Chelerythrine as a fluorescent light-up ligand for an i-motif DNA structure

Abstract

A fluorescent light-up ligand for an i-motif structure has been reported in this study. Chelerythrine (CHE) could bind to i-motif and show a significant increase in the fluorescence intensity, while exhibiting weak response towards single-stranded (ssDNA), double-stranded (dsDNA), and G-quadruplexes. This feature makes CHE advantageous in the label-free fluorescence sensing systems.

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The excellent structural and functional features of DNA have attracted extensive research attention in biological processes, developing novel sensing systems, including various functional nanomaterials and molecular computers.¹ The development of DNA structure-sensitive fluorescent probes has received considerable attention due to their numerous applications.² The fluorescent ligands exhibit low fluorescence intensity when free in the solution but are highly fluorescent upon binding with specific DNA structures.² In the recognition process, DNA could self-assemble into single-stranded (ssDNA), double-stranded (dsDNA), hairpins, cruciforms, i-motifs, G-quadruplexes, and other forms.³ Therefore, the fluorescence change of the ligands could be in response to the DNA structural transition.

i-Motif is a four-stranded nucleic acid secondary structure comprising of cytosine-rich sequences, which are formed by two parallel duplex hydrogen bonded together by intercalated C⁺–C base pairs.⁴ i-Motif formation is promoted by a slightly acidic pH, although some could be formed even under neutral conditions.⁵ i-Motif DNA structures exist in the regions of human genome including promoters and telomeric regions.⁶ An antibody fragment (iMab), with high selectivity and affinity towards i-motif, was used in the recognition of i-motif DNA structures in vivo.⁷ Numerous recent studies suggest the important roles of i-motif in gene regulation.⁸ Besides the significant roles in the biological systems, i-motifs have been utilized to construct various nanodevices.⁹ The development of new fluorescent ligands towards i-motifs is significant for research on the functions and applications of i-motifs. So far, fluorescent ligands towards the i-motifs structure is limited, such as crystal violet,¹⁰ berberine,¹¹ thioflavin T,¹² thiazole orange,¹³ quinaldine red,¹⁴ neutral red¹⁵ and DMSB.^9a,b However, most of them are easily disturbed by ssDNA, dsDNA and G-quadruplexes. Therefore, developing i-motif fluorescent ligands is an exigent need.

Natural isoquinoline alkaloids (IQAs) are produced mostly by higher plants during metabolism.¹⁶ It is proved that IQAs possess numerous important therapeutic activities including antimicrobial, anti-inflammatory, antioxidation, and anticancer properties,¹⁷ which are speculated to play their roles by binding with nucleic acids.¹⁸ Since IQAs are almost low fluorescent and share similar molecular weight and structural frame,¹⁹ screening an aptamer that can selectively target i-motif is very important to expand the application of IQAs. In a previous study, Shao et al. found that chelerythrine (CHE), which is a stabilizer and inducer to the triplex, can sense triplex via fluorescence.²⁰ However, there is a lack of investigation on the recognition of the i-motif structure by CHE. In this study, we selected CHE from several IQAs as a promising i-motif fluorophore. CHE could bind to i-motif and show a significant increase in the fluorescence intensity, while exhibited weak response towards other DNA structures including ssDNA, dsDNA and G-quadruplexes. Our study developed a new ligand for i-motif, which has promising applications in the construction of nanodevices (such as biosensors and logic gates) in systems without triplex.

Scheme 1 shows the structure of CHE and the principle of recognization of the i-motif structure by CHE. CHE exhibits weak fluorescence intensity in buffer solutions. We tested CHE with various DNA structures including ssDNA, dsDNA, i-motif and G-quadruplex. Among these DNA structures, CHE interacts with i-motif alone and exhibits increasing fluorescence intensity, while it has no response towards other DNA structures. This feature allows CHE as a fluorescent light-up ligand to i-motif.

Scheme 1 The structure of CHE and the schematic of the recognition of CHE to i-motif.

We first screened i-motif as the promising ligand from several IQAs. Natural IQAs exhibit weak fluorescence signal in free solutions. Chelerythrine (CHE), coptisine (COP), jatrorrhizine (JAT), sanguinarine (SAN) and stephania (STE) were investigated. We added dsDNA, G-quadruplex and the i-motif structure into IQAs. Among these IQAs, only the fluorescence intensity of CHE has an obvious increase after binding with i-motif and has weak increase towards other DNA structures, implying that CHE could recognize the i-motif structure (Fig. S1 and S2, ESI†). COP exhibits increasing fluorescent intensity towards i-motif, but respond similarly to dsDNA and G-quadruplex. This result indicates that CHE is expected to be a promising fluorescent light-up ligand to i-motif.

Before exploring the recognition of i-motif by CHE, the influence of pH on CHE was measured. There was no significant difference in the CHE fluorescence when CHE was placed in different pH, indicating that the CHE fluorescence is less sensitive towards pH (Fig. S3, ESI†). The interaction of CHE to various DNA structures was investigated by titration experiments. With the gradual addition of i-motifs, the fluorescence intensity of CHE increased sharply with the fluorescence peaks blue shifting from 566 nm to 558 nm, corresponding to the compact binding of CHE with i-motif (Fig. 1A and Fig. S4, ESI†). The fluorescence intensity of CHE exhibits weak variation for the titration of other nucleic acid models, implying that the combination of CHE with i-motifs is much stronger than that with other nucleic acid structures (Fig. S4, ESI†). The plots of the fluorescence intensity of CHE at 558 nm versus the increasing concentration of various DNA structures were measured (Fig. 1B). The CHE fluorescence intensity at 558 nm increases with the addition of the i-motif structure including AS1411-C,^9a,b H22-C,^13,14 H24-C, ILPR,²¹ Rb²² and c-myc-C²³ sequences. However, the CHE fluorescence intensity at 558 nm has weak fluctuation with increasing G-quadruplex (AS1411, c-myc,²⁴ VEGF,²⁵ Bcl-2²⁶ and c-kit²⁷) and dsDNA (ds20, ds22 and ds26). As shown in Fig. 2, the comparison of CHE fluorescence intensity (F/F₀) monitored at 558 nm demonstrated that i-motif enhanced the CHE fluorescence by 5–14 folds than in the absence of i-motif. In contrast, the fluorescence enhancement of CHE by ssDNA, dsDNA and G-quadruplexes was less than 2-fold (Fig. 2). While binding with the i-motif structure, the movement of CHE was inhibited and the loss of energy decreased, leading to the increase in the fluorescence intensity. In order to validate this viewpoint, we measured the fluorescence intensity of CHE with increasing viscosity in the glycerol–water system (Fig. S5, ESI†). With the increase in viscosity, the fluorescence intensity of CHE increased, which was caused by the inhibition of the movement of CHE and the loss of energy decreased in a high viscosity environment. By a Job's Plot analysis (Fig. S6, ESI†), we know that the binding stoichiometry (n) of numerous i-motif structures withCHE is 1 : 1, indicating that CHE may only bind to one i-motif molecule. Further, we computed the apparent binding equilibrium constants (K_a) of CHE to i-motifs utilizing the fluorimetric titration results. The binding stoichiometry and apparent binding equilibrium constants are listed in Table S2 (ESI†).

Fig. 1 (A) Fluorescence spectra of CHE (5 μM) with the increase in concentrations of the H22-C i-motif. (B) The plots of the fluorescence intensity of CHE at 558 nm versus the increasing concentration of numerous DNA structures. i-Motif samples were measured in a PB buffer solution (10 mM, pH 5.8), other samples were measured in a PBS buffer solution (10 mM, pH 7.2).

Fig. 2 Dependence of the CHE (5 μM) fluorescence intensity at 558 nm on various nucleic acid samples (20 μM). i-Motif samples were measured in PB buffer solution (10 mM, pH 5.8), other samples were measured in PBS buffer solution (10 mM, pH 7.2).

C-rich oligonucleotides can form the i-motif structure in an acidic environment.⁵ To validate the conformation transition of C-rich DNA in this process, we recorded their circular dichroism (CD) spectra in the pH range of 5.8–7.2 (Fig. S7, ESI†). The CD spectra of AS1411-C in pH 5.8 show a positive peak at 285 nm and a negative peak at 253 nm, indicating the characteristic signal of the i-motif structures.²⁸ When pH changes to 6.4, the CD spectra of AS1411-C show a positive peak at 277 nm and a negative peak at 242 nm, indicating that the i-motif structure turn into ssDNA. The changing rule of the CD spectra of c-myc-C and H22-C was similar to AS1411-C, indicating the formation of c-myc-C and H22-C change from i-motif to ssDNA in the pH range of 5.8–7.2. These results illustrate that these C-rich oligonucleotides can form the i-motif structure at pH 5.8.

Furthermore, we measured the fluorescence spectra of CHE in numerous pH values in the presence of AS1411-C (Fig. 3, Fig. S8A and B, ESI†). With pH changing from 5.0 to 7.5, the fluorescence intensity of CHE at 588 nm decreased, indicating that the formation of AS1411-C DNA changed from i-motif to ssDNA and CHE released from AS1411-C. Besides, we measured the fluorescence spectra of CHE with the increasing concentration of Ag⁺ in the presence of AS1411-C (Fig. 3, Fig. S8C and D, ESI†). It was found that i-motif can form with Ag⁺ present at neutral pH owing to the strong interaction of Ag⁺ with cytosine to form a C–Ag⁺–C complex.^9a,b,29 With the addition of Ag⁺, the fluorescence intensity of CHE at 588 nm increased obviously, indicating AS1411-C ssDNA form into i-motif under Ag⁺ solution and CHE binds to the AS1411-C i-motif specially. These results further manifest the selectivity of CHE towards i-motif rather than ssDNA. In order to validate the formation of AS1411-C i-motif, we compared the response of CHE to AS1411-C and ds12 (a specific double helical structure for the whole length of the sequence). The fluorescence intensity of CHE towards AS1411-C increased sharply while exhibiting weak change in the presence of ds12 (Fig. S9A, ESI†). The plot of the fluorescence intensity at 558 nm with increasing sample showed the difference of CHE to AS1411-C and ds12 (Fig. S9B, ESI†). This experiment illustrated that the CHE can bind with the i-motif structure rather than the double helical structure.

Fig. 3 The plot of the fluorescence intensity of CHE (5 μM) at 558 nm with increasing concentrations of Ag⁺ (blank) and versus pH (red) in the presence of 5 μM AS1411-C in the 10 mM PB solution.

The influence of CHE towards i-motif thermostability was discussed via the melting temperature experiment. In pH 5.8, H22-C DNA exhibits the characteristic signal of i-motif structures (a positive peak at 290 nm and a negative peak at 255 nm). With the increase in temperature, the CD signal of H22-C at 290 nm decreased and the melting temperature (T_m) is around 41 °C. After the addition of CHE, the CD signal of H22-C at 290 nm decreased and T_m was also around 41 °C, indicating that CHE has no influence on the thermostability of the H22-C structure (Fig. S10, ESI†). Moreover, in the melting fluorescence spectra, the fluorescence intensity of the CHE-H22-C complex at 558 nm decreased with the increase in temperature, indicating that the H22-C i-motif structure was destructed and CHE was released from H22-C. T_m in the melting fluorescence spectra is around 40 °C, which is consistent with that in the CD-melting experiment (Fig. S11, ESI†). The results of the melting temperature experiment exhibit that CHE has no influence on the i-motif thermostability.

In summary, we have roundly investigated the interaction of CHE with numerous DNA structures. CHE could bind to i-motif and show a significant increase in the fluorescence intensity. In contrast, CHE exhibits low fluorescence response towards ssDNA, dsDNA and G-quadruplex. We expect that our results could make a new way for research on the structures and functions of i-motif with the advantages of simplicity, label-free detection, low cost and sensitivity for future studies.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was supported by Beijing Nova Program (No. Z201100006820048), Beijing Natural Science Foundation (7182189), the Distinguished Young Scholars Fundation of China Academy of Apace Technology and the National Natural Science Foundation of China (Grant No. 21977096, 21977099, 21675162 and 21778058).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0nj04863a

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