Photocaged G-Quadruplex DNAzyme and Aptamer by Post-Synthetic

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Photocaged G-quadruplex DNAzyme and Aptamer by Postsynthetic Modification on Phosphodiester Backbone Mengli Feng, Zhiyuan Ruan, Jiachen Shang, Lu Xiao, Aijun Tong, and Yu Xiang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00646 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 9, 2016

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Bioconjugate Chemistry

Photocaged G-quadruplex DNAzyme and Aptamer by Postsynthetic Modification on Phosphodiester Backbone Mengli Feng, Zhiyuan Ruan, Jiachen Shang, Lu Xiao, Aijun Tong, and Yu Xiang* Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China. *E-mail: [email protected] ABSTRACT: G-quadruplex-containing DNAzymes and aptamers are widely applied in many research fields because of their high stability and prominent activities versus the protein counterparts. In this work, G-quadruplex DNAs were equipped with photolabile groups to construct photocaged DNAzymes and aptamers. We incorporated TEEP-OH (thioether-enol phosphate, phenol substituted) into phosphodiester backbone of G-quadruplex DNA by a facile postsynthetic method to achieve efficient photocaging of their activities. Upon light irradiation, the peroxidase-mimicking activity of the caged Gquadruplex DNAzyme was activated, through the transformation of TEEP-OH into a native DNA phosphodiester without any artificial scar. Similarly, the caged G-quadruplex thrombin-binding aptamer also showed light-induced activation of thrombin inhibition activity. This method could serve as a general strategy to prepare photocaged G-quadruplex DNA with other activities for noninvasive control of their functions.

INTRODUCTION Since the discover of DNA’s biological function other than genetic codes, many single-stranded oligonucleotides of specific sequences have been found to possess diverse catalytic or target recognition activities like proteins.1-5 These oligonucleotides, with their sequences identified by either in vitro selection2, 4 or systematic evolution of ligands by exponential enrichment (SELEX),1 are termed DNAzymes or DNA aptamers (in total called functional DNAs) because of their enzyme-like or antibody-like characteristics, respectively. So far, functional DNAs have been widely applied as enzyme mimics,5 signal amplifiers,6, 7 target binders8, 9 and molecular switches10 in multiple research fields, including chemical biology,11, 12 analytical chemistry7, 13-15 and nanotechnology.16, 17 DNA G-quadruplex, formed by G-rich oligonucleotides responsible for human telomeric DNA structures and telomerase inhibition,18, 19 is also widely present in many functional DNAs and critical for their catalytic and target recognition activities.8, 9, 20-22 These G-quadruplex-containing functional DNAs are particularly interesting because of their prominent activities versus their protein counterparts.22-24 For example, peroxidase-mimicking DNAzymes with heminbound G-quadruplex were found to show comparable or even higher catalytic robustness than some known protein peroxidases,5, 23 while DNA aptamers with G-quadruplex structures exhibit specific and high binding affinity to proteins such as thrombin,25, 26 VEGF22 and nucleolin.9 To achieve high spatial and temporal control of biological activities, photocaged biomolecules, including proteins and nucleic acids that can be activated from their

Scheme 1. (a) Introducing TEEP-OH into the phosphodiester backbone of phosphorothioate-containing G-quadruplex DNA through the reaction with bromoacetophenone, as well as the conversion of TEEP-OH into a native phosphodiester by UV light. (b) Photocaging the peroxidase activity of G-quadruplex DNAzyme by TEEP-OH and its decaging by UV light. (c) Photocaging the thrombin inhibition activity of G-quadruplex aptamer (TBA) by TEEP-OH and its decaging by UV light.

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Figure 1. (a) Sequences of PS2.M, PS2M-11-1PS and PS2M-101112-3PS (“*” indicates phosphorothioate), and 20% denatured PAGE images of PS2M-C11-1PS and PS2M-G10C11G12-3PS before (lane 1 and 4) and after TEEP-OH modifications (lane 2 and 5), as well as subsequent removal of TEEP-OH by UV light (lane 3 and 6). (b) ABTS-based peroxidase activity measurement of PS2.M (black squares), PS2M-11-TEEP (green triangles), PS2M-101112-TEEP (red diamonds), PS2M-11-TEEP+UV (purple crosses) and PS2M-101112-TEEP+UV (blue stars). The experimental condition: 0.25 μM DNAzyme, 0.5 μM hemin, 1 mM ABTS, 1 mM H2O2, 50 mM HEPES at pH 6.9, 100 mM NaCl, 20 mM KCl and 0.03% Triton X-100 at 25oC. (c) CD spectra of PS2.M (blue squares), PS2M-101112-3PS (purple stars), PS2M-101112-TEEP (green dots) and PS2M-101112-TEEP+UV (black triangles). The experimental condition: 6 μM DNA, 50 mM HEPES, pH 6.9, 100 mM NaCl and 20 mM KCl at 25oC.

inactive forms by light irradiation, have been intensively studied.27-30 Caged nucleosides were introduced into oligonucleotides for efficient photoactivation of DNA hybridization and functional DNAs’ activities.31 Using caged nucleosides32-35 in G-quadruplex DNAs, the activities of peroxidase-mimicking DNAzymes and thrombininhibition aptamers were efficiently tuned by light.36, 37 Photocleavable linkers38 and azobenzene derivatives39-41 were also incorporated into the backbone of oligonucleotides for photo-induced switching of DNA structures and functions. G-quadruplex DNA aptamers containing a photocleavable linker and an amine-reactive group showed great promise in site-specific modification of native proteins.42 These approaches, though highly efficient for photocaging and photoswitching, all require the insertion of noncanonical phosphoramidite monomers into the oligonucleotides through solid-phase synthesis,32, 35, 43 which usually needs complicated case-by-case optimization of the synthetic conditions for each type of noncanonical phosphoramidites. Postsynthetic modifications, on the other hand, are simpler, under milder conditions and compatible for a broader range of chemical functional groups.44, 45 Most of the reported methods for photocaged DNA based on postsynthetic modifications, however, were either non-specific to all nucleotides that makes rational design of photoactivation difficult,46, 47 or leaving an artificial scar instead of native DNA after light irradiation that may reduce the original activity of the native nucleic acids.48, 49 In our previous study, we developed a postsynthetic method to incorporate a photolabile modification (TEEP-OH, as shown in Scheme 1a) into DNA phosphodiester backbone through the reaction between bromoacetophenone and readily available phosphorothioate, and successfully introduced such modifications into RNA-cleaving DNAzymes for efficient photoactivation.50 In this work, we constructed photocaged G-quadruplex functional DNAs such as a peroxidase-mimicking DNAzyme and a thrombin aptamer,

through rational modifications on their critical active sites. The peroxidase and thrombin inhibition activities of these modified G-quadruplex DNAs were efficiently caged, and subsequently recovered by light irradiation that restored the native G-quadruplex DNA structures without any artificial scar. Such a method should also be applicable for photocaging other DNA G-quadruplex structures with different activities, serving as a promising tool for studying their biological functions in a spatially and temporally controlled manner.

RESULTS AND DISCUSSION Photocaged peroxidase-mimicking containing a G-quadruplex structure

DNAzyme

Peroxidase-mimicking DNAzymes containing Gquadruplex structures are a particular member of the DNAzyme family.5, 51 When bound with hemin through Gquadruplex, these DNAzymes act as robust peroxidases efficiently catalyzing the oxidation of substrates by hydrogen peroxide (H2O2).21, 23, 24 To construct a photocaged version of such G-quadruplex DNAzymes, we chose PS2.M,20, 32 one of the most studied and used peroxidase-mimicking DNAzyme as an example. The PS2.M sequence (5'G1T2G3G4G5T6A7G8G9G10C11G12G13G14T15T16G17G18-3') with several combinations of phosphorothioates adjacent to nucleobases potentially critical for the G-quadruplex structures (Figure 1a) were reacted with 2-bromo-4'hydroxyacetophenoe (BHAP) under a mild condition to afford TEEP-OH (thioether-enol phosphate, phenol substituted) modified PS2.M DNAzymes.50 The formation of TEEP-OH involves the reaction of phosphorothioate with BHAP in 1:2 ratio through an interesting intramolecular transformation from thiophosphate into phosphate ester,50 making the challenging synthesis of photocaged phosphotriester facilely achieved by readily available reagents under mild conditions (Scheme 1a). Because of the ease in introducing multiple TEEP-OH

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modifications into each DNA strand, we investigated different caging site combinations on PS2.M to identify the most efficient strategy for caging the DNAzyme’s activity. The peroxidase activity was evaluated by monitoring the kinetic curve of absorbance at 414 nm, which is a characteristic peak of the green product ABTS−• from ABTS oxidization by the peroxidase-H2O2 catalytic system.5, 52-54 As illustrated in Figure S1 (Supporting Information), among the 10 designs each containing 2 TEEP-OH modifications, those with any 2 sites (G10C11, G10G12, C11G12) within the three 3'-phosphodiesters of G10, C11 and G12 showed much higher activity inhibition (caging effect) than the others, suggesting the 3 phosphodiesters are the most vulnerable sites for caging and the 3 nucleotides must be critical to the DNAzyme’s catalytic function. This is in agreement with the finding from a previous structurefunction study.55 It’s interesting to find that PS2M-G4G8TEEP and PS2M-G13G17-TEEP almost preserved the full activity as the unmodified DNAzyme, suggesting modifications close to these important deoxyguanosines did not affect the overal structure of the G-quadrupex. Indeed, the one with 3 adjacent TEEP-OH modifications at G10C11G12 resulted in the best caging effect (Figure S1). Therefore, PS2.M with 3 TEEP-OH modifications at G10C11G12 was used as the photocaged peroxidasemimicking DNAzyme for the following study in this work. We didn’t test any design with more than 3 TEEP-OH modifications because the yield of full modifications in one oligonucleotide may be compromised. To confirm the efficient TEEP-OH modification and photolysis, PS2.M sequences containing 1 phosphorothioate at C11 (PS2M-C11-1PS) or 3 phosphorothioates at G10C11G12 (PS2M-101112-3PS) were modified by BHAP and the products before and after photolysis were then characterized. After TEEP-OH modification, PS2M-11-TEEP showed clear band upshift on 20% denatured polyacrylamide gel electrophoresis (PAGE) and PS2M-101112-TEEP had an even larger shift (Figure 1a) as a result of molecular weight increase, suggesting the successful covalent attachment of TEEP-OH to the phosphorothioate-containing oligonucleotides. After UV irradiation at 300 nm, both photolysis product PS2M-11TEEP+UV and PS2M-101112-TEEP+UV displayed very similar band shifts in PAGE as the original phosphorothioate DNA before modification, indicating the efficient removal of TEEP-OH by light (Figure 1a).50 According to the stain in the PAGE image, the yields of modification and photolysis should be both over 95%. The results of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis also demonstrated the successful removal of TEEP-OH by UV irradiation (Figure S2 and S3, Supporting Information). The multiple MS peaks for TEEP-OH modified oligonucleotides are most likely because that the high energy laser for ionization during MS measurement could also induce photolysis to yield partially decaged species (about 134 or 150 Da for the loss of each subunit of TEEPOH).50 The formation of native PS2.M instead of its phosphorothioate analogue as the final product from the

photocaged DNAs was clearly supported by the molecular weight of the photolysis product, with about −16 and −48 Da compared with that of PS2M-11-1PS and PS2M-1011123PS, respectively (Figure S2 and S3, each DNA phosphorothioate, (RO)2POS− has +16 Da over native DNA phosphodiester, (RO)2PO2−). As shown in Figure 1b, one TEEP-OH modification on PS2M-11-TEEP (kPS2M-11-TEEP = 20.4±0.7 min−1) induced about 72% peroxidase inhibition compared to native PS2.M (kPS2M = 74.0±5.2 min−1), and three TEEP-OH modifications on PS2M-101112-TEEP (kPS2M-101112-TEEP = 2.2±0.3 min−1) resulted in 97% activity loss of PS2.M min−1. Subsequently, UV light irradiation decaged both PS2M-11-TEEP and PS2M-101112TEEP to yield much more active PS2M-11-TEEP+UV (kPS2M−1 11-TEEP+UV = 66.2±3.9 min ) and PS2M-101112-TEEP+UV (kPS2M-101112-TEEP+UV = 46.3±1.9 min−1), which is a 3.2-fold and 21-fold activity enhancement, respectively. Three TEEPOH modifications were advantageous for both higher caging and decaging efficiencies. It is natural that TEEP-OH can block phosphodiester chemically to reduce the DNAzyme’s original activity. To further understand the effect of TEEP-OH on the structure of G-quadruplex, we also carried out circular dichroism (CD) spectral characterization of the photocaged Gquadruplex before and after UV irradiation, to study the possible mechanism behind the efficient activity inhibition and recovery. It has been reported that the characteristic CD peaks of parallel DNA G-quadruplex structures are at around 240 (−) and 264 (+) nm while antiparallel ones at around 260 (−) and 295 (+) nm.18 Here (−) and (+) means negative and positive peaks respectively. Peaks present at both 264 (+) and 295 (+) nm usually indicate either the coexistence of antiparallel and parallel folded species or a parallel-antiparallel hybrid G-quadruplex structure. Importantly, G-quadruplex DNAzymes with parallel structures were found to have much higher peroxidase activities than antiparallel ones.51 As illustrated in Figure 1c, the CD spectra showed that PS2.M and phosphorothioate PS2M-101112-3PS were both mainly in parallel structures because of the much stronger peaks at 240 (−) and 264 (+) nm than that at 295 (+) nm. In contrast, TEEP-OH modified PS2M-101112-TEEP did not have any apparent peak at 240 (−) or 264 (+) nm, and instead a slightly stronger peak at 295 (+) nm over the original PS2.M arose. This result indicated that TEEP-OH modification almost eliminated the peroxidase-active parallel G-quadruplex structures and dominated the antiparallel ones, in accordance with the observation activity inhibition in the above ABTS-based activity measurement. After irradiating by UV light for 20 min, PS2M-101112-TEEP+UV had peaks at 240 (−) and 264 (+) nm emerged again along with that at 295(+) nm weakened, suggesting the formation of active parallel G-quadruplex structures. Therefore, the peroxidase activation of PS2M-101112-TEEP+UV was very likely, at least partially, contributed by the formation of active parallel G-quadruplex structures by light from the photocaged antiparallel ones (Scheme 1b).



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Figure 2. (a) The sequences of TBA, TBA-G1-1PS, TBA-G1G5-2PS and TBA-T3T4-2PS (“*” indicates phosphorothioate), and The 20% denatured PAGE images of TBA-G1-1PS, TBA-G1G5-2PS and TBA-T3T4-2PS before (lane 1, 4 and 7) and after TEEP-OH modifications (lane 2, 5 and 8), as well as subsequent removal of TEEP-OH by UV light (lane 3, 6 and 9). (b) Fibrinoge-based coagulation time measurement of blank (no TBA), TBA, TBA-G1G5-TEEP and TBA-G1G5-TEEP+UV. (c) Fibrinoge-based coagulation time measurement of blank (no TBA), TBA, TBA-T3T4-TEEP and TBA-T3T4-TEEP+UV. The experimental condition: 300 nmol/L aptamer (none for blank), 0.1 U/mL thrombin (Th), 2 mg/mL fibrinogen (Fib), 20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2 at 25 o C

Table 1. Half time (t0.5Amax) of thrombin-catalyzed fibrinogen coagulation in the absence and presence of TBA and its TEEP-OH derivatives before and after UV light irradiation.

Samples

No TBA

TBA

TBA-G1TEEP

TBA-G1TEEP+UV

TBA-G1G5TEEP

TBA-G1G5TEEP+UV

TBA-T3T4TEEP

TBA-T3T4TEEP+UV

Time at A= 0.5●Amax/s

162±6

1910±8

397±19

1980±35

157±3

1240±71

463±13

1838±50

To understand why the activity of PS2M-101112-TEEP was not fully recovered by UV light while the decaging seemed complete in the gel data of Figure 1a, first, we tried fully reviving the UV-treated PS2M-101112-TEEP by unfolding/refolding via heating to 95oC and cooling down to 25oC. However, it gave little change to the activity of the UV-treated DNAzyme, as the results shown in Figure S10A (Supporting Information). Then, we also irradiated the native DNAzyme by UV light, and found the DNAzyme was almost of the same activity, excluding the possibility of UV light damage (Figure S10B). The UV-treated DNAzyme solution was subjected to ultrafiltration by Amicon-3K, and still gave little activity loss (Figure S10B). Based on these results and the data in the above paragraphs, we propose that the photolysis small molecular byproducts might interfere with G-quadruplex structure of the DNAzyme by a strong non-covalent binding interaction, which reduced its activity. Photocaged thrombin aptamer containing DNA Gquadruplex In addition to G-quadruplex DNAzymes, we further used this TEEP-OH-based caging method for developing photocaged G-quadruplex aptamers, to explore its generality in photocaging G-qudruplex DNAs with different functions. One thrombin-binding aptamer (TBA) sequence,25 a 15-mer oligonucleotide with unimolecular Gquadruplex structure,8 is well known for its outstanding thrombin binding and coagulation inhibition activities by

occupying the thrombin’s fibrinogen-binding pocket.56 We took this TBA sequence (5'G1G2T3T4G5G6T7G8T9G10G11T12T13G14G15-3') as an example to construct a photocaged G-quadruplex aptamer (Figure 2a). Its target, thrombin, known as a multifunctional serine protease, plays a pivotal role in the regulation of tissue homeostasis and anticoagulation therapy for many diseases.57 Therefore, engineering oligonucleotides based on TBA that specifically targets thrombin and is precisely modulated by light irradiation, might greatly expand its usefulness in medicine and pharmacy.31 It was reported that the TBA sequence had a highly symmetrical structure with two TT and one TGT loops, and the binding with thrombin occurred at the fibrinogen exosite through the TT loops.36, 58 We anticipated that TEEP-OH modifications near these sites might result in efficient caging effects (Scheme 1c), thus phosphorothioate-containing TBA-G1-1PS, TBA-G1G5-2PS, TBA-T3T4-2PS were selected as candidates (Figure 2a). As shown in the results of PAGE (Figure 2a) and MALDI-TOF MS (Figure S4-S6, Supporting Information) analysis, for each of the oligonucleotides, TEEP-OH modification resulted in upshift in PAGE while photolysis products had the same gel shift as the original DNA. MALDI-TOF peaks also demonstrated the formation of native TBA instead of phosphorothioate DNA from the photocaged form after UV light irradiation, suggesting the high efficiency in


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TEEP-OH modifications and their subsequent removal by light. To quantify the activity of TBA in inhibiting thrombininduced coagulation of fibrinogen, coagulation time measurement based on UV-Vis monitoring was carried out according to the previous literatures.26, 36 The method was based on the enhancement of UV absorption at 280 nm (UV280) when thrombin-catalyzed coagulation of fibrinogen occurred. As shown in Figure 2b, 2c, Table 1 and Figure S7 (Supporting Information), thrombin catalyzed very fast fibrinogen coagulation (coagulation half time, t0.5Amax = 162 s). However, in the presence native TBA as inhibitor, UV280 of the solution containing thrombin and fibrinogen increased very slowly (t0.5Amax = 1910 s), indicating efficient inhibition of thrombin by TBA. When any of the three TEEP-OH modified TBAs was used instead of native TBA, the solutions underwent much faster enhancement in UV280 (t0.5Amax = 397, 157 and 463 s for TBA-G1-TEEP (Figure S7), TBA-G1G5-TEEP (Figure 2b) and TBA-T3T4-TEEP (Figure 2c), respectively) than native TBA, suggesting the photocaged TBAs could not inhibit thrombin efficiently (TBA-G1-TEEP and TBA-T3T4-TEEP) or at all (TBA-G1G5-TEEP). After UV irradiation, the TEEPOH modifications on these caged TBAs were removed, and recovered efficient thrombin inhibition (t0.5Amax = 1980, 1240, 1838 s, e.g. 5-, 8-, 4-fold activity activation, for TBAG1-TEEP+UV, TBA-G1G5-TEEP+UV, TBA-T3T4-TEEP+UV, respectively according to Table 1). Control samples with only photocaged TBAs or fibrinogen in the absence of thrombin were also tested to exclude the effect of photocaged TBAs on the abosorbance change, whether with light irradiation or not (Figure S8, Supporting Information). The kinetics for decaging TBA-G1G5-TEEP by light was also monitored (Figure S9, Supporting Information), displaying gradual activity recovery over time under UV irradiation.

CONCLUSIONS In summary, we developed a general method for preparing photocaged G-quadruplex DNAzymes and aptamers based on postsynthetic modification of a photolabile TEEP-OH on DNA phosphodiester backbone. Through this method, both peroxidase-mimicking DNAzyme and thrombin-binding aptamer were successfully caged by the TEEP-OH modifications adjacent to their active site regions. Upon light irradiation, the photocaged G-quadruplex were converted into their native form without any artificial scar, and the original activities were recovered. The method could be further used for the temporal and spatial regulations of Gquadruplex DNAs with different functions in biological systems.

EXPERIMENTAL SECTION Synthesis of TEEP-OH-modified G-quadruplex DNAzyme and Aptamer: 30 μL solution of 333 nM Gquadruplex DNAzyme or aptamer w/wo phosphorothioates in 100 mM sodium phosphate at pH 6.0 was added 10 μL 40 mM 2-bromo-4'-hydroxyacetophenoe

in DMF. The solution was kept on a roller at room temperature for 60 h. The resulting solution was purified by Amicon-3K ultrafilters using 10 mM sodium phosphate buffer at pH 6.0 to remove excess 2-bromo-4'hydroxyacetophenoe for 6 times (or water to remove salts for MALDI-TOF analysis). The Absorption Measurement of Peroxidasemimicking DNAzyme in ABTS Oxidation: 1.5 μL of 30 μM peroxidase-mimicking PS2M DNAzyme was added into 170 μL Buffer A firstly, and then UV light irradiation at 300 nm for 20 min was applied to the DNAzymes prior to the experiment if necessary. After that, 1.5 μL of 60 μM hemin, 3 μL of 60 mM ABTS and 3 μL of 60 mM H2O2 were sequentially added. The final concentrations of DNAyme, hemin, ABTS and H2O2 were 0.25 μM, 0.5 μM, 1 mM and 1 mM, respectively. After vortexing for 10 s, the solution was transferred into a JASCO V-550 UV-Vis spectrophotometer. Time-dependent absorption measurement at 280 nm was performed at 25oC after zeroing the starting absorbance. The Absorption Measurement of Thrombin Aptamer in Thrombin Inhibition: 3.6 μL of 10 μM thrombin aptamer was added into 109 μL Buffer B, and then UV light irradiation at 300 nm for a proper time was applied to the aptamers prior to the experiment if necessary. After that, 0.6 μL of 20 U/mL thrombin and 6 μL of 40 mg/mL fibrinogen were sequentially added. The final concentrations of thrombin, aptamer, fibrinogen were 0.1 U/mL, 300 nmol/L, 2 mg/mL respectively. After vortexing for 10 s, the solution was transferred into a JASCO V-550 UV-Vis spectrophotometer. Time-dependent absorption measurement at 280 nm was performed at 25oC after zeroing the starting absorbance. Circular Dichroism Spectrum of Peroxidasemimicking DNAzyme: circular dichroism (CD) spectra of PS2M were recorded at 25oC on a Chirascan Plus spectropolarimeter over the range of 220-320 nm using a 1 cm path length quartz cuvette. In each sample, 400 μL of 6 μM DNAzyme in Buffer A were used, and UV light irradiation at 300 nm for 20 min was applied to the DNAzymes prior to the experiment if necessary. The DNAzyme samples for CD measurement were each with a 3' A6 tag (AAAAAA) to improve light polarization. PAGE Imaging of TEEP-OH-modified G-quadruplex DNAzyme and Aptamer: a solution of 10 μL 10 μM each DNA sample w/wo TEEP-OH modifications in water was mixed with 10 μL loading buffer containing 1 M sucrose and 6 M urea. UV light irradiation at 300 nm for 20 min was applied to the samples prior to the experiment if necessary. Each solution was loaded to one lane of a 20% denatured polyacrylamide gels for PAGE analysis. Calculation of Apparent Rate Constants (kcat) of Peroxidase-mimicking DNAzyme: the apparent rate constant (kcat) was quantified by the amount of substrate consumed by per DNAzyme per second. The formula used to calcluate kcat was kcat = k30 /(ε●c), where k30 is the slope of the first 30 seconds in the absorbance measurement, ε is the molar absorption coefficient of product ABTS− (31100 M/cm), and c is the concentration of DNAzyme.



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ASSOCIATED CONTENT

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Supporting Information, containing detail information of materials, instrument and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author Yu Xiang* Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China. E-mail: [email protected]

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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ACKNOWLEDGMENT

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We are thankful for the ﬁnancial support from the National Natural Science Foundation of China (Nos. 21621003, 21675097, 21405091, 20131351079 and 20131329159), National Key Scientific Instrument and Equipment Development Project of China (2012YQ030111), Tsinghua University Initiative Scientific Research Program (20131089220), and the Recruitment Program of Global Youth Experts of China.

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Graphic entry for the Table of Contents (TOC)


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TOC graphic 55x50mm (300 x 300 DPI)



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Scheme 1. (a) Introducing TEEP-OH into the phosphodiester backbone of phosphorothioate-containing Gquadruplex DNA through the reaction with bromoacetophenone, as well as the conversion of TEEP-OH into a native phosphodiester by UV light. (b) Photocaging the peroxidase activity of G-quadruplex DNAzyme by TEEP-OH and its decaging by UV light. (c) Photocaging the thrombin inhibition activity of G-quadruplex aptamer (TBA) by TEEP-OH and its decaging by UV light. 176x213mm (144 x 144 DPI)


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Figure 1. (a) Sequences of PS2.M, PS2M-11-1PS and PS2M-101112-3PS (“*” indicates phosphorothioate), and 20% denatured PAGE images of PS2M-C11-1PS and PS2M-G10C11G12-3PS before (lane 1 and 4) and after TEEP-OH modifications (lane 2 and 5), as well as subsequent removal of TEEP-OH by UV light (lane 3 and 6). (b) ABTS-based peroxidase activity measurement of PS2.M (black squares), PS2M-11-TEEP (green triangles), PS2M-101112-TEEP (red diamonds), PS2M-11-TEEP+UV (purple crosses) and PS2M-101112TEEP+UV (blue stars). The experimental condition: 0.25 µM DNAzyme, 0.5 µM hemin, 1 mM ABTS, 1 mM H2O2, 50 mM HEPES at pH 6.9, 100 mM NaCl, 20 mM KCl and 0.03% Triton X-100 at 25oC. (c) CD spectra of PS2.M (blue squares), PS2M-101112-3PS (purple stars), PS2M-101112-TEEP (green dots) and PS2M101112-TEEP+UV (black triangles). The experimental condition: 6 µM DNA, 50 mM HEPES, pH 6.9, 100 mM NaCl and 20 mM KCl at 25oC. 277x80mm (96 x 96 DPI)



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Figure 2. (a) The sequences of TBA, TBA-G1-1PS, TBA-G1G5-2PS and TBA-T3T4-2PS (“*” indicates phosphorothioate), and The 20% denatured PAGE images of TBA-G1-1PS, TBA-G1G5-2PS and TBA-T3T42PS before (lane 1, 4 and 7) and after TEEP-OH modifications (lane 2, 5 and 8), as well as subsequent removal of TEEP-OH by UV light (lane 3, 6 and 9). (b) Fibrinoge-based coagulation time measurement of blank (no TBA), TBA, TBA-G1G5-TEEP and TBA-G1G5-TEEP+UV. (c) Fibrinoge-based coagulation time measurement of blank (no TBA), TBA, TBA-T3T4-TEEP and TBA-T3T4-TEEP+UV. The experimental condition: 300 nmol/L aptamer (none for blank), 0.1 U/mL thrombin (Th), 2 mg/mL fibrinogen (Fib), 20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2 at 25 oC 177x53mm (300 x 300 DPI)


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Photocaged G-Quadruplex DNAzyme and Aptamer by Post-Synthetic

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