Ya-Ping Gongab,
Jian Yangc,
Ji-Wang Fangac,
Qian Lib,
Zhi-Yong Yu*a,
Aijiao Guan*b and
Han-Yuan Gong*c
aDepartment of Chemistry, Renmin University of China, Beijing, 100872, P. R. China
bInstitute of Chemistry, Chinese Academy of Sciences, Zhongguancunbeiyijie 2, Beijing 100190, P. R. China
cCollege of Chemistry, Beijing Normal University, Xinjiekouwaidajie 19, Beijing 100875, P. R. China. E-mail: hanyuangong@bnu.edu.cn
First published on 22nd April 2021
DNA small molecular probe study was considered as a promising approach to achieve DNA related disease diagnosis. Most related reports were performed under specific salinity. Herein, 4-imino-3-(pyridin-2-yl)-4H-quinolizine-1-carbonitrile (IPQC) was generated via a facile procedure with high yield (85%). It is found that IPQC could act as a universal probe for most tested ssDNA, dsDNA and G4 DNA in low [K+] concentration (less than 20 mM). However, IPQC showed highly selective G4 DNA binding via UV-vis and fluorescence response in increasing [K+] (e.g., 150 mM) conditions. The ion atmosphere effects are instructive for DNA probe exploration. This provides guidance for the design, selection and optimization of the probes for target DNA sensing.
Herein, we reported an unusual DNA small molecular probe, 4-imino-3-(pyridin-2-yl)-4H-quinolizine-1-carbonitrile (IPQC) with high K+ concentration dependent selectivity. In low [K+] environment (i.e., [K+] ≤ 20 mM), IPQC shows obvious UV-vis and/or fluorescence responses for most additional DNA species. The tested DNAs include ssDNA (e.g., ssVEGF, ssPS1c-a, ssPS1c-b, ssAf17, ssG-tripl and ssT30), dsDNA (including dsPS1c, ds19AT, dsDx12, ds20, ds22, ds26 and 15GC), and G4 DNA (involving VEGF, bcl-2, H22, P21, c-myc and c-kit). Surprisingly, increasing [K+] improved the DNA recognition selectivity of IPQC. 150 mM [K+] (an intracellular K+ concentration) induces IPQC response only for some additional G4 (Scheme 1). Such findings suggested that ion atmosphere effects (e.g., [K+]) in DNA detection should not be ignored in either exploring new or re-examining previous probes. The ion atmosphere effect study in the DNA detection will provide guidance for the design, selection and optimization of target DNA post-probes.
Scheme 1 The binding selectivity of previous DNA probe (left, in specific salinity (i.e., [K+])) and IPQC probe (right) with increasing salinity (e.g., [K+]) promoted selectivity for G4 DNA sensing. |
Fluorescence spectra were recorded on a HITACHI F-4600 fluorescence spectrophotometer (Hitachi Limited, Japan). Fluorescence titration experiments were carried out with the same samples used in the UV-vis titration study. Fluorescence spectroscopic Job-plot was used to determine the binding stoichiometry. The total concentration of IPQC and DNAs were maintained as 2.00 × 10−5 M, with the molar ratio between IPQC and DNAs as 1:0, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 and 1:5, respectively.
CD (circular dichroism) spectra were collected on a Jasco 815 spectropolarimeter in a 2 mm path-length quartz cell. CD spectra of DNAs (2.00 × 10−5 M) were measured in the absence or presence of 1.0 molar equiv. of IPQC. 1H-NMR and 13C-NMR spectra were recorded on Bruker AVANCE 400. The single crystals used to obtain the X-ray diffraction structure grew clear light red cube. The .cif documents are available as separate ESI† files, which provide details regarding the specific crystal used for the analysis, along with the structure in question. Diffraction grade crystals were obtained via slow evaporation from solution using a mixture of dichloromethane/n-hexane.
The UV-vis and fluorescence spectroscopic properties of IPQC were characterized. The related experiments were carried out in ethanol–Tris–HCl buffer. It is noted that 1% ethanol was added due to the limited solubility of IPQC in pure water phase. Finally, IPQC reveals a peak at λ = 392 nm with ε392 nm = 2.57 × 104 mol−1 L cm−1 in UV-vis absorption. Fluorescence spectrum recorded at λex = 392 nm showed a strong green emission peak of 528 nm, with fluorescence quantum yield Φ = 0.35 and lifetime as 4.84 ns.
Cationic H+·IPQC was believed as the form of IPQC in aqueous phase under pH around 7 (note: the pKb value of IPQC is deduced as around 9.15 from the value of analogic quinoline). The pH dependent fluorescence spectra of 2.00 × 10−5 M IPQC show obvious change with pH value scale as 2 to 12 (cf. ESI Fig. S68 and S69†). It suggests that H+·IPQC is the main form with pH around 7. The planar skeleton and protonation induce the expectation to use IPQC as a potential biological probe of anionic biological macromolecules (e.g., nucleic acids), as the well-known G-quartet ligand, berberine.36–38
As initial work, we investigated the interaction between IPQC and a series of DNA structures (cf., Table S1†). G-quadruplex VEGF (Vascular Endothelial Growth Factor, a cancer treatment target)39–41 was selected for the first test. UV-vis absorption peak of IPQC at λ = 392 nm red-shifts 19 nm with adding 4.0 molar equiv. of G4-VEGF. Meanwhile, obvious intensity decreasing was shown in fluorescence emission spectrum (carried out in ethanol–Tris–HCl buffer with [KCl] as 20 mM). The decreasing fluorescence intensity attributed mainly to the H+·IPQC binding on the surface of G4-VEGF via π–π stacking interaction via molecular simulation (cf. Fig. 4a–c). Other synergistic effects, such as electrostatic attraction between negative DNA and positive H+·IPQC, suitable shape and volume of H+·IPQC to the groove, also may lead to the fluorescence quenching of H+·IPQC.
Further CD (circular dichroism) experiments showed DNA signals are almost the same before and after adding IPQC, while IPQC has no Cotton effect in the presence or absence of DNA species (cf. ESI Fig. S73†). This phenomenon indicates DNA configuration is kept during the binding process. The stoichiometric ratio between IPQC and DNA were speculated by Job-plot analysis, despite the method is limited in some complicated cases.42–44 Fluorescence Job-plot analysis resulted in a minimum value at [IPQC]/([IPQC] + [G4-VEGF]) ratio as 0.5. The finding suggested a 1:1 binding stoichiometry (Fig. 2). Further UV-vis titration experiment suggested the association constant Ka1 = (1.3 ± 0.1) × 105 M−1 for the 1:1 complexation. The result is consistent with calculated Ka1 = (7.9 ± 0.2) × 104 M−1 from fluorescence titration.
Same methods were also used to study the interactions between IPQC and other tested DNAs. The association constants were summarized in Table 1 and Fig. 3, which exhibit both obvious UV-vis and fluorescence signal response in most cases involving ssDNA, dsDNA, and G-quadruples.
Substrate (S) | [IPQC]:[S] | Ka measurement in [K+] as 0 or 20 mM | Ka measurement in [K+] as 150 mM | ||
---|---|---|---|---|---|
Method | Method | ||||
UV-vis | Fluorescence | UV-vis | Fluorescence | ||
a (1) Equations governing the relevant equilibria: here H indicated IPQC and G for various DNA substrates. (2) ‘—’ indicates titration experiments are unsuccessful because of the weak response of IPQC to additional DNAs. (3) All the tests were carried out in buffer ([Tris] = 20 mM; the ratio between ethanol and aqueous phase as 1:99, v/v, pH = 7.2). 20 or 150 mM KCl was used in G4 DNA tests, 0 or 150 mM KCl was used in the cases of ss/ds DNAs. | |||||
G4-bcl-2 | 1:1 | (5.0 ± 0.2) × 105 | (2.5 ± 0.1) × 105 | (1.6 ± 0.1) × 105 | (2.5 ± 0.1) × 105 |
1:2 | (7.9 ± 0.4) × 104 | (1.3 ± 0.1) × 105 | (5.0 ± 0.3) × 104 | (2.5 ± 0.1) × 104 | |
2:3 | (3.2 ± 0.3) × 104 | (2.0 ± 0.2) × 103 | (1.3 ± 0.1) × 104 | (4.0 ± 0.3) × 103 | |
G4-H22 | 1:1 | (7.9 ± 0.2) × 104 | (1.0 ± 0.1) × 105 | (2.0 ± 0.1) × 104 | (1.3 ± 0.1) × 104 |
1:2 | (6.3 ± 0.3) × 104 | (2.5 ± 0.1) × 104 | — | — | |
2:3 | (2.0 ± 0.2) × 105 | (7.9 ± 0.6) × 104 | — | — | |
G4-P21 | 1:1 | (7.9 ± 0.2) × 104 | (7.9 ± 0.2) × 104 | (1.3 ± 0.1) × 104 | (1.0 ± 0.1) × 104 |
1:2 | (1.3 ± 0.1) × 105 | (1.3 ± 0.1) × 105 | — | — | |
2:3 | (2.5 ± 0.2) × 104 | (2.5 ± 0.2) × 104 | — | — | |
G4-VEGF | 1:1 | (1.3 ± 0.1) × 105 | (7.9 ± 0.2) × 104 | (2.5 ± 0.1) × 104 | (2.0 ± 0.1) × 104 |
G4-c-kit | 1:1 | (5.0 ± 0.2) × 105 | (3.2 ± 0.1) × 105 | (2.0 ± 0.1) × 105 | (1.3 ± 0.1) × 105 |
1:2 | (1.0 ± 0.1) × 104 | (7.9 ± 0.4) × 103 | (1.6 ± 0.1) × 104 | (1.6 ± 0.1) × 104 | |
2:3 | (2.5 ± 0.2) × 103 | (5.0 ± 0.4) × 103 | (4.0 ± 0.3) × 104 | (4.0 ± 0.3) × 103 | |
G4-c-myc | 1:1 | (6.3 ± 0.2) × 105 | (7.9 ± 0.2) × 105 | (1.6 ± 0.1) × 105 | (5.0 ± 0.2) × 104 |
1:2 | (1.0 ± 0.1) × 105 | (3.2 ± 0.2) × 104 | (6.3 ± 0.3) × 104 | (7.9 ± 0.4) × 104 | |
2:3 | (5.0 ± 0.4) × 104 | (2.0 ± 0.2) × 105 | (1.0 ± 0.1) × 105 | (5.0 ± 0.4) × 104 | |
ds20 | 1:1 | (1.6 ± 0.1) × 104 | (2.0 ± 0.1) × 104 | — | — |
ds22 | 1:1 | (2.0 ± 0.1) × 105 | (2.0 ± 0.1) × 105 | — | — |
1:2 | (1.6 ± 0.1) × 104 | (1.3 ± 0.1) × 104 | |||
2:3 | (1.3 ± 0.1) × 106 | (3.2 ± 0.3) × 105 | |||
ds26 | 1:1 | (5.0 ± 0.2) × 104 | (8.0 ± 0.2) × 104 | — | — |
1:2 | (1.0 ± 0.1) × 104 | (6.3 ± 0.3) × 103 | |||
2:3 | (7.9 ± 0.6) × 104 | (1.6 ± 0.1) × 104 | |||
ds19AT | 1:1 | (3.2 ± 0.1) × 104 | (3.2 ± 0.1) × 104 | — | — |
dsDx12 | 1:1 | (7.9 ± 0.2) × 104 | (6.3 ± 0.2) × 104 | — | — |
hairpin15GC | 1:1 | — | (5.0 ± 0.2) × 104 | — | — |
ssVEGF | 1:1 | (5.0 ± 0.2) × 104 | (6.3 ± 0.2) × 104 | — | — |
ssPS1c-a | 1:1 | — | — | — | — |
ssAf17 | 1:1 | — | — | — | — |
ssG-tripl | 1:1 | — | — | — | — |
dsPS1c | — | — | — | — | — |
ssPS1c-b | — | — | — | — | — |
ssT30 | — | — | — | — | — |
For further in vivo and/or in situ detection, we studied the DNA sensing behaviour of IPQC in simulated physiological environment. Specifically, the interaction between IPQC and former tested DNAs were explored in ethanol–Tris–HCl buffer with 150 mM K+ (an intracellular K+ concentration). Surprisingly, IPQC exhibits both UV-vis and fluorescence response only for G-quadruplex in this condition, while the responses for all the tested ss/ds DNA disappeared. Specifically, the association constant value Ka1 = (2.0 ± 0.1) × 104 M−1 between IPQC and G4-VEGF indicates that excess potassium do not strongly weaken combination between IPQC and G4-VEGF. As Fig. 3 shows, although the affinity between other G-quadruplex and probe IPQC may be slightly weakened with increasing [K+], they all show similar spectral response. The Ka1 value of the 1:1 binding calculated by UV-vis and fluorescence titration has no significant change compared with which in low [K+] system.
20 mM magnesium ions and 10 mM sodium ions were added to ethanol–Tris–HCl buffer with 150 mM K+ to further mimic the intracellular environment (cf. ESI Fig. S74–S79†). The final spectral responses (especially in fluorescence spectrum) are observed as the same as before adding. It implied that the high selectivity of IPQC for G4 remains after adding 20 mM MgCl2 and 10 mM NaCl. The result proves high [K+] (150 mM) promoted high selectivity of IPQC for G-quadruplex binding.
To further explore the increasing selectivity of IPQC, K+ concentrate dependent effect between IPQC and dsDNA was studied in detail via UV-vis and fluorescence methods. Additional dsDNA induced spectral response of IPQC was carried out. The spectroscopic changes decline with adding [K+], until completely disappear with [K+] as 500 mM (cf. Fig. S82–S84†). This experiment interpreted that intermolecular interactions between dsDNA and probe IPQC are acutely sensitive to ion atmosphere effects. To deeper insight of the phenomena, molecular modelling was carried out. It revealed that IPQC can stack on the surface of the terminal quartet of G4 DNA (Fig. 4) or insert into the groove of ss/dsDNA structure in low [K+]. It is suggested that excess cations can compete with protonated IPQC to bind DNA (cf. ESI Fig. S70 and S71†). Furthermore, K+ bind with polyanionic DNA helix to reduce electrostatic repulsion of the negative charges on DNA phosphate backbone. It is unfavourable for IPQC to intercalate to the groove binding sites in ss/dsDNAs. As the result, the binding between IPQC and ss/dsDNA weakens or even disappears with increasing K+. Differently, in the cases of G4 DNAs, the presence of metal cation (e.g., Li+, Na+ or K+) benefit to form a metal-ion-stabilized G4 structures (although Li+ provide inefficient induction for G4 structure formation45–47) so that the electron-rich quinoline plane of IPQC can effectively sit on the planar surface of the G-quadruplex via π–π stacking (cf. ESI Fig. S80 and S81†). Finally, the electrostatic completion of K+ and the enhancement of ion atmosphere effect shown above led a net result as little influence on the binding process between G4 and IPQC sitting on the G-quadruplex. However, the other G4 binding mode(s) of IPQC will also be weaken as the cases of ss/ds DNAs. Actually, the binding stoichiometries between IPQC and some G4 DNAs (e.g., G4-H22 or G4-P21) change from 2:3 to 1:1 corresponding to [K+] as 20 or 150 mM, respectively (cf. ESI Fig. S67†).
Footnote |
† Electronic supplementary information (ESI) available: UV-vis, fluorescence, NMR, HRMS and X-ray single crystal diffraction data. CCDC 1977107. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0ra06274j |
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