-fold with the aid of adenine-rich ssDNA (AT, AC, AG), which demonstrated that it has high specificity to adenine-containing ssDNA.

Fig. 2 (A) Emission spectra of TPE–T in ethanol–water mixture (36 : 64, v/v; 10 μM) with different ssDNA (2 μM). Excitation wavelength: 350 nm. Inset: fluorescent photographs of TPE–T containing different ssDNA (from left to right: blank, AT, TT, CC and GG) taken under UV illumination. (B) Plot of relative emission intensity (I/I₀) at 450 nm versus the concentration of different ssDNA.

In addition, the control experiments were carried out by using solutions of nucleobase-containing molecules, such as dTTP, dATP, dCTP, dGTP. As shown in Fig. S17–20,† even at the high concentration of 20 μM, the emission intensity exhibited very weak signals. The signals from the mixtures of TPE–T with dTTP, dCTP and dGTP were match to the hypothesis due to the absence of hydrogen bond formation. Interestingly, weak fluorescence signals were given even TPE–T was mixed with adenine-containing species. It shows that TPE–T could not detect the presence of adenine solely by the formation of hydrogen bond as dATP was too small to trigger RIR. To obtain more evidence, six ssDNA with different sequences were studied (Fig. S8, 9, 14, 21–23†). Among these, from TT, A9T, A4T, A2T, AT, to AA, the percentage of adenine increased gradually. As shown in Fig. 3B, the emission intensity of TPE–T remains almost unchanged in the presence of TT but increases along with the increase of the percentage of adenine in different ssDNA. These results proved that the restriction of intramolecular rotation of the phenyl groups on TPE–T is crucial for this detection system.

Fig. 3 (A) Fluorescence response of TPE–T in ethanol–water mixture (36 : 64, v/v; 10 μM) at 450 nm in the presence of different ssDNA (2 μM). Excitation wavelength: 350 nm. (B) Plot of relative emission intensity (I/I₀) at 450 nm versus the concentration of different ssDNA.

When 2 μM of AA was added in this detection system, the emission intensity enhanced about 25-fold, almost the same as in the case with AT as shown in Fig. 3A. The binding stoichiometry of TPE–T with AA and AT were then determined for rationalizing this similarity. Binding analysis was performed using the method of continuous variations (Job plot) as shown in Fig. S24.† Take AT as an example, the total concentration of TPE–T and AT was kept constant at 10.0 μM. A maximum and constant relative intensity occurs at [AT]/([AT] + [TPE–T]) = 0.1, which establishes the formation of a 1 : 9 complex of AT and TPE–T and is responsible for the observed fluorescence signals. These data indicate that the formation of the complex, and the binding stoichiometry agrees well with the known adenine–thymine base pair. Interestingly, the plots of AA with TPE–T was also peaked at 0.1, giving a 1 : 9 binding ratio for AA to TPE–T. Since the presence of the steric hindrance between TPE–T bound ssDNA, no significant signal enhancement difference was observed between TPE–T and AA or AT.

To study whether the TPE–T based fluorescent probe developed here was applicable to real systems, three random ssDNA sequences (R1, R2, R3) were analyzed using our detecting system (Table 1). The experimental results showed that this sensing system can also sense the random ssDNA. Take a random ssDNA (R2) as an example, a linear correlation existed between the fluorescence intensity and the concentration of R2 within the range from 0 to 2.0 × 10⁻⁶ M (Fig. 4). The TPE–T bound to R2 through hydrogen bond indeed shows a distinct difference in fluorescence emission intensity, which exhibits more than 4-fold higher brightness than the TPE–T probe alone. The change in fluorescence emission intensity (I/I₀) in three random ssDNA sequences (R1 = 3.4; R2 = 4.3; R3 = 5.6) is in accordance with our previous studies, which further proved that such a sensing system still worked well in real systems.

Fig. 4 (A) Fluorescence response of TPE–T in ethanol–water mixture (36 : 64, v/v; 10 μM) at 450 nm in the presence of three random ssDNA (2 μM). Excitation wavelength: 350 nm. (B) Plot of relative emission intensity (I/I₀) at 450 nm versus the concentration of different ssDNA.

In addition to this, we have also explored the biological applications of TPE–T. We found that TPE–T can work as a fluorescent visualizer for intracellular imaging. As depicted in Fig. S25,† TPE–T selectively stained the cytoplasmic regions of living HeLa cells. Bright blue fluorescence was observed from the fixed HeLa cells, presumably because the dead HeLa cells with compromised membrane open the access for the dye molecules to enter the protoplasm, which represents an indicator of cell death.

Conclusions

In summary, we have developed a fluorescent probe, TPE–T, with aggregation-induced emission characteristics for differentiating adenine-containing ssDNA from dsDNA and ssDNA without the presence of adenine. Compared with numerous commercial biosensors for dsDNA, the detection of ssDNA is much more difficult. We demonstrate that the well-known hydrogen bond of base pair is used to sense ssDNA with high sensitivity and specificity by utilizing “turn-on” method. Furthermore, this detection system shows different signal to different adenine contained ssDNA sequences, which enable TPE–T a useful probe to detect the content of adenine. As we have developed lots of AIE dyes, this piece of work might open up a new avenue for the further development of ssDNA detection systems.

Experimental

Materials, methods are described in the ESI.†

Synthesis

5-Methyl-1-(4-(1,2,2-triphenylvinyl)benzyl)pyrimidine-2,4(1H,3H)-dione (TPE–T). A solution of TPE–Br (213 mg, 0.5 mmol), thymine (194 mg, 1.5 mmol) and K₂CO₃ (207 mg, 1.5 mmol) in dry DMF (15 mL) was refluxed under nitrogen for 24 h. After cooling to ambient temperature, the solvent was evaporated under reduced pressure. The residue was purified by a silica gel column chromatography using dichloromethane and ethyl acetate (3 : 1 v/v) as eluent to give a white powder in 41% yield.

Cell culture

HeLa cells were cultured in the MEM containing 10% FBS and antibiotics (100 units per mL penicillin and 100 μg mL⁻¹ streptomycin) in a 5% CO₂ humidity incubator at 37 °C.

Cell imaging

HeLa cells were grown overnight on a 35 mm petri dish with a cover slip. The cells were fixed with 96% ethanol at −20 ^OC for 5 min. The fixed cells were stained with 5 μM of TPE–T for 30 min (by adding 2 μL of a 5 mM stock solution of TPE–T in DMSO to 2 mL culture medium). The cells were imaged under an FL microscope (BX41 Microscope) using combination of excitation and emission filters (excitation filter = 330–385 nm, dichroic mirror = 400 nm, and emission filter = 420 nm long pass).

Acknowledgements

This work was partially supported by National Basic Research Program of China (973 Program; 2013CB834701), the Research Grants Council of Hong Kong (604711, 604913, HKUST2/CRF/10 and N_HKUST620/11), and the University Grants Committee of Hong Kong (AoE/P-03/08). B.Z.T. thanks the support from Guangdong Innovative Research Team Program of China (201101C0105067115).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Details of the experimental procedure; UV-vis spectra ¹H NMR spectra; mass spectra. See DOI: 10.1039/c4ra05765a

‡ These authors contributed equally to this work.

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