Articles pubs.acs.org/acschemicalbiology
Postsynthetic Modification of DNA Phosphodiester Backbone for Photocaged DNAzyme Xiaoyan Wang,† Mengli Feng,† 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 S Supporting Information *
ABSTRACT: Photocaged (photoactivatable) biomolecules are powerful tools for noninvasive control of biochemical activities by light irradiation. DNAzymes (deoxyribozymes) are single-stranded oligonucleotides with a broad range of enzymatic activities. In this work, to construct photocaged DNAzymes, we developed a facile and mild postsynthetic method to incorporate an interesting photolabile modification (thioether-enol phosphate, phenol substituted, TEEP−OH) into readily available phosphorothioate DNA. Upon light irradiation, TEEP−OH transformed into a native DNA phosphodiester, and accordingly the DNAzymes with RNAcleaving activities were turned “on” from its inactive and caged form. Activation of the TEEP−OH-caged DNAzyme by light was also successful inside live cells.
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ight is a noninvasive method for spatially and temporally controlling the activities of biomolecules. Photocaged (photoactivatable) biomolecules,1−6 including proteins and nucleic acids with photolabile groups,7,8 undergo light-induced activation of their activities and have been widely applied for regulating a serial of important biological functions by light in vitro and in vivo.9−20 Developing new approaches and chemistry for photocaged biomolecules, therefore, is of increasing interest. DNAzyme, also called deoxyribozyme or catalytic DNA, is a family of single-stranded DNA molecules with a broad range of catalytic activities beyond genetic information.21−27 The sequence of DNAzyme with a specific activity can be obtained from a random DNA library by a combinatorial technique named in vitro selection.21−23 Since the 1990s, DNAzymes with catalytic functions such as nuclease,21,23,24,28,29 ligase,22,30 and kinase31 have been identified and subsequently utilized for biosensing,27,29,32−39 gene regulation,35,38,40,41 and nanotechnology.27,42−47 For more specific and precise control of target sensing and gene expression, DNAzymes are equipped with photoreactive groups for developing photocaged DNAzymes,48−55 whose activities can be switched on and off by light irradiation. For example, azobenzene derivatives49,51,56,57 and photocleavable linkers53 were modified on DNA backbones to tune the activities of DNAzymes by light reversibly and irreversibly, respectively. Photolabile 2-nitrobenzyl derivatives were attached on DNA nucleobases to construct light-activated DNAzymes and antisense DNA for regulation of protein expression.52,54 The 2-nitrobenzyl modification on the 2′-OH of a RNA nucleoside in the RNA substrate also indirectly inhibited the RNA-cleaving activity of DNAzymes until the 2′OH was restored by light irradiation.48,55 © XXXX American Chemical Society
Although the photocaged DNAzymes based on the above methods are highly efficient, unfortunately they all require the incorporation of modified phosphoramidite monomers containing photoreactive groups into DNA during the solid-phase synthesis.48,50,52,53 This is also the case for a series of methods used to develop photocaged DNA13,16,20 for other applications beyond DNAzymes. Unlike the standard phosphoramidite monomers routinely used for solid-phase synthesis (e.g., dA, dT, dG, and dC phosphoramidites), the noncanonical monomers are complicated to synthesize and usually need case-by-case optimization of coupling/deprotection conditions. Alternatively, postsynthetic modifications (e.g., modifications of DNA after standard solid-phase synthesis) are much simpler and can be carried out under mild conditions, which are also compatible with a broader range of chemical modifications on DNA.58,59 Previously reported techniques using postsynthetic modifications for photocaged DNA or RNA were successful in tuning the transcription and translation functions of nucleic acids by light irradiation.16,60−62 However, the diazo-based methods were nonspecific to all the nucleotides, and the exact structure of the modification on each nucleotide was not characterized,60,61 making the rational design of this method for other applications challenging. On the other hand, the methods based on site-specific postsynthetic modifications of photocaging groups on modified DNA usually left an artificial “scar” Received: October 23, 2015 Accepted: December 3, 2015
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DOI: 10.1021/acschembio.5b00867 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 1. Synthesis of DEPT−OH and TEEP−OH from diethylthiophosphate in the presence of first eq and second eq 2-bromo-4′hydroxyacetophenone, respectively.
on the DNA after light irradiation,16,62 which could significantly reduce the original activity of the native nucleic acids. In this work, we developed a postsynthetic method under mild conditions to photocage DNAzymes by site-specifically incorporating an interesting photolabile group (thioether-enol phosphotriester, phenol-substituted, TEEP−OH, Figure 1a) into the phosphodiester backbone of DNA. TEEP−OH was efficiently formed in buffer solutions via the reaction between 2bromo-4′-hydroxyacetophenone63−66 and readily available phosphorothioate DNA,67−75 which was obtained simply through replacing iodine by sulfur in the standard automated solid-phase synthesis of DNA. With TEEP−OH modifications to the “active site” regions of 8−1750,76 and 10−2352,53,76 DNAzymes, the RNA-cleaving activity of these photocaged DNAzymes was significantly inhibited or completely abolished. Upon light irradiation at 365 nm, although prepared from phosphorothioate DNA, the photocaged DNAzymes were restored to the native DNA format containing phosphodiesters instead of phosphorothioate and underwent activity “turn-on” from the completely inactive caged form. The photocaged 8− 17 DNAzyme was also delivered into HeLa cells with its substrates, and the light-induced activation was found compatible with the intracellular environment, suggesting its potential usefulness for cellular studies.
Figure 2. Crystal structure of TEEP-NO2.
phosphoric acid as the 0 ppm standard), DEPT−OH (27.05 ppm), and TEEP−OH (−5.06 ppm) supported that a phosphotriester (OP(OR)3, typically −20 to 0 ppm)77 instead of a thiophosphotriester (OP(OR)2SR, typically 24− 80 ppm)77 was present in TEEP−OH (Figure S2, Supporting Information). Upon light irradiation at 365 nm by a 12 W hand-held UV lamp, both TEEP−OH and DEPT−OH underwent photolysis as indicated by TLC (Figure S3, Supporting Information) and yielded diethylphosphate (m/z = 153.0) and diethylthiophosphate (m/z = 169.0) as the products for TEEP−OH and DEPT−OH (Figure 3), respectively, according to the ESI-MS (negative ion) analysis of the mixtures after light irradiation (Figure S4, Supporting Information). The light-induced conversion of TEEP−OH to diethylphosphate, an analogue of native DNA phosphodiester, suggested that TEEP−OH could be a photoremovable postsynthetic modification of DNA without a scar (e.g., yielding native DNA as photolysis instead of phosphorothioate DNA). Further investigation of the photolysis suggested that UV light was likely essential for the conversion of PTET (partially formed from DEPT−OH in CH3CN/Cs2CO3) to diethylphosphate, indicating PTET was a photolabile group (Figure S5, Supporting Information). The fate of the hydroxyacetophenone moiety upon light irradiation was reported to be complicated,63−66 and we currently could not get a clear major product. We proposed that the mechansim might be through either β-elimination or hydrolysis of a light-activated PTET* (Figure 3). Calculation of free energy using Gaussian (Figure S6, Supporting Information) suggested that a five-memberedring intermediate with a high free energy was present between
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RESULTS AND DISCUSSION Formation and Photolysis of TEEP−OH. We used the diethylthiophosphate salt as a small molecular mimic of phosphorothioate DNA nucleotide to study the formation and photolysis of the interesting photolabile group TEEP−OH (Figure 1). TEEP−OH was synthesized unexpectedly by reacting diethylthiophosphate with excess (2 equiv) 2-bromo4′-hydroxyacetophenone, while the usual diethylphosphothioester (DEPT−OH) was the product when diethylthiophosphate was treated with only 1 equiv of bromoacetophenone under the same conditions, suggesting DEPT−OH was the intermediate for the formation of TEEP−OH (Figure 1) in the presence of bromoacetophenone and base (K2CO3). The X-ray single crystal structure of TEEP-NO2 (Figure 2), an analogue of TEEP−OH prepared similarly using 2-bromo-4′nitroacetophenone, strongly indicated that the structure of TEEP−OH was a thioether-enol phosphotriester. We are currently unable to obtain the single crystals of TEEP−OH likely because the phenol groups complicated the crystallization. Such a proposed structure for TEEP−OH was further confirmed by ESI-MS and 1H, 13C NMR characterizations of the compound (Figure S1, Supporting Information). The 31P NMR peaks of diethylthiophosphate (53.99 ppm, using B
DOI: 10.1021/acschembio.5b00867 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 3. Proposed mechanism for the photochemical conversion of TEEP−OH and DEPT−OH into diethylphosphate and diethylthiophosphate, respectively.
Figure 4. (a) Postsynthetic modification of phosphorothioate DNA with TEEP−OH and the subsequent removal by light irradiation to form native DNA phosphodiester. (b) The sequences of 8−17 (left) and 10−23 (right) DNAzymes (green) along with their substrates (red). The blue letters with “*” indicate the phosphorothioates at the 3′ of the nucleotides. The black arrows indicate the cleavage sites of the substrates. (c) Images of 20% denatured PAGE showing the modification and removal of TEEP−OH from 8−17 (left) and 10−23 (right) DNAzymes containing 1 or 3 phosphorothioates.
PTET was favored under basic conditions (deprotonated). This energy barrier significantly inhibited the interconversion under acidic or neutral conditions and gave PTET (partially formed
the interconversion of PTET (ΔG = 75.3 kJ/mol to the intermediate) and DEPT−OH (ΔG = 107.5 kJ/mol to the intermediate) in their neutral forms, while the conversion to C
DOI: 10.1021/acschembio.5b00867 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 5. (a) Removal of TEEP−OH by light irradiation to activate photocated DNAzymes, and the fluorescence enhancement induced by DNAzyme-catalyzed cleavage of dually labeled substrates. (b) The activation of 8−17 DNAzyme with one TEEP−OH modification by UV light. (c) The activation of 8−17 DNAzyme with three TEEP−OH modifications by UV light. (d) Images of 20% denatured PAGE showing the cleavage of fluorophore-labeled substrate by photocaged DNAzyme (8−17 DNAzyme with three TEEP−OH modifications) only after light irradiation. (e) The activation of 10−23 DNAzyme with one or three TEEP−OH modifications by UV light.
Postsynthetic Modification of TEEP−OH on DNA and Its Photoactivation. The modification of TEEP−OH on phosphorothioate DNA was then carried out using a T15 DNA containing one phosphorothioate (T15−1PS) at the central nucleotide to validate the methodology (Figure 4a). The postsynthetic modification was initiated by mixing excess 2bromo-4′-hydroxyacetophenone (8 mM) with the phosphorothioate DNA (0.5 mM) in a sodium phosphate buffer/DMF solution at pH 6.0 at 37 °C for 6 h. Both PAGE and MALDITOF analysis confirmed the successful modification of TEEP− OH to the phosphorothioate DNA as well as the subsequent transformation to native DNA by UV light irradiation at 365 nm using a 12 W hand-held UV lamp (Figure S8a snd b, Supporting Information). The yield of the TEEP−OH modification on T15−1PS was over 95% according to the PAGE image (Figure S8a, Supporting Information), suggesting the high efficiency of the modification. Although the TEEP−
from TEEP−OH) and DEPT−OH completely different fates and products upon light irradiation. On the other hand, the moderate stability of thiophosphotrieste (such as DEPT−OH) in aqueous solutions has been a major limitation for the utilization of readily available phosphorothioate DNA in postsynthetic modifications.67,68 TEEP−OH was found highly stable even under basic pH and elevated temperatures, where DEPT−OH was completely hydrolyzed (Figure S7, Supporting Information). Therefore, TEEP−OH and its derivatives (e.g., products by reacting with other bromoacetophenones) could be ideal modifications on phosphorothioate DNA to overcome the stability issue. The facile synthesis from diethylthiophosphate (phosphorothioate DNA), high stability, and the light-induced transformation into diethylphosphate (native DNA phosphodiester) made TEEP−OH a very promising postsynthetic modification for photocaged DNA. D
DOI: 10.1021/acschembio.5b00867 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology Table 1. Apparent Rate Constants (kcat) of 8−17 and 10−23 DNAzymes in the Presence or Absence of TEEP−OH Modifications before and after UV Light Irradiation kcat (min−1)a
native
1PS
1PS-TEEP
1PS-TEEP+UV
3PS
8−17 DNAzyme 10−23 DNAzyme
0.47 ± 0.02 0.34 ± 0.02
0.44 ± 0.03 0.28 ± 0.02
0.19 ± 0.01 0.04 ± 0.01
0.45 ± 0.02 0.31 ± 0.04
0.32 ± 0.02 0.20 ± 0.03
3PS-TEEP b
inactive inactiveb
3PS-TEEP+UV 0.40 ± 0.01 0.24 ± 0.02
a Buffer for 8−17 DNAzymes: 0.5 mM Zn(NO3)2, 100 mM NaCl, 100 mM MOPS, pH 7.0. Buffer for 10−23 DNAzymes: 10 mM MgCl2, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. b“Inactive” means little substrate cleavage (kcat < 0.01 min−1).
OH group in DNA was a mixture of two diastereomers because of the chiral phosphorus, each diastereomer should be converted to the same native DNA phosphodiester (achiral phosphorus) after light irradiation (Figure 4a). Encouraged by the above results showing efficient modification of DNA by TEEP−OH and successful recovery of native DNA phosphodiester by light, we further incorporated TEEP−OH into 8−17 DNAzyme (Figure 4b) that catalyzed the cleavage of its nucleic acid substrate in the presence of Zn2+ as a cofactor. Considering that introducing TEEP−OH into the substrate binding arms of the DNAzyme to reduce substrate binding affinity might not be efficient for a significant lightinduced activity enhancement, we instead chose the “active site” region of the DNAzyme as the caging target. The three nucleotides in the blue “GAG” region of 8−17 DNAzyme (Figure 4b) were previously found essential for the catalytic activity, and the phosphodiesters also played a critical role.76,78 We anticipated that the modification of TEEP−OH in this region could either block the essential phosphodiester or disrupt the DNA secondary structure of the DNAzyme for efficient caging. Therefore, 8−17 DNAzymes containing one or three phosphorothioates in “GAG” were utilized as precursors for preparing phtocaged DNAzymes (Figure 4a). As illustrated in the results of PAGE (Figure 4c) and MALDI-TOF analysis (Figure S9, Supporting Information), the phosphorothioate in 8−17 DNAzyme with a phosphorothioate G (8−17−1PS) was almost completely transformed (>95% yield based on the PAGE image) into TEEP−OH (8−17−1PS-TEEP), and the TEEP−OH was then efficiently restored to a native DNA phosphodiester (8−17−1PS-TEEP+UV) by light irradiation. Labeled substrates were used to measure the activities of the unmodified, photocaged, and photoactivated DNAzymes, respectively (Figure 5a). The percentage of substrate cleavage was calculated by the fluorescence enhancement (Figure 5b), and the rate constant was estimated based on the fluorescence kinetics (Table 1). One TEEP−OH modification in 8−17− 1PS-TEEP reduced the activity to 40% of the native 8−17 DNAzyme, and 1.4-fold activity enhancement was observed after light irradiation for 15 min (Figure 5b and Table 1). Since multiple TEEP−OH modifications could be easily incorporated into one DNAzyme as long as readily available phosphorothioates were present on the nucleotides of interest, we were then more aggressive to add three TEEP−OH groups into 8− 17−3PS phosphorothioate DNAzyme for more efficient light activation (e.g., larger fold of activity enhancement, and ideally an off-on switch). With three TEEP−OH modifications validated by PAGE (with yield >95% from the image, Figure 4c) and MALDI-TOF MS (Figure S10, Supporting Information), 8−17−3PS-TEEP was completely inactive, while the activity was recovered to 85% of the native 8−17 DNAzyme after light irradiation (Figure 5c and Table 1), achieving a remarkable light-induced activity “turn on” from completely “off.” PAGE analysis of the substrate cleavage was in accordance with the fluorescence measurement (Figure 5d). In fact, we also
examined the modification of two phosphorothioates with TEEP−OH in the “GAG” site to see whether three TEEP− OH groups were all necessary to “turn off” the DNAzyme activity. As shown in Figure S11 (Supporting Information), none of the 2-TEEP−OH-modified DNAzyme was completely inactive. Similar to some other studies, it was common that photoactivation could not fully recover the activity of photocaged DNAzymes to that of the original DNAzymes, likely because the photolysis of photolabile groups could hardly go to completion.50,52 Nevertheless, we still observed that the activity of TEEP−OH-caged DNAzymes after photoactivation (8−17−1PS-TEEP+UV and 8−17−3PS-TEEP+UV in Table 1) exceeded that of its precursor DNAzyme containing one or three phosphorothioates (8−17−1PS and 8−17−3PS in Table 1), suggesting the formation of native phosphodiesters rather than phosphorothioates by light irradiation was beneficial for the activity of the DNAzymes. The sensitivity of the DNAzymes’ activity to the cofactor Zn2+ showed very similar trends for 8−17−3PS, 8−17−3PS-TEEP, and 8−17−3PSTEEP+UV (Figure S12, Supporting Information). In addition to 8−17 DNAzyme, we also modified the Mg2+dependent 10−23 DNAzyme by TEEP−OH to testify the usefulness of TEEP−OH as a general method to cage different DNAzymes (Figure 4b). The efficient modification of TEEP− OH on DNAzymes and the subsequent removal by light were confirmed by PAGE (with yield >95% from the image, Figure 4c) and MALDI-TOF MS (Figure S13 and S14, Supporting Information). Introducing one TEEP−OH group at the C nucleotide in the “active site” region “CTA” of 10−23 DNAzyme76 (10−23−1PS-TEEP) caused 88% activity loss compared to that of the native 10−23 DNAzyme, as well as 6.8fold activity enhancement after photoactivation (Figure 5d and Table 1). Similarly to the case of 8−17 DNAzyme, 3-TEEP− OH-caged 10−23 DNAzyme (10−23−3PS-TEEP) was completely inactive, while 70% activity of the native 10−23 DNAzyme was turned on after light irradiation for 15 min (Figure 5d and Table 1). The installation of three TEEP−OH groups on the DNAzyme was also found necessary for the complete turning “off” of the DNAzyme activity (Figure S11, Supporting Information). Activation of Photocaged DNAzyme Inside Live Cells. Because 3-TEEP−OH-caged 8−17 DNAzyme (8−17−3PSTEEP) was completely inactive and the TEEP−OH modification was highly stable (Figure S7, Supporting Information), we then combined the photocaged DNAzyme with its substrate and delivered the mixture with lipofectamine into live cells to study their biocompatibility in a cellular environment (Figure 6). The photocaged DNAzyme and substrate were both dually labeled to minimize their degradation and background fluorescence in a cellular environment. A fluorescence enhancement should be present when the substrate was cleaved by the DNAzyme (Figure 5a). We observed more than 8-fold activity enhancement of the photocaged DNAzyme in diluted HeLa cell lysates after light E
DOI: 10.1021/acschembio.5b00867 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00867. Materials and instruments, synthesis, experimental procedures, and additional figures (PDF) Crustallographic information (CIF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions †
Figure 6. Fluorescence images (overlap with bright field) of live HeLa cells after delivery of 3-TEEP−OH-photocaged 8−17 DNAzyme (dually labeled) and its substrate (dually labeled) by lipofetamine. (a− c) Without UV irradiation, (d−f) with UV irradiation at 365 nm for 20 min. The time label in each figure indicates how long the cells were incubated to allow the DNAzyme-catalyzed reaction to proceed after UV irradiation (or not) and the addition of 100 μM Zn2+.
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are thankful for the financial support from the National Natural Science Foundation of China (Nos. 2142200199, 21390410 and 21375074), the National Key Scientific Instrument and Equipment Development Project of China (No. 2012YQ030111), the Tsinghua University Initiative Scientific Research Program (No. 20131089220), and the Recruitment Program of Global Youth Experts of China.
irradiation for 15 min (Figure S15, Supporting Information), indicating that the photoactivation was tolerant of the cellular components such as proteins and metabolites. Next, the 3TEEP−OH-photocaged 8−17 DNAzyme were delivered with the substrate into live HeLa cells54,55 and then activated by light irradiation at 365 nm for 20 min, after which Zn2+ was added as a cofactor to accelerate the DNAzyme-catalyzed fluorescence enhancement. As shown in Figure 6a−c, almost no fluorescence enhancement was observed for the cells in the dark regardless of the incubation time after the supplement of Zn2+, while those with light irradiation displayed much faster fluorescence enhancement upon incubation after Zn2+ addition, as a result of activation of the DNAzyme by light. The small green spots were likely the particles of DNA-lipofectamine complex adsorbed on the surface of cells, and their fluorescence underwent very mild changes during the experiment. These data suggested that the photocaged DNAzyme retained its light-induced activity enhancement inside live cells; therefore it could be promising for further cellular studies such as lightcontrolled cellular biosensing and gene regulation.11 Conclusion. In summary, we developed a facile postsynthetic method to prepare photocaged DNAzymes by incorporation an interesting photolabile group TEEP−OH into readily available phosphorothioate DNA under mild conditions. The TEEP−OH molecule with an unusual structure was formed by the reaction of phosphorothioate with 2 equiv of 2-bromo-4′-hydroxyacetophenone. Upon light irradiation, TEEP−OH underwent photolysis and transformed into the native DNA phosphodiester without a scar. Both 8−17 and 10−23 DNAzymes were successfully caged by TEEP−OH modifications specifically in their “active site” regions. After light irradiation, the photocaged DNAzymes containing three TEEP−OH groups showed a significant activity “off−on” switch. The activation of the photocaged DNAzyme by light was also found compatible with an intracellular environment, suggesting the promise of TEEP−OH-caged DNAzymes and nucleic acids for cellular applications.
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