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Letter
DNA Duplex Engineering for Enantioselective Fluorescent Sensor Yuehua Hu, Fan Lin, Tao Wu, Yufeng Zhou, Qiusha Li, Yong Shao, and Zhiai Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04709 • Publication Date (Web): 27 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017
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Analytical Chemistry
DNA Duplex Engineering for Enantioselective Fluorescent Sensor Yuehua Hu,† Fan Lin,† Tao Wu,† Yufeng Zhou,† Qiusha Li,† Yong Shao,*,† and Zhiai Xu*,‡ † ‡
Institute of Physical Chemistry, Zhejiang Normal University, Jinhua 321004, Zhejiang, China School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China
* E-mail:
[email protected] (S. Y.). Fax: 86 579 82282595;
[email protected] (Z. X.)
ABSTRACT: The rapid identification of biomacromolecule structure that has a specific association with chiral enantiomers especially from natural sources will be helpful in developing enantioselective sensor and in speeding up drug exploitation. Herein, owing to its existence also in living cells, apurinic/apyrimidinic site (AP site) was first engineered into ds-DNA duplex to explore its competence in enantiomer selectivity. An AP site-specific fluorophore was utilized as enantioselective discrimination probe to develop a straightforward chiral sensor using natural tetrahydropalmatine (L- and D-THP) as enantiomer representatives. We found that only L-THP can efficiently replace the pre-bound fluorophore to cause a significant fluorescence increase due to its specific binding with the AP site (two orders magnitude higher in affinity than binding with D-THP). The AP site binding specificity of LTHP over D-THP was assessed via intrinsic fluorescence, isothermal titration calorimetry, and DNA stability. The enantioselective performance can be easily tuned by the sequences near the AP site and the number of AP sites. A single AP site provides a perfect binding pocket to differentiate the chiral atom-induced structure discrepancy. We expect that our work will inspire interest in engineering local structures into ds-DNA duplex for developing novel enantioselective sensors.
The bioactivities of some enantiomers are strongly dependent on their chirality. 1 ,2 For example, R-(+)-thalidomide is a sedative, whereas S-(-)-thalidomide is a teratogen. 3 Some chiral pharmaceuticals pollute the environment where they exhibit different degradations and toxic behaviors.4 The separation and detection of enantiomers are of a great challenge because of the nearly identical chemical and physical properties of the chiral isomers. Chromatography and electrophoresis techniques using specially designed chiral materials as the stationary phases or as mobile phase additives have been widely used to separate enantiomers and identify interconversion between enantiomers.5-11 The assembly of silver/gold nanoparticles and semiconductor quantum dots with chiral interfaces have been used as alternative rationales in colorimetric and fluorescent recognition and separation of enantiomers.12-15 Nucleic acids are promising enantiomer selectors because of their inherent chirality. 16 , 17 Some stereoselective synthesis 15, reactions can be tuned via DNA. 18 20 Furthermore, isomers that have specific interaction with structured nucleic acids can be developed as clinically important chiral drugs.21 G-quadruplexes (G4s) have been widely used as DNA chiral platforms because their polymorphisim structures provide chance to regulate their stereoselective binding with variant enantiomers. 22 -27 However, unfortunately, the G4 structure sometimes makes the enantiomer recognition unsuccessful for small molecule targets because several binding sites are available in G4 and always give similar associations to compromise the selectivity. Thus, to achieve ideal enantiomer selectivity, synthetic chiral metal complexes are usually used as conceptional targets with size comparable to the G4 tetrad in order to have sufficient binding strength.22-27 The chiral discrimination competency of more rigid ds-DNA duplex structures has also been investigated.10,11, 28 31 Of these reports, sequence-dependent enantiomer differentiation has been observed for small-sized enantiomers.10,11,28,29 However, DNA duplex-based enantiomer recognition with an ideal selectivity and
a strong binding remains a challenge since only the weak binding sites of the groove are usually available. This is especially true for small, natural chiral molecules. For example, L- and Dtetrahydropalmatine (L- and D-THP) have weak ds-DNA bindings with only a two-fold difference in binding affinity.10 More importantly, the specific enantiomer recognition event following a straightforward signal (such as fluorescence) as readout will be a promising approach to conveniently evaluate the chiral binding behavior and to develop enantioselective sensors. Herein, we first attempt to develop an enantioselective fluorescence sensor and strengthen the enantiomer binding event via simply engineering an abasic site (AP site) into a DNA duplex structure. This attempt avoids the complicated modification with DNA. 32 Following the seminal AP site strategy designed by Teramae et al. for DNA analysis,33 variant sensors have been successfully developed. 34 - 38 In this work, THP, an alkaloid usually extracted from Corydalis genus and Stephania rotunda, was selected as the naturally occurring enantiomer representative. It has been reported that THP is pharmacologically active and its in vivo bioactivity and metabolism are chirality dependent.39-42 LTHP is used in traditional Chinese medicine for its analgesic effects by blocking dopamine receptors.40 However, it is far from deeply understanding its bioactivity at nucleic acid level. Therefore, in addition to developing enantioselective sensors, this work also provides an insight into the binding specificity of natural enantiomers with the AP site of ds-DNA since this site always exists in cells with dysregulation in replication and transcription.43 We initially evaluated the binding behaviors of the THP enantiomers with ds-DNA via isothermal titration calorimetry (ITC). As shown in Figure 1 (See Figure S1 for the raw data), the L- and D-THP enantiomers have a poor binding with ds-DNA that does not contain the AP site, although L-THP exhibits a slightly higher binding than D-THP, as observed in a previous report.10 This reflects the inability of the ds-DNA without the AP site to
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Analytical Chemistry differentiate the enantiomers. However, we expect that introducing one AP site into ds-DNA (AP-DNA) can significantly strengthen the THP binding, since this vacant site should provide a strong binding pocket for THP.
0.0
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to L-THP, while the D-THP binding is less specific. The free energy change for the L-THP binding is 2.93 kcal/mol favorable relative to that for the D-THP binding (Table S2). Next, we investigated the THP intrinsic fluorescence (282 excitation/311 nm emission 44 ) to understand the affinity that contributes to THP binding specificity. As shown in Figure 2, the ds-DNA without the AP site (FM) has no effect on intrinsic fluorescence of the THP enantiomers. Note that the inner filter effect from DNA absorption seems be omitted with 282 nm excitation. However, GXG-T significantly quenches the L-THP fluorescence, as opposed to the almost unchanged fluorescence with D-THP. Thus, an efficient stacking interaction likely occurs between L-THP and the guanines that flank the AP site. This enables electron transfer between the L-THP excited state and the guanines to cause fluorescence quenching. This stacking interaction was further confirmed using the DNA melting experiments. With increasing the enantiomer concentration up to 50 µM, the GXG-T melting temperature increased up to 4 oC with L-THP, as opposed to only the 2 oC increase with D-THP (Figure S2). These experiments suggest that the L-THP binding is preferable to the AP site with specific affinity. However, although the addition of D-THP also stabilizes GXG-T (but less so than L-THP), the intrinsic fluorescence upon binding with AP-DNA (Figure 2) is almost unchanged. This indicates that the binding mode of GXG-T with D-THP is different from that with L-THP. The intrinsically fluorogenic group in L-THP has a much closer contact with the flanking guanines of the AP site, which favors electron transfer from these bases to the DNA-bound THP in the excited state.
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Figure 1. Binding of THP with DNA evaluated by ITC: (a) DTHP/FM; (b) L-THP/FM; (c) D-THP/GXG-T; (d) L-THP/GXG-T. 30 µM DNA was titrated by 300 µM THP in 0.1 M PBS buffer (pH 6.0) at 25 oC. The solid curves are the fitting results. Also shown are the L/D-THP structures.
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Figure 3. Fluorescence response of ATMND-GXG-Y system to the THP enantiomers in 0.1 M PBS buffer (pH 6.0). ATMND: 0.5 µM; GXG-Y: 1 µM; THP: 4 µM. F and Fo are the fluorescence in the presence and absence of THP. Excitation/emission: 357/404 nm. Inset: the corresponding spectra for GXG-T. Also shown are photographs of 2 µM solutions of ATMND and GXG-T under UV illumination with 20 µM THP enantiomers.
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Figure 2. Dependence of THP intrinsic fluorescence at 311 nm on the added DNA concentration in 0.1 M PBS buffer (pH 6.0). THP: 0.5 µM; Excitation wavelength: 282 nm.
The intrinsic fluorescence of THP emitting at 311 nm is not a suitable readout for a practical chiral sensor. However, the previously developed fluorescent probes specific for the AP site provide the possibility of operating the enantioselective sensor at visible emission wavelengths.33, 45 We selected an appropriate probe that experiences a fluorescence quenching upon binding to the AP site. Such a strongly binding enantiomer will efficiently replace the pre-bound probe, and the released probe should give a turn-on fluorescence response dependent on the AP site binding strength of the enantiomer. In this work, we tried to use 2-amino5,6,7-trimethyl-1,8-naphthyridine (ATMND, Scheme S1), an AP site-selective fluorophore,45 as the promising probe. To also understand the sequence effects, the AP site in the DNA was always flanked by guanines, but the opposite base Y was
Accordingly, the DNA with the AP site (X) flanked by guanines and opposed by thymine was first evaluated (GXG-T, Table S1). A two-binding-site model offered good fitting for the THP binding with GXG-T. The ITC fitting results with the stronger binding affinity reasonably ascribe to the AP site binding. L-THP exhibits two orders of magnitude stronger binding than DTHP (1.45×107 vs 1.03×105 M-1, Table S2), suggesting that the AP site can differentiate the THP enantiomers with high selectivity. Note that the binding affinities of both L- and D-THP with ds-DNA not containing the AP site (FM, Table S1) are 104 M-1 level.10 Relative to the D-THP binding, the L-THP binding causes a larger change in exothermic enthalpy and less of an increase in entropy. This indicates that specific binding happens
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introduced AP sites can be used to tune the performance of the enantioselective sensor.
systematically changed (thus named GXG-Y with Y=A, C, G, and T, Table S1). Figure 3 shows that the fluorescence response to the THP enantiomers is strongly dependent on the identity of the base Y in GXG-Y. GXG-A and GXG-G lead to negligible changes in fluorescence upon addition of either L-THP or D-THP. However, L-THP causes an 86% increase in the ATMND fluorescence at 404 nm using GXG-T as the binding field. This is 6 times higher than that obtained using D-THP (only 14% increase). For GXG-C, L-THP and D-THP results in only 42 % and 11% increase, respectively. Note that no fluorescence change was observed in the case of direct interaction of ATMND with L-THP and D-THP without AP-DNA. Thus, the resulting fluorescence increases reflect replacement of the AP site-bound ATMND with the enantiomers. Such the varied differentiation performance for the enantiomers is due to the base Y-dependent ATMND/THP binding. ATMND exhibits strong binding with GXG-C and -T versus much weaker binding with GXG-G and -A.45 This is in good agreement with our enantioselective fluorescence responses. This pyrimidine-over-purine performance is because of the perfect complementarity in hydrogen bonding pattern of protonated ATMND with C and T (Scheme S1). 45 Furthermore, ATMND has a slightly higher binding with GXG-C than with GXG-T (1.9×107 vs 9.1×106 M-1). 45 It is thus expected that the AP-DNA having an ATMND binding affinity that is even stronger than any of the enantiomers would compromise the enantioselective performance, as observed in Figure 3 for the GXG-C response to L-THP, which is lower than GXG-T. This binding affinity relevance was confirmed via the observed pH-dependent fluorescence of ATMND alone in solution and its binding with GXG-T. pH-tuned selectivity in discriminating L-THP and D-THP was observed (Figure S3). The optimized pH is 6.0 for the enantioselective fluorescent sensor. This is because only the protonated ATMND is totally complementary with T via three-point hydrogen bonds (Scheme S1). 45 The differentiation of L-THP from D-THP can be even visualized with the naked eye for the former solution in favor of a much brighter blue appearance under UV illumination (Inset of Figure 3). The ATMND replacement by THP was also confirmed via the time-dependent evolution of fluorescence (Figure S4). A faster replacement occurred to L-THP in comparison to D-THP (7.46 versus 1.39 min-1). This reflects the kinetically favored binding of L-THP with the AP site. Discrimination of L-THP from D-THP can be achieved across a wide range of enantiomer concentrations (Figure 4A). The limit of detection for L-THP is about 650 nM assuming a signal-to-noise ratio of 3. Furthermore, when keeping the total concentration of a mixture of L-THP and D-THP at 10 µM but only changing the ratio of the enantiomers, increase in the L-THP percent population rightly results in a corresponding increase in fluorescence response (Figure 4B), suggesting practical application of our proposed sensor. The enantioselective recognition ratio, defined as (FL-F0)/(FD-F0),25 is about 4.4. In order to improve the discrimination capability, we introduced two AP sites into one DNA molecule (2GXG-TT, Table S1). The two AP sites in 2GXG-TT were separated from each other to individually accommodate THP. In this case, the DNA length was increased to keep the duplex stability at room temperature (Figure S5). As shown in Figure 4A, in comparison with GXG-T, 2GXGTT exhibits an apparently increased L-THP response, as opposed to the minor change for D-THP. The limit of detection for L-THP thus decreases to about 420 nM assuming a signal-to-noise ratio of 3. This engineered DNA can also cause a more sensitive response to the L-THP population in an enantiomer mixture than GXG-T (Figure 4B). The enantioselective recognition ratio increases to about 4.9. These results suggest that the number of
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Figure 4. (A) Discrimination of L-THP (solid symbol) from D-THP (open symbol) under various enantiomer concentrations using GXG-T (triangle) and 2GXG-TT (circle). (B) Fluorescence response on the percent population of L-THP in an enantiomer mixture of 10 µM in 0.1 M PBS buffer (pH 6.0) containing 0.5 µM ATMND and 1 µM DNA. F and Fo are the fluorescence responses in the presence and absence of THP, respectively.
According to the above results, we expect that the AP siteengineered enantioselectivity of L-THP over D-THP is due to their conformation differences, which are determined solely by the chiral carbon atom (Figure 1).46,47 The binding mode of LTHP with AP-DNA experiences less hindrance with respect to DTHP because of the limited space provided by the AP site. It is noted that the structure features of both D- and L-THP should weaken their binding with the intact ds-DNA that do not contain the AP site, as confirmed in a previous report10 and with our results in Figure 1. The detailed structure modeling of D-/L-THP and their DNA docking are underway in this laboratory. In light of the role of the void space provided by the AP site, we then introduced two consecutive AP sites into ds-DNA but with one and two thymine(s) opposite the AP sites (GXXG-T and GXXG-TT in Table S1). These DNA designs aim to confirm that a single AP site is enough to provide the spatial confinement needed to perfectly define the enantioselectivity. These ds-DNAs remain stable at room temperature (Figure S5). As occurred to GXG-T, ATMND retains its binding capacity with the AP sites in these two ds-DNAs (Figure S6A, B). However, while D- and LTHP can displace the bound ATMND to cause an increase in fluorescence, the discrimination capability for the enantiomers is totally lost when using GXXG-TT as the selector, as apposed to a minor discrimination of L-THP over D-THP with GXXG-T as the selector (Figure S6C and D). These experiments suggest that, as expected, the larger space provided by the two AP sites can accommodate both D- and L-THP and resultantly weaken the enantioselectivity. Finally, to generalize the ability of the AP site design in differentiating other enantiomers, we also measured the interaction of R-(+)-thalidomide and S-(-)-thalidomide (R-THA and S-THA) with AP-DNA using the THA’s intrinsic fluorescence (305 excitation/401 nm emission48). We found that the AP-DNA of AXA-A (Table S1) with adenines flanking and opposite the AP site can increase the intrinsic fluorescence of RTHA, as opposed to the negligible fluorescence change for STHA (Figure S7). The AP-DNA binding can also retard the hydrolysis of R-THA.49 These facts suggest the competency of AP-DNA in differentiating R-THA and S-THA. In conclusion, the engineered AP site provides a pocket to strengthen the binding of D- and L-THP with ds-DNA. However, the limited space provided by the AP site endows an L-THP-overD-THP discrimination capability. The specific binding of L-THP
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with the AP site releases the pre-bound probe of ATMND to solution and a fluorescent turn-on response is followed as a convenient readout for an enantioselective fluorescent sensor based on the rigid ds-DNA. Our work also provides insight into the possible L-THP bioactivity at nucleic acid level due to existence of the AP site in living cells. We expect that our work will inspire interest in engineering a local structure into ds-DNA duplex (e.g., besides the AP site, also including bulge site, gap site, etc) for constructing next-generation enantioselective sensors.
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(23) Shi, S.; Xu, J.; Gao, X.; Huang, H.; Yao, T. Chem. - Eur. J. 2015, 21, 11435-11445. (24) Wang, J.; Chen, Y.; Ren, J.; Zhao, C.; Qu, X. Nucleic Acids Res. 2014, 42, 3792-3802. (25) Feng, L.; Xu, B.; Ren, J.; Zhao, C.; Qu, X. Chem. Commun. 2012, 48, 9068-9070. (26) Zhao, C.; Geng J.; Feng, L.; Ren, J.; Qu, X. Chem. - Eur. J. 2011, 17, 8209-8215. (27) Yu, H.; Wang, X.; Fu, M.; Ren, J.; Qu, X. Nucleic Acids Res. 2008, 36, 5695-5703. (28) Benedetti, M.; Malina, J.; Kasparkova, J.; Brabec, V.; Natile, G. Environ. Health Perspect. 2002, 110, 779-782. (29) Hu, Q.; Xu, S. Angew. Chem. Int. Ed. 2014, 53, 14135-14138. (30) Zhao, C.; Ren, J.; Gregolinski, J.; Lisowski, J.; Qu, X. Nucleic Acids Res. 2012, 40, 8186-8196. (31) Dubois, M.; Grandbois, A.; Collins, S. K.; Schmitzera, A. R. J. Mol. Recognit. 2011, 24, 288-294. (32) Shoji, A.; Kuwahara, M.; Ozaki, H.; Sawai, H. J. Am. Chem. Soc. 2007, 129, 1456-1464. (33) Yoshimoto, K.; Nishizawa, S.; Minagawa, M.; Teramae, N. J. Am. Chem. Soc. 2003, 125, 8982-8983. (34) Xiang, Y.; Tong, A. J.; Lu, Y. J. Am. Chem. Soc. 2009, 131, 15352-15357. (35) Sankaran, N. B.; Nishizawa, S.; Seino, T.; Yoshimoto, K.; Teramae, N. Angew. Chem. Int. Ed. 2006, 45, 1563-1568. 36 Song, P. S.; Xiang, Y., Xing, H.; Zhou, Z. J.; Tong, A. J.; Lu, Y. Anal. Chem. 2012, 84, 2916-2922. (37) M. J. Li, Y. Sato, S. Nishizawa, T. Seino, K. Nakamura and N. Teramae, J. Am. Chem. Soc. 2009, 131, 2448-2449. (38) Xiang, Y.; Wang, Z. D.; Xing, H.; Wong, N. Y.; Lu, Y. Anal. Chem. 2010, 82, 4122-4129. (39) Sun, S.; Wang, Y.; Li, L.; Wang, L.; Zeng, S.; Zhou, H.; Jiang, H. Chirality 2013, 25, 43-47. (40) Chu, H.; Jin, G.; Friedman, E.; Zhen, X. Cell. Mol. Neurobiol. 2008, 28, 491-499. (41) Iranshahy, M.; Quinn, R. J.; Iranshahi, M. RSC Adv. 2014, 4, 15900-15913. (42) Wang, C.; Zhou, J.; Wang, S.; Ye, M.; Jiang, C.; Fan, G.; Zou, H. J. Proteome Res. 2010, 9, 3225-3234. (43) Boiteux, S.; Guillet, M. DNA repair 2004, 3, 1-12. (44) Li, C.; Xu, Q.; Li, J.; Jia, X. J. Inclusion Phenom. Mol. Recognit. Chem. 2009, 64, 37-42. (45) Sato, Y.; Nishizawa, S.; Yoshimoto, K.; Seino, T.; Ichihashi, T.; Morita, K.; Teramae, N. Nucleic Acids Res. 2009, 37, 1411-1422. (46) Luger, P.; Weber, M.; Dung, N. X.; Moi, L. D.; Khoi, T. T.; Phuong, D. L. Acta Crystallogr., Sect. C 1998, 54, 1977-1980. (47) Ribar, B.; Lazar, D.; Gasic, O.; Kanyo, I.; Simonov, Y. A.; Kravtsov, V. C. Acta Crystallogr., Sect. C 1993, 49, 1691-1693. (48) Cardoso, C. E.; Martins, R. O. R.; Aucelio, R. Q. Microchem. J. 2004, 77, 1-7. (49) Reist, M.; Carrupt, P.-A.; Francotte, E.; Testa, B. Chem. Res. Toxicol. 1998, 11, 1521-1528.
ASSOCIATED CONTENT Supporting Information Experimental details; DNA sequences; ITC results; Tm measurements; pH- and AP site number-dependent fluorescence; reaction kinetics; thalidomide experiments. This material is available free of charge via the Internet at http://pubs.acs.org.
ORCID Yong Shao: 0000-0003-0834-6244 Zhiai Xu: 0000-0002-4391-2507
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant No. 21675142 and 21545009).
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