Efficient DNA-Catalyzed Porphyrin Metalation for Fluorescent

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Efficient DNA-Catalyzed Porphyrin Metalation for Fluorescent Ratiometric Pb2+ Detection Dong Peng, Yuqing Li, Zhicheng Huang, Ru-Ping Liang, Jian-Ding Qiu, and Juewen Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02759 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Analytical Chemistry

Efficient DNA-Catalyzed Porphyrin Metalation for Fluorescent Ratiometric Pb2+ Detection Dong Penga,b, Yuqing Lib, Zhicheng Huangb, Ru-Ping Lianga, Jian-Ding Qiua,c* and Juewen Liub* a

College of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang, 330031 Jiangxi, China Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada c Environmental Protection Materials and Equipment Engineering Technology Center of Jiangxi, Department of Materials and Chemical Engineering, Pingxiang University, 211 Pingan North Road, Pingxiang, 337055 Jiangxi, China b

ABSTRACT: Developing biosensors for Pb2+ is an important analytical topic. DNA-based Pb2+ sensors have been designed mainly based on RNA-cleaving DNAzymes and Pb2+-induced folding of G-quadruplex (G4) DNA. Porphyrin metalation is a key reaction in biology and catalysis. Many enzyme mimics have been developed to catalyze this reaction, and some metalation DNAzymes were reported with a G4 structure. Inspired by the excellent G4 binding properties of certain divalent metal ions, we herein screened a few metals and G-rich DNA sequences. The metalation activity of a DNA named T30695 (sequence: G3T)4 was significantly accelerated by Pb2+. The reaction of Cu2+ insertion into the mesoporphyrin IX had a kcat of 0.89 min-1 and a Km of 9.8 μM, representing a catalytic efficiency similar to that of human ferrochelatase. The reason for the acceleration was attributed to Pb2+ binding of the G4 DNA and the catalytic activity of the large Pb2+ ion for this reaction. A ratiometric sensor for Pb2+ was developed by inserting Zn2+ with a detection limit of 23.5 nM Pb2+. This work has established a new DNA-based reaction that can be used for Pb2+ detection, and it also provides a highly efficient new DNAzyme for porphyrin metalation, which might be used for signal production for other biosensors.

INTRODUCTION Being a highly toxic heavy metal, Pb2+ has attracted a lot of attention of analytical chemists.1 In particular, DNA-based Pb2+ sensors have been extensively researched for their high sensitivity, selectivity and the excellent programmability of DNA.2-4 Some well-known Pb2+ sensors used RNA-cleaving DNAzymes,5, 6 which relied on substrate cleavage and involved multiple DNA strands. Pb2+ sensors were also designed based on its strong binding to G-quadruplex (G4) DNA,7-9 but these binding based sensors may suffer from interference such as nonspecific DNA folding in the presence of other metal ions. Metalloporphyrins are critical cofactors in many enzymatic reactions from photosynthesis to detoxification.10, 11 Insertion of metal ions into porphyrins is called porphyrin metalation, which is kinetically slow.12 Enzymes are responsible for porphyrin metalation in biological systems. For example, ferrochelatase inserts Fe2+ into protoporphyrin IX (PPIX), which is the final step for the biosynthesis of heme. Many efforts were made to develop enzyme mimics, such as catalytic antibodies,13 ribozymes,14 DNAzymes,15 and nanozymes.16 Schultz and coworkers isolated a 35-mer ribozyme for inserting Cu2+ into mesoporphyrin IX (MPIX) with a kcat/Km of 2100 M-1s-1, which is close to that of the recombinant human ferrochelatase.14 Sen and coworkers selected a DNAzyme with a G4 structure,15 and DNA can overcome the poor stability and high cost of RNA. This DNAzyme catalyzed MPIX metalation

with a kcat of 13.7 h-1 and Km of 2.9 mM. Their further work selected DNAzymes with a minimal and optimal catalytic unit, which exhibited an improved catalytic efficiency of ~540 M-1s1.17 Recently, other aptamers against the NMM porphyrin were reported, and some non-G4 structures also had catalytic activities.18-20 Some aptamer/hemin complexes showed excellent peroxidase-like activity,21, 22 which were widely used for signal amplification in biosensors.23 Metal ions are highly important for DNAzyme catalysis, as demonstrated in many RNA-cleaving DNAzymes.2, 4, 24-28 Some other DNAzymes for DNA cleavage,29 DNA ligation,30 and RNA ligation31 also require specific metal ions for their activity. However, metal ions for porphyrin metalation have yet to be explored, and most previous works studied only Na+, K+ and Mg2+. Some heavy metal ions (e.g. Sr2+, Ba2+, and Pb2+) can strongly bind and stabilize G4 structures.32-34 Since many metalation DNAzymes are G-rich, we herein screened various metal ions along with a few common G4 sequences. We report that Pb2+ can drastically improve the metalation rate of a particular G4 sequence, allowing efficient multiple turnover reactions, and label-free fluorescent ratiometric Pb2+ detection. MATERIALS AND METHODS Materials. All of the DNA samples were from Integrated DNA Technologies (IDT, Coralville, IA, USA). Mesoporphyrin IX (MPIX) dihydrochloride, methyl sulfoxide (DMSO), sodium acetate, Potassium acetate, magnesium acetate tetrahydrate,

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copper(II) chloride, zinc chloride, strontium chloride, lead(II) acetate trihydrate, thallium(I) chloride, barium chloride, cadmium chloride, manganese(II) chloride, nickel(II) chloride, gadolinium(III) chloride, and cerium(III) chloride were purchased from Sigma-Aldrich. Milli-Q water was used for all of the experiments. Apparatus. Colorimetric and fluorescence measurements were performed by a Tecan Spark Multimode microplate reader (Switzerland) using either Costar 96-well flat transparent plates (USA) or Corning 96-well Flat Black half area plates (USA). CD spectroscopy was performed in a 1 cm UV−vis quartz cuvette using a Jasco J-715 spectrophotometer. Kinetics of Cu2+ insertion into MPIX. To study the kinetics in the SB buffer, 0.2 μM of the G-rich DNAs were added into 50 μL of SB buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaOAc, 25 mM KOAc, and 10 mM Mg(OAc)2).15 The samples were heated at 90 ℃ for 10 min and then gradually cooled to room temperature to fold the DNAzymes. 2.5 μM MPIX and 1 mM of Cu2+ were then sequentially added. All of the mixtures were diluted with ultrapure water to a final volume of 100 μL. Because of the poor solubility of MPIX, 5% DMSO and 0.5% Triton X-100 were presence in all reaction solutions. The fluorescence intensity at 623 nm (λex= 400 nm) was followed to study the reaction kinetics. To study the catalytic effects of the Pb2+-stabilized DNAzymes, 0.2 μM of the G-rich DNAs were incubated in 50 μL Tris-HCl buffer (100 mM, pH 7.5) with the addition of 1.2 μM of Pb2+ and then heating and cooling to form the Pb2+stabilized DNAzymes. 5% DMSO and 0.5% Triton X-100 were then added before MPIX was added. Finally, the reaction was initiated by the addition of Cu2+. To compare the effects of different metal ions, the final concentrations of Sr2+ and Ba2+ were 100 μM, K+ and Na+ were 1 mM, Tl+, Mg2+, Ca2+, Ni2+, Mn2+, Ce3+, and Gd3+ were 50 μM, Hg2+ and Cd2+ were 5 μM, and Pb2+ was 1.2 μM. To study the kinetic parameters of the Pb2+-stabilized T30695 DNAzyme, different concentrations of MPIX (varied from 0.5 to 3.5 μM) were added into the reaction solutions. The fluorescence intensities at 623 nm were recorded with 5 min interval. The reaction rates were calculated for the initial 40 min. To ensure the calculated concentrations of the metal-free MPIX, a new calibration curve was made under the same condition with every experiment. CD spectroscopy. T30695 (10 μM) was dissolved in 50 mM Tris-HCl (pH = 7.5), and 60 μM of Pb2+ (or 100 mM of K+) were added with a final volume of 200 μL. The samples were heated to 90 ℃ for 10 min, and then gradually cooled to room temperature. The CD spectra were obtained by taking the average of five scans made from 210 nm to 340 nm. Pb2+ detection. T30695 (0.2 μM) was mixed with different concentrations of Pb2+ in 50 μL of Tris-HCl buffer (500 mM, pH 7.5). The mixtures were heated at 90 ℃ for 10 min, and gradually cooled to room temperature. A final of 2.5 μM MPIX and 0.4 mM Zn2+ were sequentially added and the samples were diluted with ultrapure water to 100 μL. The fluorescence spectra (λex = 400 nm) of these samples were recorded after reaction for 3 h. RESULTS AND DISCUSSION Metalation of MPIX. In this work, we used the MPIX porphyrin (Figure 1A) as the substrate for its good photostability and solubility in aqueous solutions.14 Free MPIX

appeared faint yellow and showed a typical Soret band at 399 nm with a small shoulder at 375 nm and three Q bonds (~510 nm, 540 nm, and 570 nm) (Figure 1B).35 After incubating Cu2+ (1 mM) with MPIX (2.5 µM) for 7 days, a sharp and strong Soret band and two weaker Q bands were observed, indicating the formation of the metalation product Cu2+(MPIX).36 The redshift of the Soret band to 410 nm also demonstrated the insertion of Zn2+.37 When excited at 400 nm, the free MPIX has two strong red fluorescence peaks at 623 nm and 683 nm, respectively (Figure 1C). The fluorescence of MPIX was fully quenched after insertion of Cu2+. For Zn2+, the fluorescence at 623 nm decreased, while a new peak emerged at 583 nm. Further studies found that the fluorescence intensity of MPIX showed a good liner relationship with the metal concentration from 0 to 2.5 μM, allowing us to quantitatively follow the insertion of Cu2+ and Zn2+ (Figure S1, Supporting Information). Initially, we selected Cu2+ to study MPIX metalation. Fe2+ is not a good candidate for analytical applications because of its sensitivity to air.14

Figure 1. (A) A scheme of metalation of MPIX. (B) UV-vis and (C) fluorescence spectra of 2.5 µM metal-free MPIX in 50 mM pH 7.5 Tris-HCl buffer and after insertion of Zn2+ and Cu2+. Insets: the corresponding photographs and the fluorescence photographs (excited at 365 nm).

Pb2+ promotes the T30695 DNA activity. Since G4 DNA can bind hemin in general,38, 39 they may also have a high affinity for other porphyrins by - stacking. For example, the anionic MPIX porphyrin has a high affinity to many G4 DNAs.40 A total of six common G4 sequences were tested here (Figure 2A). We first mixed MPIX with each of the G4 DNA and Cu2+ in the high salt 0.5 SB buffer (final 50 mM Tris-HCl, pH 7.5, 100 mM Na+, 12.5 mM K+, and 5 mM Mg2+), and followed the kinetics of fluorescence quenching (Figure 2B). While all the DNAs promoted Cu2+ insertion, the improvements compared to the background rate without DNA were quite small (within 2-fold). After a long incubation time (19 h), the difference became larger, where PS2.M and T30695 showed the best efficiency, consistent with the literature (Figure S2).15, 17 Overall, these reactions were very slow, even with the DNAs. To test if a stronger G4 binding metal can facilitate the reaction, we then tried Pb2+ (Figure 2C). For this reaction, we removed the other metal ions in the SB buffer and only left 50 mM Tris-HCl (pH 7.5). This way, we could better test the effect


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Analytical Chemistry of Pb2+. Quite interestingly, the T39695 DNA showed a significantly higher rate with just 0.6 µM of Pb2+ (Figure 2D, and Figure S3), while the other DNAs were still close to the background rate (Figure 2C). To confirm our results, we did a few controls (Figure 2E). Adding Pb2+ alone to MPIX (without DNA or Cu2+) did not show noticeable quenching indicating that Pb2+ alone was not responsible for the reaction (the red line). We only used 0.6 µM Pb2+, while nearly 2 µM porphyrin was inserted with Cu2+ (the green line), also confirming that quenching was not due to insertion of Pb2+. Pb2+ could slightly accelerate Cu2+ insertion without the DNA (the blue line), and this is consistent with the literature.41 Thus, T39695 can be considered as a metallo-DNAzyme with Pb2+ being its cofactor.

Figure 2. (A) The six G-rich DNA sequences studied (from 5 to 3). (B) Kinetics of Cu2+ insertion into MPIX in the 0.5 SB buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaOAc, 12.5 mM KOAc, 5 mM Mg(OAc)2) in the presence of different DNA sequences. (C) Reaction kinetics with these DNAs in 50 mM of Tris-HCl, pH 7.5 in the presence of 0.6 μM of Pb2+. All the buffers also contained 5% DMSO and 0.5% Triton X-100 to help dissolve MPIX. (D) Kinetics of Cu2+ insertion with T30695 and various concentrations of Pb2+ (0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 μM). (E) A few control experiments. The concentrations of MPIX, Cu2+, and DNA were 2.5 μM, 1 mM and 0.1 μM, respectively.

Only Pb2+ promoted the reaction. Inspired by the results from Pb2+, we also tried a few other G4 binding metals including Sr2+ and Ba2+,42, 43 but they were ineffective (Figure S4). Therefore, simple stabilization of the G4 structure cannot help the reaction, and Pb2+ must have additional chemical roles. Some large metals (e.g. Pb2+, Hg2+, and Cd2+) cannot fit well into the porphyrin pocket, but rather they can be on top of the porphyrin ring and deform its structure (‘sitting-atop’) to favor the attack by smaller metal ions (like Cu2+) from the back.41 Normally, the catalytic effects of these large metal ions follow the order of Hg2+ > Pb2+ > Cd2+ and we also observed this in the DNA-free reactions (Figure S5).44 We then compared these metal ions with the Cu2+/MPIX/T30695 system (Figure 3A). With 5 μM Hg2+ or Cd2+, their reactions were much slower than

that with 0.6 µM Pb2+. Among them, only Pb2+ can stabilize the G4 structure. Therefore, forming a G4 structure is required. The unique DNAzyme structure folded by Pb2+ and the intrinsic catalytic effect of Pb2+ are likely both responsible for the accelerated reaction. We further compared a few more mono-, di- and trivalent metal ions. Again, only Pb2+ showed an obvious acceleration (Figure 3A). Tl+, K+, and Pb2+ form another interesting group for comparison. Tl+ is similar to K+ and it can also stabilize G4 structures, but Tl+ has a stronger binding strength.33, 45 Tl+ is also a heavy metal and in this regard, it is similar to Pb2+. However, Tl+ did not accelerate the reaction with T30695 either.

Figure 3. (A) The fluorescence intensities of MPIX mixed with Cu2+ in the presence of T30695 and different metal ions for 3 h. The concentrations of MPIX, Cu2+ and DNAs were 2.5 μM, 1 mM and 0.1 μM, respectively. The final concentrations of Hg2+ and Cd2+ were 5 μM, K+ and Na+ were 1 mM, Tl+, Mg2+, Ca2+, Ni2+, Mn2+, Ce3+, and Gd3+ were 50 μM, and Pb2+ was only 0.6 μM. (B) CD spectra of 10 μM T30695 in the presence of 60 μM Pb2+ or 100 mM K+. (C) The proposed secondary structures of T30695 binding with K+ or Pb2+. (D) A scheme of DNAzyme-catalyzed multiple turnover MPIX metalation.

Folding of G4 DNAs including T30695 has been extensively studied using circular dichroism (CD) spectroscopy.46-48 We also measured the CD spectra in our buffer conditions with K+ and Pb2+ (Figure 3B). The K+stabilized T30695 showed a strong positive peak at ~265 nm, suggesting a parallel G4 structure (Figure 3C).49, 50 The Pb2+stabilized T30695 exhibited a similar strong positive peak at 265 nm and a negative peak at 240 nm, which was quite similar to the K+-stabilized parallel structure. The additional but smaller positive band at ~312 nm suggested the coexistence of Pb2+-stabilized antiparallel structures.8, 48 Therefore, most of the T30695 adopted a parallel conformation in the presence of Pb2+, but a small fraction was antiparallel.47, 48 By using UV-vis and CD spectroscopy and simulation, it has been generally accepted that this DNA binds two K+ ions,51 while based on mass spectrometry,50 and fluorescence spectroscopy,47 it binds only one Pb2+. We reason that a parallel G4 structure may provide an



ideal binding geometry for MPIX, allowing potential MPIX/Pb2+ interactions yet still exposing its backside for attack by Cu2+ (Figure 3D). Therefore, the enhanced catalytic activity might mainly come from the chemical property of Pb2+ and its sitting in a uniquely folded G4 DNA. The other tested G4 DNAs may not satisfy these requirements and thus they showed low activities. Rational modification of the DNAzyme. To further understand the effect of DNA sequence and folding, we modified T30695 by adding three A or T bases to its 3 and 5 ends, respectively (Figure 4A). All these sequences retained high activities (Figure 4B), indicating that the T30695 core is highly robust in the presence of Pb2+. A careful comparison indicated that capping the 3-end slightly decreased the rate, while capping the 5-end had no effect. From the proposed structure above, the 3-end of the Pb2+-folded T30695 is close to the porphyrin binding site, and this result further supported this model. The ability to modify the sequence with retained activity is useful for designing biosensors and stimuliresponsive hybrid nanomaterials.52-54 Enzyme kinetic parameters. To quantitatively understand the enzyme kinetics, we then measured the reaction rates with various concentrations of MPIX. A Lineweaver– Burk plot was made to extract the kinetic parameters (Figure 4C). With just 0.6 µM Pb2+, the DNAzyme had a kcat of 0.89 min-1, 4.3-fold higher than that in the SB buffer. Notably, the Km was only 9.8 μM, 296-fold smaller than that of the reported PS5.ST1 DNAzyme (2.9 mM), and it also compared favorably with catalytic antibodies (50 μM). Thus, Pb2+ not only improved catalytic rate, but also enhanced substrate binding. The specificity of the DNAzyme (kcat/Km) was 1513 M-1 s-1, comparable with protein-based metalation enzymes.

Figure 4. (A) Extending the T30695 DNA by adding three A or T bases to the ends. (B) Kinetics of Cu2+ insertion into 2.5 μM MPIX by these five DNAs in the presence of Pb2+. (C) The Lineweaver– Burk plot for extracting enzyme kinetic constants. The concentrations of the DNA, Pb2+, and Cu2+ were 0.1 μM, 0.6 μM, and 1 mM, respectively.

Selective and sensitive Pb2+ sensing. Our porphyrin metalation showed a surprisingly high specificity for Pb2+. We then tested the feasibility of using it for Pb2+ detection. Since Cu2+ only quenched the emission, using it for detection would create a ‘signal-off’ sensor with limited sensitivity and susceptibility to false signals. On the other hand, metalation of Zn2+ has an increased peak at 583 nm, allowing more reliable ratiometric detection.55 The Pb2+-stabilized DNAzyme

exhibited best catalytic effect in the presence of 0.4 mM of Zn2+, and the value of kcat/Km reached 1675.3 M-1 s-1 (Figure S6). With increasing concentration of Pb2+, the fluorescence spectra from the inserted product gradually evolved (Figure 5A). We chose the fluorescence ratio at 583 nm and 623 nm for quantification of Pb2+ (Figure 5B), and this ratio gradually increased with Pb2+. We determined the detection limit to be 23.5 nM Pb2+ (calculated for signal beyond three times of background variation). This is below the toxic limit of 75 nM Pb2+ defined by the US Environmental Protection Agency (EPA). It is interesting to note that the calibration curve showed two slopes, initially with a lower sensitivity and later a higher sensitivity. It typically implies that more than one Pb2+ is involved in the catalysis, although most reports showed that the T30695 DNA only binds one Pb2+. At this moment, it is unclear what might have caused this. It cannot be ruled out that one Pb2+ was for stabilizing the G4 structure, while another Pb2+ is needed at the same time for catalysis. Further studies are needed to find out the exact way of metal binding and catalysis. This sensor also has good selectivity and other tested metal ions did not accelerate this reaction (Figure 5C). Furthermore, we tested Pb2+ detection in Lake Ontario water (Table S1), the good recoveries suggested its feasibility in practical analytical applications. Although compared to some Pb2+ sensors based on RNA-cleaving DNAzymes,5, 6, 56, 57 this metalation-based sensor is slightly less sensitive, it does not require covalent labels and the reaction itself can generate a ratiometric fluorescence signal. Compared to sensors based on Pb2+-induced G4 DNA formation,7 this sensor is more selective with a comparable sensitive (Table S2).

Figure 5. (A) Fluorescence spectra of MPIX reacted with Zn2+ for 3 h in the presence of 0.1 μM T30695 and different concentrations of Pb2+. (B) The fluorescence intensity ratio (F583/F623) versus Pb2+ concentration for insertion of Zn2+. (C) Selectivity of Pb2+ analysis. The concentrations of Zn2+, T30695, and Pb2+ were 0.4 mM, 0.1 μM and 0.6 μM, respectively. For other metal ions, Hg2+ and Cd2+ were 5 μM; K+ and Na+ were 1 mM; and Tl+, Mg2+, Ca2+, Ni2+, Mn2+, Fe2+, Ce3+, and Gd3+ were 50 μM.

CONCLUTIONS In summary, we discovered that Pb2+ can significantly accelerate metalation of both Cu2+ and Zn2+ into MPIX using the T30695 sequence as a DNAzyme. The reaction with Zn2+


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Analytical Chemistry showed an interesting fluorescent response allowing ratiometric detection of Pb2+. Many G4/hemin complexes have peroxidaselike activities and found interesting applications in signal amplification (e.g. replacing protein-based peroxidases).23, 54 We show here that MPIX can directly produce another type of sensitive optical signal without the need of unstable H2O2. Therefore, this accelerated fluorogenic reaction might also find applications for signaling other biosensors. This DNAzyme already has a catalytic efficiency close to that of protein-based enzymes. Notably, T30695 was not obtained from the typical aptamer selection process and we discovered it by screening just a small set of DNA sequences. Further aptamer or DNAzyme selections can be carried out with Pb2+ and even more effective DNAzymes might be obtained for this important reaction. ASSOCIATED CONTENT

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Supporting Information

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Experimental details, fluorescence spectra for control experiments, quantitative response to Cu2+ and Zn2+, data for measuring spiked Lake Ontario water, and a table for comparing various DNA-based Pb2+ sensors. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION

ACKNOWLEDGMENT

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REFERENCES

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Funding for work is from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the National Natural Science Foundation of China (21675078). D. Peng was supported by the 2018 Nanchang University doctoral students abroad visiting scholar program to visit the University of Waterloo.

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Notes The authors declare no competing financial interests.

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*Email: [email protected]; [email protected]

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Corresponding Author

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