An optical-logic system based on cationic conjugated polymer/DNA/intercalating dyes assembly for label-free detection of conformational conversion of DNA i-motif structure

Abstract

In this paper, we described our strategy for the design, construction, and characterization of a novel supramolecular optical-logic system based on cationic conjugated polymer/DNA/intercalating dyes assembly. Multiple logic gates operating in parallel were simulated by taking advantage of the pH-driven conformational conversion of DNA i-motif structure and the two-step fluorescence resonance energy transfer (FRET) process in this assembly. This logic system does not require any chemical modification or oligonucleotide labeling, which offers the advantages of simplicity and cost efficiency; and it can be switched back and forth by addition of H⁺ and OH⁻, which makes the logic gates operate easily and feasible to realize system reset. The strategy also gives rise to a new method for label-free detection of conformational conversion of DNA i-motif structure by working in a simple “mix-and-detect” manner, which provides some inspiration for the investigation of other biomolecular conformational conversions upon environmental changes or binding to their targets.

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Introduction

Towards the ambitions to miniaturize and integrate information-processing electronics and optoelectronics devices for their widespread applications, the development of molecular logic gates capable of computation at molecular level has been growing at a tremendous pace recently.^1–5 By making use of single-molecule systems, a number of interesting logic gates have been reported.^6–9 However, to achieve future “computer-like” logic functions of enhanced complexity,^10–13 carefully designed multimolecular systems are needed and should be sought due to the intricate design and synthesis limitation of unimolecular switches.¹⁴ Recently, supramolecular systems have been developing towards such systems capable of mimicking complex and integrated logic operations. So far, there have appeared supramolecular platforms that provide alternative modes of function to unimolecular systems by coordinating multiple molecular components, such as organic molecules,¹⁵ DNA,^16–18 RNA,¹⁹ peptides²⁰ and proteins.²¹

Of note, for a new generation of molecular logic gates, much research attention has been focused on DNA owing to its structural and functional features. DNA is well-known to possess well-regulated structures and molecular recognition abilities which can be used to provide amplification and sensor functions. Moreover, the straightforward hybridization rules enable convenient interface with other molecular computation devices based on DNA.^22–24 During the past decade, many molecular logic gates have been designed on the basis of inspiration provided by some valuable features of DNA;^18,25,26 however, there are still many potential advantages that deserve to be exploited for the design of complex and integrated logic gates, especially those based on supramolecular systems. In this paper, we will show our attempt to construct a novel supramolecular optical-logic system related to the pH-driven conformational conversion of a complex DNA geometric structure known as i-motif, a closely packed quadruplex structure of C-rich single-stranded DNA (ssDNA) based on C–C⁺ base pairs.²⁷ This unique structure has become an attractive target of detection because it often participates in important biological processes, such as the multiplication of cancer cells and modulation of gene transcription.^28,29 Herein, a cationic water-soluble conjugated polymer (CCP), DNA, and two DNA intercalating dyes (thiazole orange (TO) and ethidium bromide (EB)) were combined to make our optical-logic system by taking advantage of the fluorescence resonance energy transfer (FRET) process, which can be modulated by the pH-driven conformational conversion of the i-motif structure. This optical-logic system can be switched back and forth by additions of H⁺ and OH⁻ (chemical inputs), and the fluorescence variation behavior (outputs) can not only mimic INH and NINH logic operations, but can also be used for label-free detection of conformational conversion of the i-motif structure.

Water-soluble conjugated polymers are known to function as light harvesting materials due to large, delocalized molecular structures and can exhibit optical amplification of fluorescent signals via FRET.³⁰ They can also form complexes with oppositely charged biomolecules through strong electrostatic interactions and thus avoid covalent labeling. Because of these exceptional properties, they have been recently used as sensitive and convenient optical platforms for the detection of biomolecules, such as DNA and proteins.^31–34 For example, our recent work showed a colorimetric strategy based on cationic polythiophene for sensing of the DNA i-motif structure; nevertheless, it could not provide multiple signals for logic expressions.²⁷ By contrast, in this strategy, C-rich ssDNA can undergo a swift and reversible conformational conversion between i-motif, duplex, and random-coiled state on the pH change of buffer solution; moreover, intercalating dyes are known to exhibit different affinities for these different conformations of DNA.^35–38 Therefore, the pH value can also modulate the affinities of intercalating dyes for DNA as well as the CCP/DNA electrostatic interactions,³⁹ and consequently regulate the distance-dependent two-step FRET sequence (energy transfer from the CCP to the TO, followed by FRET from the TO to the EB) in our CCP/DNA/intercalating dyes assembly. Different types of logic gate can thus be operated in this system by simultaneously observing the emission intensities of the CCP (blue), TO (green), and EB (red) upon a single excitation of the CCP. Meanwhile, this strategy also gives rise to a new pathway for the label-free detection of the i-motif structure by working in a simple “mix-and-detect” manner, avoiding the time-consuming, inconvenient and costly processes needed by traditional fluorescence spectroscopy assays, or some other traditional methods including circular dichroism, UV-vis adsorption spectroscopy and nuclear magnetic resonance.^30,40–47

Experimental

Poly{[9,9-bis(6′-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3′-(N,N,N-diethylmethylammonium)-1′-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP) was synthesized according to the literature.⁴⁸ All DNA oligonucleotides were ordered from Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. (China). EB was purchased from Shanghai Genebase Gene-Tech Co., Ltd. (China). TO was ordered from Sigma-Aldrich. Both EB and TO were used without further purification. All solutions were prepared with MilliQ water (18.2 MQ cm) from a Millipore system.

DNA concentrations were determined by measuring the UV-vis absorbance at 260 nm in 3 mL quartz cuvettes. The concentration of PFEP refers to the concentration of polymer structure unit, which was calculated according to the molecular weight and monomer weights of its neutral polymer.⁴⁸ The molecular weight and polydispersity of its neutral polymer are 12600 and 1.28, respectively. UV-vis absorption spectra were recorded on a SHIMADZU UV-3600 spectrophotometer. The double-stranded DNA (dsDNA) was obtained by annealing the mixtures of complementary strands in a buffer solution (10 mM tris-HCl, 100 mM NaCl, pH 8.5) at 2 °C below the melting temperature T_m for 20 min and then slowly cooled to room temperature. Photoluminescence (PL) and FRET measurements were carried out using a SHIMADZU RF-5301PC spectrofluorophotometer with a xenon lamp as a light source. For logic operations at different pH, the pH value of buffer solution was adjusted by additions of hydrogen chloride or sodium hydroxide.

Results and discussion

FRET experiments were carried out using PFEP as the light harvesting material and two intercalating dyes (TO and EB) as the other optical components. Their molecular structures are shown in Fig. 1a, and relevant absorption and photoluminescence (PL) spectra are given in Fig. 1b. Good overlap can be observed between the emission of PFEP and the absorption of each dye. Despite the good spectral overlap, FRET between CCP and EB intercalated into dsDNA showed poor efficiency due to a nonoptimal (orthogonal) orientation between the transition moment of the CCP backbone and that of the EB within the self-assembled CCP/dsDNA complex.^49,50 Hence, fluorescein appended to the 5′-terminus of the dsDNA has been used as a chromophore of intermediate energy to provide a resonance gate for FRET from CCP to EB in previous sensing assays. On the other hand, we noted the obvious spectral overlap between the emission of TO and the absorption of EB (Fig. 1b); moreover, a label-free sequence specific DNA detection with good sensitivity and single nucleotide polymorphism (SNP) selectivity has recently been realized by taking advantage of CCP sensitized TO emission.⁵¹ Therefore, it is reasonable to consider that TO may also be capable of providing a resonance gate for FRET from CCP to EB in the CCP/dsDNA/TO/EB assembly.

Fig. 1 a) Molecular structures of PFEP, TO and EB. b) Photoluminescence (PL) spectra of PFEP (solid line, λ_ex = 404 nm), TO (dashed line, λ_ex = 500 nm) and absorption spectra of TO (dotted line) and EB (dash dotted line) in water.

More importantly, TO exhibits some special properties in terms of its affinities for DNA; that is, it not only shows very high fluorescence enhancement upon dsDNA intercalation like other intercalating dyes, but also associates with G-rich ssDNA through hydrophobic interactions to generate strong fluorescence signals.³⁶ Furthermore, preferential FRET was observed in the CCP/G-rich ssDNA/TO assembly, relative to the CCP/dsDNA/TO assembly. This phenomenon was proposed to arise most likely because TO dyes associated with G-rich ssDNA would have more geometric freedom to conform to the CCP structure, as compared with those intercalated within the more rigid dsDNA.³⁸ Therefore, in respect that one C-rich ssDNA can be complementary to another G-rich ssDNA, we anticipated that it would be possible to detect the conformational conversion between i-motif and duplex state of C-rich ssDNA by using CCP/G-rich ssDNA/TO assembly, and that if EB was added to this assembly, multiple signals for logic expressions could be subsequently generated.

Thus, a C-rich ssDNA was chosen for this study with the 21-base sequence 5′-CCCTAACCCTAACCCTAACCC-3′ (ssDNA₁). The dsDNA was obtained by hybridization of the ssDNA₁ with its complementary strand, a G-rich ssDNA (ssDNA₂, 5′-GGGTTAGGGTTAGGGTTAGGG-3′). First, we obtained the FRET efficiencies of the PFEP/ssDNA₁/ssDNA₂/TO assembly at different pH values by monitoring the dye emission intensity upon excitation of PFEP. As shown in Fig. 2a, we observed a much higher FRET efficiency (about 3.6 times) at pH 5.0 than that at pH 8.5, implying that the two complementary strands might exhibit different state of binding at different pH values. In order to demonstrate the effect of DNA bases on the FRET efficiency, we also chose two other 21-base complementary strands, ssDNA₃ (5′-TTTTTTTTTTTTTTTTTTTTT-3′) and ssDNA₄ (5′-AAAAAAAAAAAAAAAAAAAAA-3′) to study the FRET efficiencies in the PFEP/ssDNA₃/ssDNA₄/TO assembly. In comparison to the results for the former assembly, the FRET efficiency at pH 5.0 was only a little bit higher than that at pH 8.5 (Fig. 2b), and both were approximate to the value obtained for the former assembly at pH 8.5. As the weak association for TO/poly(dA) and TO/poly(dT) has been previously documented,³⁶ it is believed that a dsDNA₍₃₊₄₎ was obtained by the hybridization of ssDNA₃ and ssDNA₄ at either pH 5.0 or pH 8.5, so that energy transfer could occur from the PFEP to the TO intercalated into dsDNA₍₃₊₄₎. Therefore, it is rational to propose the hybridization of ssDNA₁ and ssDNA₂ in the PFEP/ssDNA₁/ssDNA₂/TO (i.e., PFEP/dsDNA_(1 + 2)/TO) assembly at pH 8.5 due to its FRET efficiency similar to those obtained for the PFEP/dsDNA_(3 + 4)/TO assembly. Based on this point, we could safely reach a further conclusion that ssDNA₁ and ssDNA₂ were not hybridized at pH 5.0; specifically, the C-rich ssDNA₁ adopted an i-motif structure owing to the formation of C C⁺ base pairs through protonation while the G-rich ssDNA₂ appeared as a random-coil which associated strongly with TO.³⁸ Thus, the much higher FRET efficiency in the PFEP/ssDNA₁/ssDNA₂/TO assembly at pH 5.0 than that at pH 8.5 could be easily explained. That is to say, the conformational conversion between i-motif and duplex state of C-rich ssDNA could be simply detected by comparing the FRET efficiency in the PFEP/ssDNA₁/ssDNA₂/TO assembly at pH 5.0 with that at pH 8.5.

Fig. 2 The spectra were normalized with respect to PFEP emission. a) PL spectra of the solution containing ssDNA₁/ssDNA₂/TO in the presence of PFEP at three different pH, upon excitation at 404 nm. b) PL spectra of the solution containing ssDNA₃/ssDNA₄/TO in the presence of PFEP at three different pH, upon excitation at 404 nm. c) PL spectra of the solution containing ssDNA₂/TO in the absence of PFEP at two different pH, upon excitation at 500 nm. Conditions: [PFEP] = 2.5 × 10⁻⁹ M, [ssDNA] = 3 × 10⁻⁷ M, [TO] = 2 × 10⁻⁷ M, measurements were conducted in 10 mM tris- HCl/NaOH, 100 mM NaCl buffer.

Moreover, we also found another interesting phenomenon. When the pH value of the solution was changed to 12.0, no energy transfer was observed in either of the above two assemblies (Fig. 2a and 2b). It is well-known that dsDNA will denature and break down into ssDNAs at high pH values like pH 12.0,⁵² and previous studies in the literature generally revealed that the positive charges on the ammonium side groups of CCPs could be partially neutralized by OH⁻ (R₄N⁺ + OH⁻ ↔ R₄NOH),^39,53 which may attenuate the electrostatic attractions between PFEP and ssDNAs, and thereby decrease the FRET efficiencies in the assemblies. Additionally, we studied the TO emission in ssDNA₂/TO system at pH 8.5 and pH 12 upon direct excitation at the absorption maximum of TO. The results showed an extremely low TO emission at pH 12, which displayed a sharp decrease relative to that at pH 8.5 (Fig. 2c). Considering that TO showed a very low fluorescence intensity in the free state,³⁶ this phenomenon indicated that TO and G-rich ssDNA₂ was most likely associated at pH 8.5, but became separated at pH 12, presumably also due to the partial neutralization of positively charged TO by OH⁻. Hence, taking all the above points together, all the components in the PFEP/ssDNA₁/ssDNA₂/TO assembly or the PFEP/ssDNA₃/ssDNA₄/TO assembly were almost separated at pH 12, thereby cutting off the energy transfer from PFEP to TO.

A reversible conformational conversion between i-motif, duplex, and random-coiled state of C-rich ssDNA could thus be detected by simply comparing the FRET efficiency from PFEP to TO in the PFEP/ssDNA₁/ssDNA₂/TO assembly at different pH value of the buffer solution. On such a basis, we introduced another positively charged intercalating dye—EB into this assembly and expected to generate multiple signals for logic expressions. As shown in Fig. 3a, when adding EB to the solutions of PFEP/ssDNA₁/ssDNA₂/TO assembly at three different pH values, the fluorescence spectra obtained upon excitation at the absorption maximum of PFEP indicated evidently that the FRET from PFEP to TO and then to EB took place at pH 5.0 and 8.5 while no FRET occurred at pH 12; moreover, the FRET efficiency from TO to EB at pH 8.5 was observed to be much higher than that at pH 5.0. These phenomena could be rationally explained in terms of the properties of EB. Free EB also exhibits a low fluorescence intensity in buffer solution, but it shows an obvious enhancement in fluorescence intensity upon intercalating into dsDNA;^54,55 nevertheless, fluorescence enhancement is not evident when EB is mixed with ssDNA⁵⁶ or quadruplex DNA,⁵⁷ implying the weak association between EB and ssDNA⁵⁶ or quadruplex DNA. Therefore, as shown in Fig. 3b, at pH 8.5, EB and TO could both intercalate into dsDNA₍₁₊₂₎, resulting the close distance which allowed efficient FRET between them. However, the C-rich ssDNA₁ adopted an i-motif structure at pH 5.0, so that TO could associate strongly with the random-coiled G-rich ssDNA₂, while EB only associated weakly with ssDNA₂, likely through some electrostatic attractions, which was unfavorable for efficient FRET. Finally, all the components in the PFEP/ssDNA₁/ssDNA₂/TO/EB assembly were almost separated at pH 12, and therefore no FRET occurred at all.

Fig. 3 a) Fluorescence spectra of the following solutions: PFEP/ssDNA₁/ssDNA₂/TO/EB at three different pH values (pH 5, solid line; pH 8.5, dashed line; pH 12.0, dotted line), upon excitation at 404 nm. Conditions: [PFEP] = 2.5 × 10⁻⁸ M, [ssDNA₁] = [ssDNA₂] = 3 × 10⁻⁷ M, [TO] = 2 × 10⁻⁷ M, [EB] = 3 × 10⁻⁶ M. b) Schematic representation of the molecular basis of INH and NINH logic operations. c) Emission intensity of the output of INH or NINH logic gates at 450 nm (c-1), 530 nm (c-2), 590 nm (c-3) from the four input combinations. d) Combinatorial logic scheme.

Thus, this supramolecular PFEP/ssDNA₁/ssDNA₂/TO/EB system allowed us to simultaneously integrate two INH and one NINH logic gates operating in parallel. The two inputs were hydrogen chloride (I₁) and sodium hydroxide (I₂). This logic system could be switched back and forth by adding H⁺ and OH⁻. The four possible input combinations of H⁺ and OH⁻ ions ((0,0), (0,1), (1,0), (1,1)) were shown in Fig. 3b (we postulated that for both (0,0) and (1,1) states, the solutions were mildly alkaline, pH 8.5). When the output relied on the fluorescence intensity of PFEP (450nm, O₁), we obtained one INH logic gate. INH gates are AND gates with one of the inputs inverted through a NOT function.^58,59 The output of PFEP emission was a strong signal only in the presence of OH⁻ (pH 12) and in the absence of H⁺. This combination thus corresponded to the INH logic gate (Fig. 3c-1, d). Simultaneously, when monitoring TO emission at 530 nm (O₂), the fluorescence intensity was strong only in the presence of H⁺ (pH 5.0) and in the absence of OH⁻ (Fig. 3c-2, d), corresponding to another INH logic. By measuring the fluorescence intensity of EB at 590 nm (O₃), we could achieve an NINH logic. The NINH logic gate combines the NOT and INH operations. For (0,1) state, all the components in this supramolecular system were almost separated and no FRET occurred (Fig. 3b). For (0,0), (1,0), (1,1) states, the FRET from PFEP to TO and then to EB took place, resulting in obvious EB emission (Fig. 3c-3, d); specifically, as we discussed above, the FRET from PFEP to TO was stronger for (1,0) state than that for (0,0) and (1,1) states while the subsequent FRET from TO to EB was stronger for (0,0) and (1,1) states than that for (1,0) state (Fig. 3b). Hence, weak EB emission was present only for (0,1) state, corresponding to the NINH gate in the presence of H⁺ (pH 3).

The reversibility of this system was also testified by observing how the fluorescence intensity at different wavelengths changed with the multiple-cycling of pH values. Some of the data have been shown in Fig. 4. The fluorescence intensity at 590 nm was monitored with the pH change upon the addition of HCl or NaOH (Fig. 4a). It was clearly that slightly decreased intensity appeared after changing the pH value five times, whereas the intensity was still in its function as the same logic gate. The decreased intensity could be attributed to the dilution of the solution with the pH change.⁶⁰ Moreover, the changes of fluorescence intensity at 450 nm and 530 nm with the cycle of pH values presented results similar to those at 590 nm (Fig. 4b). All these data demonstrated that this logic system could be switched back and forth by additions of H⁺ and OH⁻. When the pH value of the system was changed, all the fluorescence intensity data could be obtained in one or two minutes, implying good speed of this logic system. Thus, with a single excitation frequency, different, reversible, logic-gate types could operate concurrently by emitting at different wavelengths; meanwhile, they could also be used to detect the conformational conversion between i-motif, duplex, and random-coiled state of C-rich ssDNA.

Fig. 4 a) Reversible cycling of fluorescence monitored at 590 nm while the pH value of the solution oscillated between 5.0 and 12.0. b) Reversible cycling of fluorescence monitored at 450 nm and 530 nm while the pH value of solution oscillated between 5.0 and 12.0. Conditions: [PFEP] = 2.5 × 10⁻⁸ M, [ssDNA₁] = [ssDNA₂] = 3 × 10⁻⁷ M, [TO] = 2 × 10⁻⁷ M, [EB] = 3 × 10⁻⁶ M.

Conclusions

In summary, a novel supramolecular system based on CCP/DNA/TO/EB assembly was designed as a two-step FRET sequence to enable multiple logic gates. With H⁺ and OH⁻ as inputs, reversible logic gates capable of INH and NINH could operate in parallel by simply observing the fluorescence variation behaviors of the CCP, TO, and EB. Hence, the present logic gates show the inherent advantages including easy operations, multifunctionality, and reversibility, over other DNA-based logic gates. This logic system also gives rise to a new pathway for the label-free detection of DNA i-motif structures by working in a simple “mix-and-detect” manner, which provides greater convenience and accuracy than previously reported approaches. More importantly, the findings of connections between the conformational conversion of DNA secondary structure, and the photophysics and supramolecular properties of water-soluble conjugated polymers and intercalating dyes constituted the basis of this logic system. Therefore, in view of the designed principles, this strategy is not limited to probing the formation of i-motif structures, it provides some inspiration for the investigation of other biomolecular conformational conversions upon environmental changes or binding to their targets. We expect that this work can promote exploration toward supramolecular logic systems that will be beneficial in future biochemical, information-processing and optoelectronic applications.

Acknowledgements

The authors gratefully acknowledge financial support provided by the National 973 Project (No. 2009CB930601), the National Natural Science Foundation of China (20804018, 51073078, 21005040), the Excellent Group of Scientific Research & Originality of Colleges in Jiangsu Province (No. TJ207035), and the Natural Science Basic Research Program for University of Jiangsu Province (10KJB150014).

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