Exploration of Catalytic Nucleic Acids on Porphyrin Metalation and

Subscriber access provided by La Trobe University Library

Article

Exploration of catalytic nucleic acids on porphyrin metalation and peroxidase activity by in vitro selection of aptamers for N-methyl mesoporphyrin IX Luyan Yang, Pi Ding, Yu Luo, Jine Wang, Haiyin Lv, Wenjing Li, Yanwei Cao, and Renjun Pei ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00129 • Publication Date (Web): 02 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Combinatorial Science

Exploration of catalytic nucleic acids on porphyrin metalation and peroxidase activity by in vitro selection of aptamers for N-methyl mesoporphyrin IX

Luyan Yang1,2, Pi Ding1, Yu Luo1,2, Jine Wang1, Haiyin Lv1, Wenjing Li1,2, Yanwei Cao1*, Renjun Pei1,3*

1

CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and

Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China. E-mail address: [email protected], Tel.: +86-512-62872776. 2

Nano Science and Technology Institute, University of Science and Technology of

China, Suzhou 215123, China. 3

School of Nano Technology and Nano Bionics, University of Science and

Technology of China, Hefei 230026, China.

1 / 20

ACS Paragon Plus Environment

ACS Combinatorial Science 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT In order to develop a novel light-up probe and DNAzyme, we selected aptamers for N-methyl mesoporphyrin IX (NMM), a common fluorogenic analogue of coenzyme hemin, by a modified affinity chromatography-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Two truncated aptamers Nm1 and Nm2 with low micromolar dissociation constants (0.75 and 13.27 μM) were obtained after 11 rounds of selection and the final minimized 39-mer aptamer Nm2.1 showed 24-fold fluorescence enhancement for NMM at saturated concentration. Study of the interactions between aptamers and other porphyrin compounds by circular dichroism (CD) and absorption spectroscopy showed that Nm1 mainly assembled as a stem-loop structure, which exhibited a catalytic activity for the metal insertion reaction of mesoporphyrin IX with 3.3-fold rate enhancement. In contrast, the G-rich Nm2 and Nm2.1 were likely to form G-quadruplexes in the presence of alkali metal cations (K+ and Na+), which displayed excellent peroxidase activity exhibiting 19-fold higher catalytic efficiency than hemin alone. The selected aptamers could therefore be used as novel light-up fluorescent probes and DNAzymes by pairing with porphyrin compounds that have potential to construct sensors for various applications.

Keywords: SELEX, N-methyl mesoporphyrin IX, aptamer, peroxidase, metalation

2 / 20


Page 2 of 27

Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Nucleic acid aptamers are short single-strand DNA or RNA molecules produced in vitro by the systematic evolution of ligands by exponential enrichment (SELEX) technology initiated by Ellington and Tuerk.1,2 Aptamers, usually approximately 6-30 kDa, can adopt various flexible three-dimensional structures, such as stems, loops, bulges, hairpins, etc.,3,4 owing to hydrogen bonding, electrostatic interaction, shape complementarity and van der Waals forces.5 Such structural features contribute to the desired target-binding affinity and specificity. Aptamers, which have advantages of small size, relatively high stability, low cost, and easily chemical modification for conjugation and labeling,5,6 have been selected to recognize a wide range of targets, including metal ions,6 organic molecules,7 entire cells,8,9 and bacteria.10 In addition, these binders have found application in biosensors,11 biomarker discovery,12 cancer diagnostic,13 integration with nanotechnology, and other areas.14 N-methyl mesoporphyrin IX (NMM) (Figure 1, right), belongs to the class of fluorogenic dyes which have low autofluorescence and strong fluorescence enhancement when combined with ligands.15,16 Aptamer binders of this biocompatible molecule would therefore have potential as sensitive and convenient light-up fluorescent probes.17 Currently, the most well-used ligands of this type are G-quadruplexes (G4s), formed spontaneously by four adjacent guanines from G-rich sequences held together by Hoogsteen hydrogen bond and stabilized by metal cations (K+, Na+).18 Many label-free fluorescent probes based on G4-NMM pairs have been reported to detect Pb2+, adenosine triphosphate (ATP), DNA, protein, and other 3 / 20



molecules.16, 19-21 However, recent studies have suggested some disadvantages of G4 structures,22,23 including strong sensitivity to differences in pH, metal ion concentration, and the presence of small compounds and various proteins.24,25 In addition, G4 probes engage in interactions with a wide variety of other molecules, which may result in higher background signal. In addition to its fluorogenic properties, as a naturally distorted porphyrin NMM is a strong inhibitor of ferrochelatases and is considered as a stable transition-state analogue for the mesoporphyrin IX (MPIX) metalation reaction.26 Li and coworkers have previously selected NMM-binding aptamers by immobilization of the target on oxirane-acrylic beads.27 The two best aptamers developed from this work (designated PS2.M28,29 and PS5.M30) appeared to be specific biocatalysts via the formation of intramolecular G4 structures. In addition, RNA aptamers selected for NMM that catalyze the metalation of MPIX have been reported.31,32 We seek to duplicate this activity with more stable DNA aptamers. Recently, Yang and Browser33 performed capillary electrophoresis SELEX platform to isolate DNA aptamers for NMM, and the full-length aptamers were tested for metalation catalytic activity with 1.7- and 2.0-fold rate enhancement without the measurement of DNA-enhanced peroxidase activity. In addition, the structural conformations of these aptamers were not analyzed and characterized in detail. Here we describe the use of modified affinity chromatography SELEX to obtain optimized catalytic DNA aptamers for NMM. The aptamer candidates were chosen on the basis of enhanced binding-dependent fluorescence, followed by structural analysis 4 / 20


Page 4 of 27

Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by circular dichroic (CD) and predictive tools. After that, the catalytic abilities of selected aptamers were further investigated. These studies resulted in two DNA aptamers, designated Nm1 and Nm2, targeting NMM with low micromolar dissociation constants. The former, a truncated 41-nt DNA aptamer was found to be a stable stem-loop structure that exhibited a 3.3-fold rate enhancement for porphyrin metalation, and the later, a truncated 45-nt DNA aptamer was proved to assemble into a G4 that displayed a 19-fold enhancement for catalytic activity.

RESULTS AND DISCUSSION Enrichment of DNA library against NMM Aptamers against NMM were selected from a primary combinatorial ssDNA library containing approximately 1015 different sequences by an in vitro selection procedure. Schematic illustration of the overall SELEX process is shown in Figure 1. To immobilize the DNA library on streptavidin-coated agarose beads indirectly, a short biotinylated strand (named capture-nmm: 5’-GTCGTCCCGAGAGCCATA-biotin-3’) was designed, which could be complementary partially to one end of the ssDNA library. For each round of selection, the ssDNA pool immobilized on the agarose beads was performed to a total of eight washing steps by SB buffer (3 times), NMM solution (3 times) and SB buffer (2 times). During these processes, unbound or low binding sequences were allowed to be washed out of the column by SB buffer. Meanwhile, DNA molecules having specific secondary/tertiary structures bound to NMM were released from the agarose beads by NMM solution and collected into the 5 / 20



fraction tubes. Subsequently, these collections were PCR amplified to generate a new DNA pool for the next round of selection. Such SELEX process was repeated several rounds, and the selection pressure was gradually increased by reducing the amount of sub-ssDNA pool and the concentration of NMM in order to obtain aptamers with better affinity. This modified affinity chromatography selection method has two advantages: (i) it can make up the deficiency of classic chromatography method based on fixing target molecules on the solid matrix in isolating aptamers for small molecule targets with inadequate active groups for coupling; (ii) it allows the specific binding interaction to occur in a free environment between flexible oligonucleotides and small target molecules encompassing fully potential binding sites.

Figure 1. Schematic illustration of the selection procedure.

The efficiency of selection procedure was monitored by electrophoresis and Q-PCR method after each round of the selection. In general, the stronger binding of the ssDNA pools to NMM was obtained, the more sequences released from the agarose beads, correspondingly the band in gel would be brighter. As shown in Figure S1, the NMM eluted bands of the 11th round have clear-cut distinction compared with the 4th round, indicating the successful enrichment of NMM binding sequences. Therefore, 6 / 20


Page 6 of 27

Page 7 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


we basically decided that the enriched ssDNA pool from the 11th round was enough to proceed to cloning and sequencing. However, gel electrophoresis of PCR amplified wash fractions based on different PCR cycle numbers may have limitations since the difference between selection rounds will not be distinguished efficiently. Thus, we further performed a Q-PCR assay for the NMM elutions from each round to monitor the selection progress according to the previous report.34 The amplification curves for all eleven rounds of SELEX selection in Figure 2A show that the calibrated fluorescence enhancement changes gradually with different selection rounds and reaches a plateau at the eleven round, indicative of the acquirement of sufficient enrichment of the ssDNA pool. The melting curves in Figure 2B show that the proportion of Tm (melting temperature) peaks display hetero-duplex (71-73 °C) decreasing and homo-duplex (82-84 °C) increasing with the number of selection rounds. Such an observation was in accordance with previous results,33 indicating successful enrichment of NMM binding sequences during the SELEX procedure. The Q-PCR results further confirmed that the 11th ssDNA pool had been enriched for NMM.

Figure 2. Q-PCR monitoring of the selection process. (A) Amplification curves and (B) Melting curves of Q-PCR for different selection rounds; the vertical axis of figure 2A is the 7 / 20



calibrated fluorescence (∆Rn) generated by the reporter dye (SYBR) relative to reference dye ROX, and the horizontal axis is the number of Q-PCR cycles. The vertical axis of figure 2B is the opposite of first derivative of the calibrated fluorescence (Derivative (-∆Rn)), and the horizontal axis is the temperature of Q-PCR.

Analysis and characterization of aptamers Based on the results of sequencing and modeling, the chosen candidate aptamers were mainly based on the homology analysis (Figure S2) and simulated secondary structures, and then nine of them with more representative and stable secondary structures (Figure S3) were optimized and synthesized for further verification (Table S2). The binding affinity of these sequences with NMM was initially determined by fluorescence spectroscopy (Figure S4). According to the result of the fluorescence assay, two sequences, designated Nm1 and Nm2, were chosen as potential aptamers which exhibited remarkable fluorescence enhancement. Their predicted secondary structures were roughly modeled and shown in Figures 3A and 3B. Nm1 contains a central multi-loop with two small hairpins and a large connecting hairpin, and Nm2 includes two interior loops within one long hairpin at the top. Additionally, Nm2.1 (Figure 3C) was designed on the basis of Nm2 by cutting the red frame and exchanging the position of 5’ G-base and 3’ C-base to avoid the contribution of the terminal bases to form G4 assemblies. And Nm2.2 (Figure 3D) was designed by substituting GGG with ATT in the top loop of Nm2.1 to evaluate the contribution of central G-repeat fragments to the formation of G4s. The fluorescence responses for 8 / 20


Page 8 of 27

Page 9 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NMM of four aptamers were shown in Figure S5. Three of them except for Nm2.2 showed great increase on fluorescence intensity, which may have potential to design NMM-aptamer biosensors.35.

Figure 3. Predicted secondary structures of NMM aptamers by using M-fold software, (A) Nm1, (B) Nm2, (C) Nm2.1 and (D) Nm2.2. Nm2.1 is made from Nm2 by cutting the red frame and exchanging the position of 5’ G-base and 3’ C-base, and Nm2.2 is designed by replacing GGG with ATT in the top loop of Nm2.1.

Subsequently, binding properties of the three truncated fluorescence-responsive aptamers were further examined. Continuous-variation experiments were first performed by recording the fluorescence enhancement of different ratios of DNA aptamers and NMM at 610 nm. Figures 4A, 4B and S6A show that Nm1, Nm2 and Nm2.1 mainly form 1:1, 4:1 and 4:1 complexes with NMM, respectively. Fluorescence titration experiments further confirmed that the Kd values of the three aptamers of Nm1, Nm2 and Nm2.1 are 0.75 μM, 13.27 μM and 16.36 μM respectively, as shown in Figures 4C, 4D and S6B. These experimental results indicated that the DNA conformation formed by Nm1 has a better binding affinity to NMM, which is different form the DNA secondary structures formed by Nm2 and Nm2.1. 9 / 20



Figure 4. Job-Plot and Binding curve. (A) Nm1 forms a 1:1 complex with NMM. (B) Nm2 forms a 4:1 complex with NMM. (C) Binding curve for Nm1. (D) Binding curve for Nm2. Data points (fluorescence Intensity) and error bars represent the mean and standard deviation from three separate trials.

Circular dichroism (CD) experiments were performed to determine detailed structural conformations of aptamers under different experimental conditions. Figure 5A shows that the CD spectra of Nm1 display a positive peak at around 280 nm and a negative peak at around 245 nm when it was incubated in Tris-HCl buffer, SB buffer and SB buffer supplemented with NMM, respectively, indicating the formation of a B-type duplex structure. Combining with the CD pattern of Nm1 and the binding ratio of NMM-Nm1 complexes (Figure 4A), we can conclude that Nm1 mainly forms a stem-loop structure rather than a G4 under our experimental conditions, which has a good affinity for NMM, as illustrated in Figure 4C. In comparison, Figure 5B shows that the CD spectrum of Nm2 displays a positive peak at around 280 nm and a 10 / 20


Page 10 of 27

Page 11 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


negative peak at around 245 nm in Tris-HCl buffer, while the positive peak has a blue shift and exhibits an obvious positive peak at around 265 nm in SB buffers in the presence and absence of NMM. These results demonstrated that aptamer Nm2 can be induced to form parallel G4 by alkali metal ions (including Na+ and K+) in SB buffer, according to reports in literature24 and the fluorescence enhancement of the mixture of Nm2 and NMM was caused by the formation of parallel G4.36 By using this approach, we also analyzed the conformation formed by Nm2.1 and Nm2.2. Figure S7A shows that the tailored aptamer of Nm2.1 displays the same characteristics as Nm2, which further confirms the formation of G4 structures for Nm2 and Nm2.1. However, the converted aptamer of Nm2.2 can maintain the stem-loop structure in different buffer solutions (Figure S7B), indicating the significant role of the middle G-repeat on the formation of G4. The maintenance of stem-loop structures for Nm1 and Nm2.2 was further supported by native-polyacrylamide gel electrophoresis (PAGE) experiments (Figure S8), in which the mobility of them approximated that of unstructured sequences half their length, suggesting the presence of stable folded conformations.

Figure 5. Circular dichroism (CD) spectra for characterizing Nm1 (A) and Nm2 (B) structural conversion in different solutions. Black square curves represent 3 μM aptamers in 20 11 / 20



mM Tris-HCl (pH 7.4), and red circle curves and blue triangle curves represent 3 μM aptamers in the absence and presence of 3 μM NMM in SB buffer, respectively.

Measurement of catalytic activity Natural porphyrins and metalloporphyrins participate in oxygen transport, electron transfer, and a variety of redox chemistries, including in catalases, peroxidases, and monooxygenases. Meanwhile, porphyrin-binding aptamers from in vitro selection have attracted some interest as potential catalysts for similar processes, such as peroxidase activity and metal insertion reaction.37,38 Some investigation of the function of these metallo-DNAzymes in living cells has also been reported. For example, the combination of G4/hemin mimicking DNAzyme and hybridization chain reaction has provided a type of electrochemical biosensor to detect the p53 gene;39 early reports also revealed that the formation of G4/hemin DNAzymes at some genetic regions might play significant roles in the gene regulation40 or disease diagnosis.41 We assessed the peroxidase activity of aptamer-bound hemin (Figures 6A and 6B) by following the oxidative conversion of ABTS to its highly colored radical cation (ABTS•+). Figure 6C shows that the changes at 414 nm in absorbance spectra that reflect the oxidation rate of ABTS by H2O2 are minimal for hemin alone, the mixtures of Nm1 and hemin, and of Nm2.2 and hemin, indicating their weak peroxidase activities. However, the mixtures of Nm2 and hemin, and Nm2.1 and hemin exhibited obvious absorbance changes at 414 nm, indicating the enhancement of peroxidase 12 / 20


Page 12 of 27

Page 13 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activity, which gave initial catalytic rates of 61.07 nM/s and 62.04 nM/s, respectively, as shown in Figure 6D. These results further confirmed the formation of G4s for Nm2 and Nm2.1, which could generate stable catalytic complexes with hemin to accelerate the oxidizing reaction.

Figure 6. Time-dependent absorbance (414 nm) change upon analysis of peroxidase activity of hemin alone and aptamer-hemin complexes. (A) Chemical structure of hemin. (B) Peroxidase-catalyzed reaction diagram. (C) Plots of absorbance at 414 nm versus reaction time for various samples: hemin alone, black; Nm1 and hemin, green; Nm2 and hemin, magenta; Nm2.1 and hemin, red; and Nm2.2 and hemin, orange. (D) The initial reaction rate V0 calculated from the changes during the first 20 s (∆ε = 36000 M-1cm-1) of each sample, and data values and error limits were from three separate trials. In all experiment, aptamers (2 μM) were complexed with hemin (2 μM), and 2 mM 2,2’-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) and 0.8 mM H2O2 were in KB buffer (pH 7.4, 25 mM Tris-HAc, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 0.05% Triton x-100 (w/v) and 1% DMSO (v/v)).

13 / 20



Notably, these aptamers selected for NMM also have the ability to catalyze the insertion of various divalent metal ions (including Cu2+, Zn2+, Co2+, and Mn2+) into MPIX as functional mimics of ferrochelatase.27 Here, we chose to explore the metalation of MPIX with Cu2+ to assess the activity of selected aptamers in this regard. Figure 7A displays the metalation-catalyzed reaction from MPIX to Cu-MPIX triggered by Cu2+. Figure 7B illustrates that the UV-visible absorption spectra of the mixture of MPIX and Cu2+ after stored in dark for 24 h, and the mixture of MPIX, Cu(OAc)2 and Nm1 after annealing for 12 h exhibit obvious difference in the Soret band and Q band regions of MPIX, indicating the formation of Cu-MPIX. Our results and previous reports also revealed that the absorbance change at 561 nm as a function of time could reflect the overall reaction rate (Figure S9 and the insert in Figure 7B).30,32 Figure 7C shows that absorbance changes at 561 nm are improved with different degrees after the addition of various DNA sequences to the mixture of MPIX and Cu2+, indicating various catalytic efficiencies of different aptamers. The Vobs values exhibited in Table S3 demonstrated that, compared with the catalytic rate in the absence of aptamers, the addition of aptamer Nm1 can increase the catalytic rate to 3.3-fold, while the addition of aptamers Nm2, Nm2.1 and Nm2.2 can only increase to about 2-fold. These results indicated that aptamer Nm1 is more suitable as a new type DNAzyme for catalytic metallization.

14 / 20


Page 14 of 27

Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Time-dependent absorbance (561 nm) change upon analysis of Cu-MPIX produced in the presence of Cu(OAc)2. (A) Metalation-catalyzed reaction diagram. (B) UV-vis spectra of MPIX (black curve), the mixture of Cu(OAc)2 and MPIX after incubating for 24 h (blue curve), and the mixture of Nm1, Cu(OAc)2 and MPIX after incubating for 12 h (red curve) in MB buffer. (C) The formation of metalloporphyrin was monitored by the spectral value change at 561 nm (∆ε=18600 M-1cm-1) over the first 60 min of each sample, and data values and error limits were from three separate trials. No aptamer, black square; Nm1, red circle; Nm2, green star; Nm2.1, blue triangle; Nm2.2, cyan diamond. In experiments, a 100 µL mixture contained MPIX (100 µM), Cu(OAc)2 (200 μM) and aptamer (5 μM) was measured in MB buffer (pH 7.4, 25 mM Tris-HAc, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1% DMSO (v/v) and 0.5% Triton X-100 (w/v)).

CONCLUSIONS In summary, we obtained two types of DNA aptamers (Nm1 and Nm2) which have high binding affinity with NMM through a modified affinity chromatogram SELEX strategy in vitro selection. And the detailed conformations of Nm1 and Nm2 were 15 / 20



analyzed here by CD, PAGE experiments and predictive tool, which demonstrate that Nm1 mainly forms a stem-loop structure under our experimental conditions and Nm2 can assemble to be G4 structures in the presence of Na+ or/and K+. In addition, Nm2-hemin complex displayed a DNA-enhanced peroxidase activity with the substrate of ABTS while the metalation rate of MPIX with Cu2+ can be enhanced effectively after titrated with Nm1. The selection method used here is proved to be an effective way to acquire and design functional aptamers for various potential applications. Meanwhile, our study provides a new insight to analyze the conformation of aptamers, which is meaningful to synthesize and design powerful and widely-used aptasensors based on the specific properties of selected aptamers.

SUPPORTING INFORMATION Gel electrophoresis, Sequence alignment analysis, Secondary structures predicted by M-fold, Enhancement in fluorescence for nine aptamer candidates, Dissociation constant (Kd) curve and job-plot for Nm2.1, Native-PAGE images, Circular dichroism (CD) spectra for Nm2.1 and Nm2.2, Metalation activity of the selected DNA sequences.

ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (21575154, 21775160 and 31800685), the CAS/SAFEA International Innovation Teams program and the China Postdoctoral Science Foundation (2017M620228, 16 / 20


Page 16 of 27

Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2018T110550).

REFERENCES (1) Ellington, A. D.; Szostak, J. W., In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818-822. (2) Tuerk, C.; Gold, L., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505-510. (3) McKeague, M.; McConnell, E. M.; Cruz-Toledo, J.; Bernard, E. D.; Pach, A.; Mastronardi, E.; Zhang, X.; Beking, M.; Francis, T.; Giamberardino, A.; Cabecinha, A.; Ruscito, A.; Aranda-Rodriguez, R.; Dumontier, M.; DeRosa, M. C., Analysis of In Vitro Aptamer Selection Parameters. J. Mol. Evol. 2015, 81, 150-161. (4) Weeks, K. M., Review toward all RNA structures, concisely. Biopolymers 2015, 103, 438-448. (5) Zhou, J.; Rossi, J., Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov. 2017, 16, 181-202. (6) Wu, J.; Zhu, Y.; Xue, F.; Mei, Z.; Yao, L.; Wang, X.; Zheng, L.; Liu, J.; Liu, G.; Peng, C.; Chen, W., Recent trends in SELEX technique and its application to food safety monitoring. Mikrochim. Acta 2014, 181, 479-491. (7) Behrouzi, G.; Rasaee, M. J.; Mousavi, M. L., Separating and utilizing DNA aptamers in detection of morphine. Clin. Biochem. 2011, 44, S315-S315. (8) Yazdian-Robati, R.; Ramezani, M.; Khedri, M.; Ansari, N.; Abnous, K.; Taghdisi, S. M., An aptamer for recognizing the transmembrane protein PDL-1 (programmed death-ligand 1), and its application to fluorometric single cell detection of human ovarian carcinoma cells. Microchim. Acta 2017, 184, 4029-4035. (9) Civit, L.; Taghdisi, S. M.; Jonczyk, A.; Hassel, S. K.; Grober, C.; Blank, M.; Stunden, H. J.; Beyer, M.; Schultze, J.; Latz, E.; Mayer, G., Systematic evaluation of cell-SELEX enriched aptamers binding to breast cancer cells. Biochimie 2018, 145, 53-62. (10) Chen, F.; Zhou, J.; Luo, F.; Mohammed, A. B.; Zhang, X. L., Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis. Biochem. Biophys. Res. Commun. 2007, 357, 743-748. (11) Wang, J.; Hou, J.; Zhang, H. C.; Tian, Y.; Jiang, L., Single Nanochannel-Aptamer-Based Biosensor for Ultrasensitive and Selective Cocaine Detection. ACS Appl. Mater. Inter. 2018, 10, 2033-2039. (12) Ostroff, R. M.; Bigbee, W. L.; Franklin, W.; Gold, L.; Mehan, M.; Miller, Y. E.; Pass, H. I.; Rom, W. N.; Siegfried, J. M.; Stewart, A.; Walker, J. J.; Weissfeld, J. L.; Williams, S.; Zichi, D.; Brody, E. N., Unlocking biomarker discovery: large scale application of aptamer proteomic technology for early detection of lung cancer. PLoS One 2010, 5, e15003. (13) Stein, C. A.; Castanotto, D., FDA-Approved Oligonucleotide Therapies in 2017. Mol. Ther. 2017, 25, 1069-1075. (14) He, M. Q.; Wang, K.; Wang, J.; Yu, Y. L.; He, R. H., A sensitive aptasensor based on molybdenum carbide nanotubes and label-free aptamer for detection of bisphenol A. Anal. Bioanal. Chem. 2017, 409, 1797-1803. (15) Arthanari, H.; Basu, S.; Kawano, T. L.; Bolton, P. H., Fluorescent dyes specific for quadruplex 17 / 20



DNA. Nucleic Acids Res. 1998, 26, 3724-3728. (16) Chen, Q.; Guo, Q.; Chen, Y.; Pang, J.; Fu, F.; Guo, L., An enzyme-free and label-free fluorescent biosensor for small molecules by G-quadruplex based hybridization chain reaction. Talanta 2015, 138, 15-19. (17) Largy, E.; Granzhan, A.; Hamon, F.; Verga, D.; Teulade-Fichou, M.-P., Visualizing the Quadruplex: From Fluorescent Ligands to Light-Up Probes. Top. Curr. Chem. 2012, 330, 111-178. (18) Collie, G. W.; Parkinson, G. N., The application of DNA and RNA G-quadruplexes to therapeutic medicines. Chem. Soc. Rev. 2011, 40, 5867-5892. (19) Guo, L.; Nie, D.; Qiu, C.; Zheng, Q.; Wu, H.; Ye, P.; Hao, Y.; Fu, F.; Chen, G., A G-quadruplex based label-free fluorescent biosensor for lead ion. Biosens. Bioelectron. 2012, 35, 123-127. (20) Zhao, C.; Wu, L.; Ren, J.; Qu, X., A label-free fluorescent turn-on enzymatic amplification assay for DNA detection using ligand-responsive G-quadruplex formation. Chem. Commun. 2011, 47, 5461-5463. (21) Li, W.; Jiang, W.; Wang, L., Self-locked aptamer probe mediated cascade amplification strategy for highly sensitive and selective detection of protein and small molecule. Anal. Chim. Acta. 2016, 940, 1-7. (22) Lee, C. Y.; Jang, H.; Park, K. S.; Park, H. G., A label-free and enzyme-free signal amplification strategy for a sensitive RNase H activity assay. Nanoscale 2017, 9, 16149-16153. (23) Liu, Y.; Liao, R.; Wang, H.; Gong, H.; Chen, C.; Chen, X.; Cai, C., Accurate and sensitive fluorescence detection of DNA based on G-quadruplex hairpin DNA. Talanta 2018, 176, 422-427. (24) Bhattacharyya, D.; Mirihana Arachchilage, G.; Basu, S., Metal Cations in G-Quadruplex Folding and Stability. Front Chem. 2016, 4, 38. (25) Zhang, S.; Wu, Y.; Zhang, W., G-quadruplex structures and their interaction diversity with ligands. Chem. Med. Chem. 2014, 9, 899-911. (26) Sen D., Poon L.C.H., RNA and DNA complexes with hemin [Fe(III) heme] are efficient peroxidases and peroxygenases: how do they do it and what does it mean? Crit. Rev. Biochem. Mol. 2011, 46, 478-492. (27) Li, Y.; Geyer, C. R.; Sen, D., Recognition of anionic porphyrins by DNA aptamers. Biochemistry 1996, 35, 6911-6922. (28) Travascio, P.; Li, Y.; Sen, D., DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chem. Biol. 1998, 5, 505-517. (29) Travascio, P.; Bennet, A. J.; Wang, D. Y.; Sen, D., A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites. Chem. Biol. 1999, 6, 779-787. (30) Li, Y.; Sen, D., Toward an efficient DNAzyme. Biochemistry 1997, 36, 5589-5599. (31) Conn, M. M.; Prudent, J. R.; Schultz, P. G., Porphyrin metalation catalyzed by a small RNA molecule. J. Am. Chem. Soc. 1996, 118, 7012-7013. (32) Kawazoe, N.; Teramoto, N.; Ichinari, H.; Imanishi, Y.; Ito, Y., In vitro selection of nonnatural ribozyme-catalyzing porphyrin metalation. Biomacromolecules 2001, 2, 681-686. (33) Yang, J.; Bowser, M. T., Capillary electrophoresis-SELEX selection of catalytic DNA aptamers for a small-molecule porphyrin target. Anal. Chem. 2013, 85, 1525-1530. (34) Luo, Z.; He, L.; Wang, J.; Fang, X.; Zhang, L., Developing a combined strategy for monitoring the progress of aptamer selection. Analyst 2017, 142, 3136-3139. (35) Li, M.; Zhao, A.; Ren, J.; Qu, X., N-Methyl Mesoporphyrin IX as an Effective Probe for Monitoring Alzheimer's Disease beta-Amyloid Aggregation in Living Cells. ACS Chem. Neurosci. 2017, 8, 18 / 20


Page 18 of 27

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1299-1304. (36) Kypr, J.; Kejnovska, I.; Renciuk, D.; Vorlickova, M., Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 2009, 37, 1713-1725. (37) Hollenstein, M., DNA Catalysis: The Chemical Repertoire of DNAzymes. Molecules 2015, 20, 20777-20804. (38) Breaker, R. R., DNA aptamers and DNA enzymes. Curr. Opin. Chem. Biol. 1997, 1, 26-31. (39) Ding, L.; Zhang, L.; Yang, H.; Liu, H.; Ge, S.; Yu, J., Electrochemical biosensor for p53 gene based on HRP-mimicking DNAzyme-catalyzed deposition of polyaniline coupled with hybridization chain reaction. Sensors and Actuat. B-Chem. 2018, 268, 210-216. (40) Wang, D.; Guo, R.; Wei, Y.; Zhang, Y.; Zhao, X.; Xu, Z., Sensitive multicolor visual detection of telomerase activity based on catalytic hairpin assembly and etching of Au nanorods. Biosens. Bioelectron. 2018, 122, 247-253. (41) Grigg, J. C.; Shumayrikh, N.; Sen, D., G-quadruplex structures formed by expanded hexanucleotide repeat RNA and DNA from the neurodegenerative disease-linked C9orf72 gene efficiently sequester and activate heme. PLoS One 2014, 9, e106449.

19 / 20



For Table of Contents Use Only Exploration of catalytic nucleic acids on porphyrin metalation and peroxidase activity by in vitro selection of aptamers for N-methyl mesoporphyrin IX Luyan Yang1,2, Pi Ding1, Yu Luo1,2, Jine Wang1, Haiyin Lv1, Wenjing Li1,2, Yanwei Cao1*, Renjun Pei1,3*

20 / 20


Page 20 of 27

Page 21 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1 109x41mm (300 x 300 DPI)



Figure 2 119x42mm (600 x 600 DPI)


Page 22 of 27

Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3 109x48mm (300 x 300 DPI)



Figure 4 119x81mm (600 x 600 DPI)


Page 24 of 27

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5 119x44mm (600 x 600 DPI)



Figure 6 109x74mm (600 x 600 DPI)


Page 26 of 27

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7 109x76mm (600 x 600 DPI)


Exploration of Catalytic Nucleic Acids on Porphyrin Metalation and

Recommend Documents