Beidou
Feng
a,
Kui
Wang
a,
Yonggang
Yang
a,
Ge
Wang
b,
Hua
Zhang
*a,
Yufang
Liu
a and
Kai
Jiang
a
aHenan Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Key Laboratory of Green Chemical Media and Reactions; Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Henan Key Laboratory of Organic Functional Molecules and Drug Innovation, School of Chemistry and Chemical Engineering, School of Environment, College of Physics and Materials Science, Henan Normal University, Xinxiang 453007, China. E-mail: zhanghua1106@163.com
bXinxiang Medical University, Xinxiang 453000, P. R. China
First published on 20th September 2019
The AP site is a primary form of DNA damage. Its presence alters the genetic structure and eventually causes malignant diseases. AP sites generally present a high-speed dynamic change in the DNA sequence. Thus, precisely recognizing AP sites is difficult, especially at the single-cell level. To address this issue, we provide a broad-spectrum strategy to design a group of molecular rotors, that is, a series of nonfluorescent 2-(4-vinylbenzylidene)malononitrile derivatives (BMN-Fluors), which constantly display molecular rotation in a free state. Interestingly, after activating the relevant specific-recognition reaction (i.e., hydrolysis reaction of benzylidenemalononitrile) only in the AP-site cavity within a short time (approximately 300 s), each of these molecules can be fixed into this cavity and can sequentially self-regulate to form different stable conformations in accordance with the cavity size. The different stable conformations possess various HOMO–LUMO energy gaps in their excited state. This condition enables the AP site to emit different fluorescence signals at various wavelengths. Given the different self-regulation abilities of the conformations, the series of molecules, BMN-Fluors, can emit different types of signals, including an “OFF–ON” single-channel signal, a “ratio” double-channel signal, and even a precise multichannel signal. Among the BMN-Fluors derivatives, d1-BMN can sequentially self-regulate to form five stable conformations, thereby resulting in the emission of a five-channel signal for different AP sites in situ. Thus, d1-BMN can be used as a probe to ultrasensitively recognize the AP site with precise fluorescent signals at the single-cell level. This design strategy can be generalized to develop additional single-channel to multichannel signal probes to recognize other specific sites in DNA sequences in living organisms.
AP sites possess a special structure and reactivities that can spontaneously induce some chemical reactions in living biological systems.9,10 Thus, developing active substrate molecules that can take part in the relevant chemical reactions induced by the AP site is immensely conducive for such site-specific recognition in a DNA sequence.11,12 To date, combined with nuclear magnetic resonance (NMR) spectrometry,13,14 mass spectrometry15,16 and optical techniques,17–20 some substrate molecules have been successfully implemented for quantifying AP sites in DNA.13–20 NMR spectrometry for detecting AP sites is characterized by highly selective signals. However, the sensitivity of signals is low.13,14 Other substrate molecules, under the condition of mass spectrometry, can emit a highly sensitive mass spectrometry signal but cannot in situ detect the AP sites in living cells.15,16 Still other substrate molecules with optical signals, such as UV-visible and fluorescence signals, only exhibit a single optical signal change at one wavelength.17–20 Thus, the stability of the recognition signal (especially fluorescence recognition signal) for the AP sites is relatively poor in living cells. In addition, inherent interference from complex living organisms (e.g., pH, hydrophilicity and hydrophobicity) for the stability of the recognition signal becomes difficult to eliminate. Furthermore, the response times are longer in these reported substrate molecules for AP sites than the formation time of the AP site. Thus, precisely detecting AP sites that appear with rapid and dynamic real-time change is extremely difficult in living cells, especially at the single-cell level. Moreover, these substrate molecules cannot recognize the differences in cavity size of the AP site and thus cannot provide feedback on the degree of DNA damage in real time. Therefore, the current major challenge is to enhance the sensitivity and fidelity of recognition for AP sites, especially for differentiating the cavity sizes of AP sites at the single-cell level.21–23
To enhance the recognition sensitivity and fidelity, inducing molecules to specifically generate a multichannel signal is an effective solution.24–26 Therefore, we constructed a series of 2-(4-vinylbenzylidene)malononitrile derivatives (BMN-Fluors), because BMN derivatives are typically chosen as the basic dye parent structure in the synthesis of chemosensors for nucleic acids. In addition, their spectra can be easily adjusted and are diverse.27,28 This series of BMN-Fluors, which are in constant rotation in a free state, can sequentially self-regulate their conformations to form different stable conformations when they encounter the AP site. BMN-Fluors can emit different types of signals, including an “OFF–ON” single-channel signal, a “ratio” double-channel signal, and even a precise multichannel signal. BMN-Fluors can ultrasensitively recognize AP sites with a high-fidelity multichannel signal at the single-cell level. This broad-spectrum molecular design strategy can be generalized to design additional probes, from single-channel probes to multichannel probes, to recognize specific sites in a DNA sequence in living organisms.
d1-BMN in all the molecules (BMN-Fluors) is a typical multichannel signal-emitting molecule used to explain the recognition behavior and signal for AP sites in PBS buffer. In the absence of AP sites, the experiment results showed that d1-BMN (3.0 μM, λex = 400 nm, ε = 1.5 × 105 M−1 cm−1) emits an extremely low signal (Fig. 2a, ΦFreeF = 0.021) at its maximum emission wavelength of 605 nm in PBS buffer. d1-BMN exhibited a unique spectral change when it encountered different AP sites in the DNA. Within the first few seconds of this process, d1-BMN immediately emitted a strong signal at 605 nm, and its fluorescence quantum yield increased to 0.66 (31.4-fold, Table S1† and Fig. 2a). However, fluorescence signal intensity at 605 nm decreased after approximately 300 seconds (Fig. 2b). In addition, a new relevant signal was emitted at different wavelengths when d1-BMN encountered various amounts of AP sites. Fig. 2a shows that new signals appeared, in turn, at 414 nm (Φ = 0.93), 441 nm (Φ = 0.88), 468 nm (Φ = 0.84), 498 nm (Φ = 0.80), 524 nm (Φ = 0.78) and 552 nm (Φ = 0.71), excited at 354 nm (Table S1†), when the number of AP sites were 2, 4, 6, 8, 10 and 14 in double-stranded DNA segments (20 bp), respectively. Their signal intensities decreased in sequence. In other words, d1-BMN exhibited a “multichannel” recognition signal for the different amounts of AP sites. During the abovementioned processes, the recognition signal changes can be observed in the solution by the naked eye (Fig. 2c). However, other BMN-Fluors, such as a2-BMN and d2-BMN, only exhibited a single signal change at one wavelength, that is, an OFF–ON single-channel signal for the AP site (Fig. S1 and Table S1†). Moreover, a1-BMN, b-BMN, c-BMN, and e-BMN displayed a ratio double-channel signal for the AP site (Fig. S1 and Table S1†). Nevertheless, all BMN-Fluors derivatives only showed the relevant types of fluorescence signal changes for AP sites in DNA, even when they encounter other biomacromolecules with aldehyde groups (e.g., 5-formyluracil or 8-OHDG in DNA) and living cell substances in the testing system (Fig. S2†); BMN-Fluors derivatives cannot emit such ratio double-channel and multichannel recognition signal.
Fig. 2 (a) Spectral data of d1-BMN (3.0 μM) for AP sites (2, 4, 6, 8, 10 and 14 AP sites/20 bp DNA, according to the given DNA sequence) in PBS buffer (pH = 7.4) at different reaction times (0, few seconds and 300 s). All DNA sequences with AP sites were synthesized by Thermo Fisher Scientific; the DNA sequence information is listed in the Procedures section of ESI.† Excitation wavelength = 400 nm and 354 nm. (b) The reaction time of d1-BMN (3.0 μM) for AP sites (14 AP sites/20 bp DNA) in PBS buffer (pH = 7.4). (c) d1-BMN emitting multicolor fluorescence, visible to the naked eye, for different contents of AP sites (6, 8, 10 and 14 AP sites/20 bp DNA) in PBS buffer (pH = 7.4) at different reaction times (0, few seconds and 300 s).23 The number of AP sites in the DNA sequence was quantitatively detected using an AP-site counting kit (Dojindo, Japan, see ESI†). |
Gaussian 16 simulation (ESI-2.avi†) indicated that the molecular skeleton of the serial derivatives BMN-Fluors are constantly in rotation under unconstrained environments. In addition, their molecular excited-state energy is nearly lost by nonradiative transitions. This phenomenon resulted in an extremely low fluorescence (Fig. 2a, S1 and Table S1†) and is the basic reason for the OFF–ON single-channel signal.32,33
Fig. 3a demonstrates the –ONH2 group of d1-BMN initially reacted with the –CHO group of HDM when it encountered an AP site, forming the new intermediate product d1-BMN-NO through aldehyde–ammonia condensation reaction. On the basis of this kind of structural transformation, d1-BMN can be bound to a biomolecule possessing the –CHO groups, such as an AP site and 5-formyluracil. This binding partly limits the molecular rotation, which is the main reason that all BMN-Fluors derivatives, including d1-BMN, emit enhanced red fluorescence for the AP site and 5-formyluracil (Fig. S2d†). Furthermore, 1H NMR spectra (Fig. 3b) showed that d1-BMN-NO subsequently generates the peculiar structure changes only in a simulation environment similar to the AP-site cavity, but not to 5-formyluracil. First, the –ph–CHC(CN)2 group of d1-BMN-NO reacts to generate a –ph–CH(OH)–CH(CN)2 group and form another new intermediate product d1-BMN-ts (Fig. 3c). d1-BMN-ts generation is the key point that effectively accelerates the rate-limiting step of the entire recognition reaction, that is, the benzylidenemalononitrile hydrolysis reaction.21–23 With this kind of acceleration, the –ph–CH(OH)–CH(CN)2 group of d1-BMN-ts instantly changes to the –ph–CHO group to form the final product (d1-BMN-CHO, Fig. 3c). The molecular docking (Fig. 4a and b) results indicated that only d1-BMN-CHO can be forced to steadily stay in one DNA crystal structure that has many AP sites in a certain conformation by the hydrogen bonds (the classical hydrogen bonds, indicated by the green dotted line, and the non-classical hydrogen bonds, indicated by the white dotted line), the charge interaction (orange dotted line), and π–π stacking (purple dotted line). However, d1-BMN (Fig. 4b) cannot enter this cavity given the insufficient binding force, such as hydrogen bonds and the charge interaction. Furthermore, the molecular docking (Fig. 4a) and the molecular dynamics simulations (Fig. 4c and d) showed that d1-BMN-CHO exhibited six different representative conformations (Fig. 4c) with various intramolecular torsion angles in the different AP-site cavities of one DNA crystal structure (Fig. 4d and S3,† 17.9°, 41.8°, 53.8°, 65.7°, 71.8° and 77.6°). The aldehyde-group metabolites of all other BMN-Fluors derivatives also have different numbers of conformations. The molecular docking indicated that a2-BMN-CHO and d2-BMN-CHO only have one conformation, whereas a1-BMN-CHO, b-BMN-CHO, c-BMN-CHO and e-BMN-CHO have two conformations. This result was mainly due to the lack of sufficient –ONH2 and –NH– groups in the molecule to form hydrogen bonds and interact with the AP site.
Fig. 3 (a) NMR titration between d1-BMN (3.0 mM) and HDM (3.0 mM) in D2O. Black line: d1-BMN; dark yellow line: HDM; red line: d1-BMN-NO. (b) NMR spectra monitoring the molecular structure changes during the recognition process in Fig. 1a and c. Red line: d1-BMN-NO; orange line: d1-BMN-ts; blue line: d1-BMN-CHO. (c) The inferred molecular structures of BMN-Fluors change during the recognition process. |
Fig. 4 The molecular docking results of (a) d1-BMN-CHO and (b) d1-BMN. (c) The RMSD analysis between the different molecular conformations obtained by the molecular dynamics simulations. The PDB code is 4Q45. (d) The six preferential conformations during the RMSD analysis. (e) and (f) The orbital energy of HOMO and LUMO using Gaussian 16 at different intermolecular torsion angles (obtained by the molecular docking) when the number of AP sites are 2, 4, 6, 8, 10 and 14. (g) The simulated fluorescence emission spectra using Gaussian 16 at different intermolecular torsion angles when the number of AP sites are 2, 4, 6, 8, 10 and 14. |
Gaussian 16 was used to calculate the ground and excited states of these different representative conformations collected by the molecular docking (Fig. 4a) and the molecular dynamics simulations (Fig. 4c and d). The energy gap (ΔE) between HOMO and LUMO of d1-BMN-CHO in the excited state gradually decreased (Fig. 4e and f). The simulated fluorescence emission spectra (Fig. 4g) were consistent with those of Fig. 2a, that is, a multichannel signal. Furthermore, through the same method, the simulated fluorescence emission spectra of other aldehyde-group metabolites (i.e.a2-BMN-CHO, d2-BMN-CHO, a1-BMN-CHO, b-BMN-CHO, c-BMN-CHO, and e-BMN-CHO) were basically consistent with their experimental data (Fig. S4†). In other words, they emitted OFF–ON single-channel signal and ratio double-channel signal.
The results mentioned above indicated that the different types of signal (i.e. OFF–ON single-channel signal, ratio double-channel signal, and multichannel signal) are mainly attributed to the sequential self-regulation of one molecule between the unstable and stable states of molecular conformations. In the free state, the aldehyde-group metabolites (BMN-Fluors-CHO, Fig. 3c) of BMN-Fluors derivatives were in a real-time rotation state, that is, molecular conformations are in an unstable state. Furthermore, these molecules are bound in an AP site cavity by different binding effects (i.e., hydrogen bonds, charge interaction, and π–π stacking). In accordance with the strength of binding effect, the degree of molecular torsion, and the cavity size of AP sites, one molecule could sequentially self-regulate from one unstable state to form different stable-state conformations. The molecules in different stable-state conformations possessed different energy gaps (ΔE) between their HOMO and LUMO orbits in the excited state. Thus, different types of signals were emitted.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc04140k |
This journal is © The Royal Society of Chemistry 2019 |