uril-driven host-guest chemistry for reversible intervention of 5

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Cucurbit[7]uril-driven host-guest chemistry for reversible intervention of 5-formylcytosine-targeted biochemical reactions Shao-Ru Wang, Yan-Yan Song, Lai Wei, Chao-Xing Liu, Bo-Shi Fu, JiaQi Wang, Xi-Ran Yang, Yi-Nong Liu, Si-Min Liu, Tian Tian, and Xiang Zhou J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09635 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Journal of the American Chemical Society

Cucurbit[7]uril-driven host-guest chemistry for reversible intervention of 5-formylcytosine-targeted biochemical reactions Shao-Ru Wang‡†, Yan-Yan Song‡†, Lai Wei†, Chao-Xing Liu†, Bo-Shi Fu†, Jia-Qi Wang†, Xi-Ran Yang§, Yi-Nong Liu†, Si-Min Liu§, Tian Tian*†, Xiang Zhou*† †College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Wuhan 430072, Hubei Province, China. Fax: (+)86-27-68756663 §College of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan 430081, Hubei Province, China KEYWORDS: cucurbit[7]uril · host-guest chemistry · 5-formylcytosine · restriction endonuclease digestion · DNA polymerase elongation · polymerase chain reaction ·

ABSTRACT: The 5-formylcytosine (5fC) is identified as one of key players in active DNA demethylation and also an epigenetic mark in mammals, thus representing a novel attractive target to chemical intervention. The current study represents an attempt to develop a reversible 5fC-targeted intervention tool. A supramolecular aldehyde reactive probe was therefore introduced for selective conversion of the 5fC to 5fC-AD nucleotide. Using various methods, we demonstrate that, the cucurbit[7]uril (CB7) selectively targets the 5fC-AD nucleotide in DNA, however, the binding of CB7 to 5fC-AD does not affect the hydrogen bonding properties of natural nucleobases in duplex DNA. Importantly, CB7-driven host-guest chemistry has been applied for reversible intervention of a variety of 5fC-targeted biochemical reactions, including restriction endonuclease digestion, DNA polymerase elongation and polymerase chain reaction. Based on the current study, the macrocyclic CB7 creates obstructions that, through steric hindrance, prevent the enzyme from binding to the substrate, whereas the CB7/5fC-AD host-guest interactions can be reversed by treatment with adamantanamine. Moreover, fragmentand site-specific identification of 5fC modification in DNA has been accomplished without sequence restrictions. These findings thus show promising potential of host-guest chemistry for DNA/RNA epigenetics.

Introduction The methylation of cytosine at the C5 position forms 5methylcytosine (5mC), which represents a primary epigenetic modification in mammalian genome1. The stepwise oxidation of 5mC by the Ten-eleven translocation (TET) family of DNA dioxygenases leads to the formation of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)2-5. The mammalian thymine DNA glycosylase (TDG) has been found to excise 5fC and 5caC (but not 5hmC and 5mC) from DNA6-8, revealing an active DNA demethylation pathway. Of particular interest, the 5fC nucleotide has been found to play essential cellular functions9-14, thus representing a novel attractive target to chemical intervention. It is therefore important to develop a reversible intervention tool capable of targeting 5fC as well as probing and manipulating 5fC-associated biochemical reactions. Additionally, reversible intervention to 5fC may further aid in our understanding of its functional roles.

drophilic exterior and hydrophobic cavity with a good structural rigidity, they can bind to their guests with a high binding affinity26-28. Of the known CBn homologues, CB7 (structure in Figure 1A) displays a variety of advantages, such as the remarkable biocompatibility, high water solubility and intermediary size29-33. Recently, a number of significant advances have been made in protein research using CB7-based tool34-38. However, applications of macrocyclic host-guest chemistry as improved tool for bioapplications still remained to be explored, especially in areas of nucleic acid research. It therefore prompted us to investigate the use of CB7-driven host-guest chemistry for studying the 5fC modification.

Here we for the first time introduced a supramolecular aldehyde reactive probe (SARP, structure in Figure 1A), in which the adamantane (AD) moiety was functionalized with the hydroxylamine group. This molecule can serve as a site-selective tag for converting the 5fC to 5fC-AD nucleotide (structure in Figure 1A). Using various methods, Supramolecular science focuses on the study of the hostwe demonstrate that, the CB7 selectively targets the 5fCguest systems that are composed of a variety of assembled AD nucleotide in DNA, however, the binding of CB7 to components15-20. The cucurbit[n]uril (CBn) family of 5fC-AD does not significantly affect the hydrogen bonding macrocycles represents a particularly attractive class of properties of natural nucleobases in duplex DNA. host molecules21-25. Because CBn molecules possess a hyImportantly, CB7-driven host-guest chemistry has been ACS Paragon Plus Environment


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applied for reversible intervention of a variety of 5fCtargeted biochemical reactions, including restriction endonuclease (RE) digestion, DNA polymerase (DNA pol) elongation and polymerase chain reaction (PCR) (schematic illustration in Figure 1). Based on the current study, the macrocyclic CB7 creates obstructions that, through steric hindrance, prevents the enzyme from binding to the substrate, whereas the CB7/5fC-AD host-guest interactions can be reversed via treatment with adamantanamine (AM, structure in Figure 1A). Moreover, fragment- and site-specific identification of DNA 5fC has been accomplished without sequence restrictions. These findings thus show promising potential of host-guest chemistry for future DNA/RNA epigenetics.

FIGURE1. Schematic illustration of the design and workflow (A) and (B) Reversible intervention of 5fC-targeted DNA pol elongation and PCR reaction. The binding of CB7 to 5fC-AD nucleotide will probably generate big steric hindrance to prevent the approaching of DNA pol and inhibit enzyme activity. Moreover, the CB7/5fC-AD host-guest interaction can be reversed by treatment with AM.

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adamantane (details in Supporting information)41. Structure determination of the synthesized compounds were performed using nuclear magnetic resonance spectroscopy (NMR) and high resolution mass spectrometry (HRMS). Next, we examined whether SARP can react with 5fC nucleoside through aldoxime ligation, which was performed under acidic conditions using p-anisidine as nucleophilic catalyst40. Importantly, this reaction occurred rapidly and the structure of expected oxime product (5fCAD nucleoside in Figure 2B) was verified. Moreover, SARP did not react with a variety of other nucleosides (Figure S1). It is known that the CB7 binding tends to lead to substantial upfield shifts for the protons located inside the cavity42. The 1H-NMR experiment was then conducted to confirm the noncovalent binding of CB7 to 5fC-AD nucleoside31. Figure S2 showed the overlay view of upfield spectrum (δ = 6.3 - 0.5 ppm) of 5fC-AD nucleoside, SARP and 5fC nucleoside in the absence or presence of CB7. At the bottom is the NMR spectrum of the molecule that was dissolved in DMSO-d6 in the absence of CB7. The green box indicated the range of proton signals characteristic of the AD moiety43, and the blue one indicated the range in further higher field. As expected, the proton signals of the AD moiety appear upfield of all other peaks for SARP and 5fCAD nucleoside (Figures S2A and S2B), whereas the spectrum of the 5fC nucleoside demonstrated no proton signals in the green box (Figure S2C). At the top is the 1H-NMR spectrum of the sample obtained after the direct addition of CB7 solid until saturated. The binding of CB7 to SARP is demonstrated by the appearance of several new proton peaks in the blue box, compared with the untreated sample (Figure S2B). The expected upfield shifts of the AD proton signals shown above were also observed in the CB7-treated 5fC-AD sample (Figure S2A). Moreover, these processes were accompanied by an evident decrease in signal intensity of protons in green box, which were associated with the free AD motif (Figures S2A and S2B). On the basis of these results, CB7 is probably able to form intermolecular complex with 5fC-AD nucleoside by binding to the AD moiety42, 44.

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Supramolecular interaction between CB7 and 5fCAD nucleoside The present study aims to develop a supramolecular strategy capable of intervening in the 5fCtargeted biochemical reactions. To achieve this goal, we designed a SARP molecule, which bears two essential moieties binding to each other. The AD moiety serves as the high affinity binding motif to CB739, while the 2(adamantyl)ethoxyamine moiety serves as the labelling group targeting 5fC aldehyde40-41. As indicated in Figure 2A, the SARP molecule was readily prepared by a convenient two-step procedure, starting from 1-(2-Bromo-ethyl)-

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FIGURE2. The synthesis of SARP and 5fC-AD nucleoside (A) The synthesis route to SARP. 1) N-hydroxyphthalimide, 1,8diazabicyclo[5.4.0]undec-7-ene, dimethylformamide, 50 °C; 2)

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hydrazine hydrate, room temperature; (B) The SARP is reacted with 5fC nucleoside. 1) p-anisidine, methanol, 50 °C.

The binding of CB7 to 5fC-AD nucleotide in SARPreacted DNA Next, we were prompted to label 5fC in DNA context. In this study, the DNA containing 5fC (fCDNA1 in Table S1) was reacted with SARP in an acidic buffer (pH = 5.0) containing p-anisidine for 12 h (Figure 3A)40, 45. The DNA in the reaction mixture was then recovered by ethanol precipitation and subjected to denaturing electrophoresis. During this process, the polyacrylamide gel matrix is responsible for the separation of DNA molecules with different molecular weight. From the observations, the SARP-reacted fC-DNA1 showed an evident slower mobility (lane 2 in Figure 3B), indicating that it had a larger molecular weight compared to the unreacted DNA. These observations have also shown that the SARP labelling reaction occurs in a quantitative yield. The MALDITOF assay further indicated that the SARP-reacted DNA does contain the desired 5fC-AD nucleotide (Figure S3). Because the different modified forms of cytosine along with lesions, especially the AP site (apurinic/apyrimidinic site), are present in the DNA genome46-47, it is necessary to examine the reaction selectivity of SARP40. Hence, five individual experiments were performed using the control oligomers (C-DNA1, mC-DNA1, hmC-DNA1, caC-DNA1 or AP-DNA1 in Table S1), in which 5fC was substituted by cytosine, 5mC, 5hmC, 5caC or AP site. After the sample of these control oligomers was treated with SARP for 20 h, the recovered DNA products were analyzed. Importantly, the SARP treatment did not slow down the migrations of these DNA strands (lanes 3 -12, Figure 3B). On the basis of these results, SARP exhibits good selectivity to 5fC nucleotide in DNA. We next investigated whether CB7 can recognize 5fCAD nucleotide in DNA context. In this study, native electrophoresis was performed to examine the impact of CB7 on the apparent mobility of DNA strands. The results indicated that the CB7 significantly retards the migration of the SARP-reacted fC-DNA1 strand on a polyacrylamide gel (lane 8 in Figure 3C). By binding to 5fC-AD nucleotide the bulky CB7 led to a greater chance of collisions between the DNA molecule and the gel matrix. Those CB7-bound DNA molecules therefore experienced greater resistance and move more slowly during electrophoresis. Importantly, the CB7 treatment did not affect the movement of unreacted fC-DNA1 and also the movement of other control DNAs after SARP treatment (lanes 2, 5 in Figures 3C; lanes 2, 5, 8, 11, 14 in Figure S4). It is known that the AM molecule presents a high binding affinity with CB748-49. We therefore anticipate that this molecule can drive the removal of CB7 obstacle from 5fCAD nucleotide in DNA. Our strategic direction is fully supported and the band migration shifted back after treatment with AM (line 9 in Figure 3C). Such a phenomenon further confirmed the supramolecular interactions between CB7 and 5fC-AD nucleotide in DNA.

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FIGURE3. The CB7 binding to 5fC-AD nucleotide in DNA (A) Schematic representation of the SARP labeling of 5fC nucleotide in DNA. The reaction was performed in 40 mM sodium acetate buffer (pH = 5.0) containing p-anisidine as the catalyst. (B) Denaturing PAGE analysis of DNA strands before and after treatment with SARP. lanes 1, 3, 5, 7, 9, 11: the untreated DNA; lanes 2, 4, 6, 8, 10, 12: the SARP-treated DNA. (C) Native PAGE analysis of DNA with different treatments. The samples were prepared as described in ‘Materials and Methods’ section. DNA has been seen with GelRed staining. The binding of CB7 to 5fC-AD nucleotide significantly impedes the movement of DNA on the gel, whereas this host-guest interaction was reversed by AM treatment. lanes 1, 4, 7: no CB7/AM control; lanes 2, 5, 8: 40 µM CB7; lanes 3, 6, 9: 40 µM CB7, 50 µM AM.

Reversible 5fC-targeted intervention of RE digestion Having demonstrated host-guest interaction between CB7 and 5fC-AD nucleotide, we became interested in investigating this interaction in a biologically relevant context. RE enzymes are known to recognize and cleave duplex DNA through the sequence-specific mechanism of action50. On the basis of fact that some DNA modifications can function to sterically hinder the RE cleavage51, we hypothesized that it might be feasible to perturb RE digestion by manipulating CB7/5fC-AD binding in DNA templates. To test this, the SnaBI was employed which has CG dinucleotide in its recognition sequence. We designed and synthesized a series of double-stranded (ds) DNA substrates consisting of 5’ fluorophore-labeled oligonucleotides bearing 5fC and the complements that possess the SnaBI restriction site (fCds20mer1, fC-ds20mer2 and fC-ds20mer3 in Table S1). In these constructs, the 5fC nucleotides were located at different loci. Specifically, substrate fC-ds20mer1 possesses a single 5fC within the SnaBI recognition site. The 5fC nucleotides in fC-ds20mer2 lie outside but immediately adjacent to the SnaBI recognition site, whereas fC-ds20mer3 possesses a 5fC nucleotide outside and two bases downstream of the enzymatic recognition.



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An initial study was performed to demonstrate the efficient cleavage of the untreated substrates with SnaBI following the manufacturer’s protocol. The cleaved products were analyzed on a denaturing polyacrylamide gel. The results indicated that the SnaBI can efficiently digest the control duplexes (C-ds20mer1, C-ds20mer2 and C-ds20mer3) and also 5fC-bearing constructs (lanes 2, 5, 8, 11, 14, 17 in Figure S5), whereas the treatment with CB7 does not abrogate the cleavage (lanes 3, 6, 9, 12, 15, 18 in Figure S5). Additionally, the cleavage of all these constructs was not influenced by the treatment with AM (lanes 4, 7, 10, 13, 16, 19 in Figure S5).

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Figure 4C). Not surprsingly, the cleavage of fC-ds20mer3 was not influenced by the AM and CB7 treatment (lanes 12 - 14 in Figure 4C). Taken together, these results indicate that CB7 acts as an OFF switch of RE digestion by binding to 5fC-AD nucleotide, whereas AM serves as an ON switch through supramolecular competition.

We next examined the cleavage of the SARP-treated DNA constructs. Serial position effects were observed for the digestion of these constructs (Figure 4A). Not surprisingly, the SnaBI can efficiently digest all these constructs in the absence of CB7 (lanes 3, 7, 11 in Figures 4B; lanes 2, 5, 8 in Figure S6). And the treatment with a high concentration of CB7 (65 µM) did not affect the cleavage of the control duplexes without 5fC (lanes 3, 6, 9 in Figure S6). In contrast, the cleavage of SARP-reacted fC-ds20mer1 and fC-ds20mer2 was decreased by the CB7 treatment in a concentration-dependent manner (Figure 4B). Importantly, higher concentrations of CB7 were required to inhibit the digestion of SARP-reacted fCds20mer2 compared to that of fC-ds20mer1. Specifically, the treatment with 0.5 µM CB7 significantly decreased the cleavage efficiency of the fC-ds20mer1 (lane 4 in Figure 4B) and this influence became more evident with the 1.0 µM treatment (lane 5 in Figure 4B). Moreover, the digestion of fC-ds20mer1 was almost completely diminished by the treatment with 2.0 µM CB7 (lane 6 in Figure 4B). For the SARP-reacted fC-ds20mer2, the cleavage reaction was substantially affected with 4.0 µM CB7 (lane 9 in Figure 4B) and the total inhibition was observed with 32 µM CB7 (lane 10 in Figure 4B). However, the digestion of SARP-reacted fC-ds20mer3 construct was insensitive to CB7 concentration as high as 65 µM (lane 12 in Figure 4B). Although the molecular basis of this differential sensitivity of SARP-reacted substrates to CB7 needs further examination, we suggest that there may be a direct interaction between the hostguest complex and RE enzyme around the recognition site. Next, we examined whether AM can relieve the inhibition of RE digestion by CB7. To this purpose, increasing amounts of AM were added to the above preparation and further incubated before the RE enzyme was added. The representative data are shown in Figure 4C. For SARP-reacted fC-ds20mer1, the CB7-induced blockage of digestion was gradually reduced in response to increasing concentrations of AM. In particular, the treatment with 7.5 µM AM resulted in an obvious recovery of the SnaBI cleavage in the presence of 8.0 µM CB7 (lane 6 in Figure 4C), and complete cleavage was observed after the treatment with 10 µM AM (lane 7 in Figure 4C). Similar phenomenon was also observed in the performance of AM on the cleavage of SARP-reacted fC-ds20mer2, while appreciably higher concentrations of compound were required to control the SnaBI cleavage (lanes 8 - 11 in

FIGURE4. Reversible intervention of RE digestion (A) Schematic illustration of the design and workflow. The adjacent regions on the ds DNA substrates are indicated with different colors: region colored in red corresponds to the SnaBI recognition sequence; regions colored in blue and green correspond to the upstream and downstream sequences. (B) The influence of CB7 on the cleavage of the SARP-reacted DNA constructs. In this assay, the DNA duplex (100 ng) was treated with increasing amounts of CB7, and then subjected to SnaBI digestion. lanes 1, 3, 7, 11: no CB7 control; lane 2: 65 µM CB7; lane 4: 0.5 µM CB7; lane 5: 1.0 µM CB7; lane 6: 2.0 µM CB7; lane 8: 1.0 µM


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CB7; lane 9: 4.0 µM CB7; lane 10: 32 µM CB7; lane 12: 65 µM CB7; lane 13: no enzyme control. (C) The influence of AM on the cleavage of DNA constructs. In this assay, the DNA duplex (100 ng) was treated with increasing amounts of AM in the presence of CB7, and then subjected to SnaBI digestion. lanes 1, 4, 8, 12: no CB7/AM control; lane 2: 65 µM CB7; lane 3: 65 µM CB7, 80 µM AM; lane 5: 8.0 µM CB7; lane 6: 8.0 µM CB7, 7.5 µM AM; lane 7: 8.0 µM CB7, 10 µM AM; lane 9: 32 µM CB7; lane 10: 32 µM CB7, 30 µM AM; lane 11: 32 µM CB7, 40 µM AM; lane 13: 65 µM CB7; lane 14: 65 µM CB7, 80 µM AM; lane 15: no enzyme control.

Reversible 5fC-targeted intervention of DNA pol elongation After achieving reversible intervention of RE digestion, we were encouraged to further explore the application of our tool. In molecular biology, DNA pol enzymes are responsible for synthesizing DNA molecules. Because chemically control of DNA synthesis is of great importance52-53, we sought to create a 5fC-targeted roadblock. With the aim of a proof of concept study, this investigation uses Bst DNA pol as the elongating enzyme and the unmodified or modified DNAs as the templates (Ctemplate1, mC-template1, hmC-template1, fC-template1 and caC-template1 in Table S1). Complementary DNA primers (primer1, primer2 and primer3 in Table S1) were designed such that their 3’ ends lie near one another and adjacent to the target residue in template.

We anticipated that the AM treatment can drive the release of CB7 roadblock and subsequent reactivation of DNA pol. Our strategic direction is fully supported by the following results, which demonstrated a gradual recovery of DNA pol elongation in response to increasing amounts of AM (Figure 5B). Particularly, the treatment with 3.5 µM AM resulted in an evident recovery of the primer extension in the presence of 4.0 µM CB7 (lanes 4, 8, 12 in Figure 5B) and a majority of the primer extension was recovered from inhibition by treatment with 4.5 µM AM (lanes 5, 9, 13 in Figure 5B). From these observations, the reversible control of DNA pol elongation would probably originate from the supramolecular competitions between AM and 5fC-AD for binding to CB7.

To investigate the effect of CB7 on DNA synthesis, the elongation scaffold was set up by assembling Bst DNA pol with each primer/template duplex. Prior to SARP treatment, each template strand in the duplex can be efficiently used to guide DNA synthesis by Bst DNA pol (lanes 2, 4, 6, 8, 10 in Figure S7), whereas the CB7 treatment did not influence the primer extension along these templates ((lanes 3, 5, 7, 9, 11 in Figure S7). These results clearly indicated that there was no direct interaction between CB7 and DNA pol. Additionally, the AM guest did not affect DNA pol elongation in the absence or presence of CB7 host (Figure S8). However after the template strands were reacted with SARP, quite different behaviors were observed. Bst DNA pol can efficiently read and bypass the 5fC-AD site in DNA (lane 8 in Figure 5A; lanes 2, 6, 10 in Figure S9), whereas the CB7 treatment impeded DNA pol elongation at 5fC-AD site in a concentration-dependent manner (lanes 9 - 11 in Figures 5A; lanes 3 - 5, 7 - 9, 11 - 13 in Figure S9). Specifically, DNA pol elongation opposite fC-template1 was obviously impeded by the 0.25 µM CB7 treatment, and a higher level of impedance was observed with the 1.0 µM treatment. Furthermore, the 4.0 µM CB7 treatment almost completely aborted DNA pol elongation. Moreover, the CB7 treatment did not affect the DNA pol elongation along other SARP-treated DNA templates (lanes 3, 5, 7, 13 in Figure 5A). Additionally, the primer extension assay indicated that the SARP labeling does not change the basepairing properties of 5fC toward guanosine (Figure S10). These observations suggest that the binding of CB7 may induce a dramatic increase in the size of 5fC-AD nucleotide, thus leading to specific stalling of DNA pol at 5fC-AD site in template DNA.

FIGURE5. Reversible intervention of DNA pol elongation (A) The influence of CB7 on the DNA pol elongation. In this assay, the SARP-treated duplex DNA construct (100 ng) was treated with varied amounts of CB7 in the reaction buffer, followed by the addition of dNTP mix and Bst DNA pol. After incubation, extension products were separated on a denaturing polyacrylamide gel. lane 1: no enzyme control; lanes 2, 4, 6, 8, 12: no CB7 control; lanes 3, 5, 7: 4.0 µM CB7; lane 9: 0.25 µM CB7; lane 10: 1.0 µM CB7; lane 11: 4.0 µM CB7; lane 13: 4.0 µM CB7; lane 14: the DNA size marker (primer3, primer3+1, primer3+2, primer3+3, primer3+4, primer3+5 and primer3+6). (B) The influence of AM on the DNA pol elongation. Reactions were carried out as described in ‘Materials and Methods’ section. lane 1: no enzyme control; lanes 2, 6, 10: no CB7/AM control; lanes 3, 7, 11: 4.0 µM CB7; lanes 4, 8, 12: 4.0 µM CB7, 3.5 µM AM; lanes 5, 9, 13: 4.0 µM CB7 4.5 µM AM; lane 14: the DNA size marker.

Reversible 5fC-targeted intervention of PCR In the above study, one step of DNA synthesis has been examined. The PCR is a multi-step process which involves re-



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petitive cycles of DNA synthesis. Due to the ability of PCR to specifically amplify minute amounts of nucleic acid, it has been widely used in scientific research. We next explored the application of our tool for reversible intervention of this reaction52, 54. For this purpose, the starting strand (C80mer-S1 in Table S1) was designed and obtained through commercial synthesis. A series of ds templates (Cds80mer1, mC-ds80mer1, hmC-ds80mer1, fC-ds80mer1 and caC-ds80mer1 in Table S2) were then prepared by incorporating the cytosine or modified base (cytosine, 5mC, 5hmC, 5fC or 5caC) into the indicated positions of starting strand55-56. Quantitative PCR (qPCR) was performed to determine the cycle threshold (Ct) whose level is inversely proportional to the amount of amplifiable DNA template in the sample54.

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respectively (Figures S13B and S14B). In the presence of CB7, only the amplification of SARP-reacted fC-ds80mer1 template was significantly inhibited (∆Ct = 8.52, red line in Figure S13A). Moreover, the inhibition of PCR by CB7 was almost fully reversed by the following treatment with AM (blue line in Figure S13A). Not surprisingly, the treatment with CB7 and AM had no significant effect on the amplification of C-ds122mer1 template in the presence of SARP-reacted fC-ds80mer1 (∆Ct ≤ 0.26, red and blue lines in Figure S14A). Collectively, these results elucidate the high specificity of CB7 targeting the 5fC-DNAs with SARP labelling and illustrate a general method for the reversible 5fC-targeted intervention of PCR reaction.

The first issue we studied is whether CB7 can inhibit PCR process by binding to 5fC-AD nucleotide in DNA templates. To test this, the respective SARP-treated DNA template was purified and subjected to qPCR analysis in the absence or presence of CB7. The representative data are shown in Figure 6A. Specifically, the treatment with 1.56 µM CB7 resulted in an evident increase in the Ct values of SARP-reacted fC-ds80mer1 (∆Ct = 2.02), and this effect was more evident with the 6.25 and 12.5 µM treatments. Moreover, the treatment with 100 µM CB7 significantly increased Ct values (∆Ct = 8.32), reflecting a pronounced reduction (320-fold) in amount of templates to be amplified. In contrast, all other SARP-treated templates (Cds80mer1, mC-ds80mer1, hmC-ds80mer1 or caCds80mer1) were amplified without evident inhibition by the presence of CB7 (∆Ct ≤ 0.14, Figure S11). We further investigated the effect of CB7 using the untreated templates. Not surprisingly, there was almost no variations in Ct values upon CB7 treatment (∆Ct ≤ 0.24, Figure S12), displaying no significant influence on amplification efficiencies. The second issue we studied is whether the inhibition of PCR by CB7 can be reversed by AM. To test this, respective SARP-reacted fC-ds80mer1 samples were treated with CB7 as above before increasing concentrations of AM were added. The representative qPCR data are shown in Figure 6B. In particular, the treatment with 80 µM AM significantly reduced the Ct values of the sample and this influence became more evident with the 90 µM treatment. Moreover, the 200 µM AM treatment largely diminished the CB7-driven inhibition of PCR and the efficiency was very close to that observed for the starting sample. From these observations, the twice-excess of AM can almost fully relieve CB7-driven suppression of PCR. It was therefore highly probable that the host-guest interplay is responsible for manipulating PCR process. We further studied whether the reversible intervention of PCR remains to be specific on SARP-reacted DNAs when the mixed templates were used. In this assay, the SARPreacted fC-ds80mer1 template was mixed with excess of unmodified C-ds122mer1 template (Table S2) at the molar ratio of 1:4. The melting curve analysis demonstrated that the SARP-reacted fC-ds80mer1 and the C-ds122mer1 have very distinct melting temperature of 74.5 °C and 71.5 °C,

FIGURE6. Reversible 5fC-targeted intervention of PCR reaction (A) CB7 specifically inhibit the PCR amplification by binding to 5fC-AD nucleotide in template DNA. (B) Reactivation efficacy of AM on the inhibited PCR reaction. For (A) and(B), reactions were carried out as described in ‘Materials and Methods’ section. All samples were tested in three biological replicates. At the bottom is the representative qPCR amplification curves under different conditions. At the top is bar plot of ∆Ct showing the difference in the amount of amplifiable DNA template in the sample.


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The site-specific identification of 5fC in DNA We next pursued to explore the application of our finding, such as in identifying the underlying 5fC modification in DNA. To illustrate the proof-of-concept, a series of SARP-reacted DNA samples were prepared (Table S3). After the primer extension step in the presence of CB7, each sample was analysed on a denaturing polyacrylamide gel. The gel image shows how extended and unextended bands change with 5fC content from 0 to 100% (Figure S15A). Specifically, an evident stop was observed for the sample containing 20% 5fC (lane 5 in Figure S15A), and higher levels of pausing were observed for the samples with increasing 5fC levels (lanes 6 - 13 in Figure S15A). Figure S15B shows the plot of the fraction of extenion versus the 5fC content. These data show an inverse linear relationship, suggesting that our strategy can be used in examination of the 5fC modification at a particular position of target DNAs. The supramolecular intervention reaction also has the potential to be explored for genome-wide analysis of 5fC modification. UV melting study We next explored potential mechanisms by which CB7 functions to slow or halt 5fC-targeted biochemical reactions. Considering the steric bulk of CB7 molecule, there are two possible mechanisms it may use: disruption of the hydrogen bonding of nucleobases in duplex DNA, or just the steric block mechanism. Previous studies have revealed significant correlations between the base pairing strength and the stability of DNA duplex57-58. The UV melting study was therefore performed to evaluate the effects of CB7 binding on the stability of DNA duplexes52. We designed and prepared a variety of DNA duplexes with the same sequence except the examined residue (sequences in Tables S1 and S4). The duplexes consisted of the 15-base pair sequences: 5’-ATCGTAG-X-ACTCGTC-3’ 3’-TAGCATC-G-TGAGCAG-5’ for the corresponding DNA duplex where X = cytosine, 5mC, 5hmC, 5fC or 5caC, respectively. In this investigation, UV melting curves were recorded at 260 nm to monitor the dissociation of DNA duplexes. We first investigated the effect of CB7 on the untreated DNA duplexes. Figure S16 showed normalized UV melting curves and corresponding melting temperatures of different duplexes in the absence or presence of CB7. For these DNA samples, there were only small variations in Tm values upon CB7 treatment (∆Tm ≤ 0.92 °C) except for hmCds15mer (∆Tm = 1.78 °C), displaying no significant effects on the duplex stability. We further investigated the effect of CB7 on the SARP-reacted fC-ds15mer. Importantly, this CB7-treated sample showed a slightly lower Tm as compared with that of the no-CB7 control (∆Tm = 0.85 °C, Figure 7). Our study demonstrates that the CB7 binding to 5fC-AD nucleotide does not significantly destabilizes the Watson-Crick base pairs in paired region of DNA duplexes. It seems reasonable to suggest that the CB7 binding does not significantly disrupt the hydrogen bonding of nucleobases in duplex DNA, but instead might prevent the recog-

nition and binding of the enzyme to the substrate around 5fC-AD site.

FIGURE7. The CB7 binding to 5fC-AD nucleotide does not significantly destabilizes the Watson-Crick base pairs Representative melting profiles of the SARP-reacted fC-ds15mer were recorded in 10 mM Tris-HCl buffer (pH 7.0, 100 mM NaCl).

Discussion Site-specific intervention of biochemical reactions can help people precisely manipulate some important biological processes59. The RE and DNA pol enzymes have been employed extensively in molecular biology and have facilitated the development of recombinant DNA technology. Several important researches have shown photochemical activation/deactivation of RE and DNA pol activities through the installation of a photolabile protecting group on DNA substrate51-52. However, chemical regulation of these enzymes was achieved in a permanent and irreversible manner in these studies. To the best of our knowledge, very few studies have been conducted on supramolecular control of biochemical reactions, especially in modulating the cell progression60. The current study aims to develop a reversible intervention tool to control the enzymatic recognition at 5fC sites by manipulating the host-guest interactions between CB7 and 5fC-AD. Based on our RE digestion results, it appears that the CB7 binding can largely inhibit enzyme cleavage when located in the enzyme recognition site or in direct proximity to this site; however, the cleavage was not affected when the binding group is located in a short region (2 bp) proximal to the recognition site. Our primer extension assay demonstrates that DNA pol can efficiently incorporate nucleotide dGTP opposite 5fC-AD site, whereas the binding of CB7 to 5fCAD functions as the roadblock to elongating DNA pol. The subsequent AM treatment can remove the caging CB7 through competitive complex formation (AM-CB7). Therefore, the interactions between CB7 and 5fC-AD can readily break and re-form, so that the enzyme recognition can be reversibly regulated. The control experiments indicate that CB or AM does not directly affect the activity of RE and DNA pol enzymes. The CD melting studies further demonstrate that the binding of CB7 to 5fC-AD nucleotide does not significantly affect the hydrogen bonding properties of natural nucleobases in duplex DNA. These results together suggest that interruption of the restriction and strand elongation is due to the steric challenge posed by the host-guest complex and is not a consequence of the



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disruption of secondary or tertiary structure at the site of the SARP/CB7 complex. Our supramolecular strategy is advantageous over other approaches. As an example, imagine a situation in which the CB7 is covalently attached to the 5fC nucleotide. In this case, the CB7 may protrude into the major groove of the DNA and thus prevent the enzyme from binding to that site. This steric hindrance depends on covalent modifications of both CB7 and 5fC site and is thus irreversible. Moreover, the covalent functionalization of CB7 remains to be a challenge. Our reversible intervention strategy also offers a general and facile approach toward the control of some other 5fC-associated biochemical reactions, such as DNA transcription, gene expression and/or base excision repair (BER) pathway61-62. To date, a variety of chemical methods have been developed to identify 5fC modifications in mammalian genomes63-64. However, some of these methods rely heavily on bisulfite treatment. Since this process required the deamination of the unmethylated cytosine, a series of problems limit its application, such as a great extend of DNA degradation. Hence, there is a pressing need to develop bisulfite conversion-free methods for selective identification of 5fC modifications65-66. The current study represents the first one on the host-guest complex blocking the PCR process by interacting with modified nucleotides. The PCR amplification is significantly blocked only when macrocyclic CB7 molecule is present and when the 5fC in template DNA is modified through SARP labelling. Therefore, the observed blocking of PCR amplification can be attributed to the specific interaction of CB7 with the 5fCAD nucleotide, which most likely leads to the formation of a bulky noncovalent complex. The application of this approach for fragment- and site-selective detection of DNA 5fC is performed. Our approach therefore has the potential to be explored for mapping 5fC iland along genome-wide scale and also initiates a promising step into the applications in vivo.

Conclusion In summary, the current study developed a chemical approach for an epigenetic target using supramolecular-tag based labelling. Moreover, we provide three different examples, which demonstrate that the reversible nature of the host-guest chemistry employed allows blocking and deblocking of protein-nucleic acid interactions at 5fC site. We believe that this finding has the advantage to provide a quick and potentially high-throughput analysis of 5fC modification in genomic DNA to gain initial epigenetic information.

Experimental section Materials Glycoluril (CAS# 496-46-8), sulfuric acid (CAS# 7664-93-9), formaldehyde solution (36.5-38% in H2O, CAS# 50-00-0), acetone (CAS# 67-64-1), adamantanamine hydrochloride (CAS# 665-66-7), Tris base (CAS# 77-86-1), 2-(N-morpholino)ethanesulfonic acid (CAS# 4432-31-9) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The oligonucleotides were obtained from TaKaRa company (Dalian, China). Hot Start Taq DNA Pol were purchased from New England Biolabs, Inc. The nucleic acid stains Super GelRed (NO.: S-2001) was

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bought from US Everbright Inc. (Suzhou, China). The 2'deoxycytidine-5'-triphosphate (dCTP), 5-methyl-2'deoxycytidine-5'-triphosphate (5-mdCTP), 5hydroxymethyl-2'-deoxycytidine-5'-triphosphate (5hmdCTP), 5-formyl-2'-deoxycytidine-5'-triphosphate (5fdCTP) and 5-carboxyl-2'-deoxycytidine-5'-triphosphate (5-cadCTP) were purchased from Trilink Biotechnologies Inc. (San Diego, California, USA). The pH was measured with Mettler Toledo, FE20-Five Easy™ pH (Mettler Toledo, Switzerland). DNA MALDI-TOF Mass Spectra were collected on MALDI-TOF-MS (Shimadzu, Japan). APDNA1 was synthesized through U-DNA1 (one T site was replaced by uracil) treating with Uracil DNA Glycosylase (Invitrogen™, USA). DNA concentration was quantified by NanoDrop 2000c (Thermo Scientific, USA). Gel Imaging was performed using Pharos FX Molecular imager (Bio-Rad, USA). Chemical synthesis For synthesis of SARP and 5fC-AD nucleoside, full experimental procedures and structure characterization data are provided in supporting information. The synthesis of CB7 and 5fC nucleoside was performed according to previous literatures67-69. The HRMS was recorded with Thermo Scientific™ Dionex Ultimate 3000 hybrid LTQ Orbitrap Elite Velos Pro (Thermo Scientific, USA). Since SARP is easily oxidized on standing for long periods of time, it is prepared fresh just prior to each use. 1

H-NMR assay The 5fC-AD nucleoside (0.005 mmol) was dissolved in 500 µL d6-DMSO and the spectrum of this sample was recorded. The sample is analyzed again after the direct addition of CB7 solid until saturated. The 1HNMR spectra of the samples were determined at 298 K using a 400-MHz Varian Mercury-VX400 spectrometer. Assignments of the signals are based on the chemical shifts and intensity patterns. The MestReNova program was used to process 1D-NMR spectrum obtained from the original data. The labelling of 5fC nucleotide in DNA The labelling reaction was performed in 40 mM sodium acetate buffer (pH = 5.0), 500 µM SARP (newly synthesized), 10 mM panisidine, and 0.5 µM DNA at room temperature for 12 h. The DNA product was recovered from the reaction mixture by ethanol precipitation. HPLC analysis The HPLC analysis was performed on a Shimadzu LC-6AD system equipped with a UV SPD-20A detector (Shimadzu Corp., Ltd., Japan), using an Inertsil ODS-SP column (5 µm, 250 × 4.6 mm) (GL Science lnc., Japan). The separation conditions were as follows: a twocomponent mobile phase with solution A (CH3CN) and B (100 mM TEAA buffer, pH=7.0); a flow rate of 1 mL/min at 25 °C (A Conc.: 5-5-30% / 0-5-30 min). Native polyacrylamide gel electrophoresis The gel electrophoresis was run in a temperature-controlled vertical electrophoretic apparatus (DYCZ-22A, Liuyi Instrument Factory, Beijing, China). The acrylamide concentration of the separating gel was 20 % (19 : 1 monomer to bis ratio). About one hundred nanograms of DNA with different


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treatment were loaded on the gel. Electrophoresis was run at 10 °C for 1 h at 200 V and 3 h at 300 V. DNA has been seen with GelRed staining. After electrophoresis, the DNA oligomers in the gel were visualized using a Pharos FX Molecular imager (Bio-Rad, USA) in the fluorescence mode (λex = 590 nm). RE digestion The 5’ fluorophore-labeled strand with or without SARP treatment was incubated with the complement at equal molar ratio at 90 °C for 2 min, and then gradually cooled to 15 °C over 2 h. For CB7 intervention assay, the duplex DNA construct (100 ng) was then incubated with various amounts of CB7 for 20 min at room temperature, and subjected to an enzymatic digest (10 µL total volume) with SnaBI (1.0 U) at 37 °C for 1 h according to the manufacturer’s protocol. As a control without any CB7, the same volume (0.5 µL) of water was added to the solution instead. Reactions were stopped by addition of a 4.5-fold excess of quenching solution (95% formamide, 25 mM EDTA at pH 8.0), and digests were analyzed on a denaturing 20% polyacrylamide gel (400 V, 4 h). After electrophoresis, the DNA oligomers in the gel were visualized using a Pharos FX Molecular imager (Bio-Rad, USA) in the fluorescence mode (λex = 488 nm). For AM intervention assay, the sample was prepared in assay buffer containing duplex DNA and CB7, followed by the addition of increasing concentrations of AM. The preparation was heated at 80 °C for 2 min, and then cooled to room temperature for 5 min. The following procedure is similar to the above one. DNA pol elongation assay. The elongation scaffold was prepared by incubating the 5’-FAM-labeled primer with template (with or without SARP treatment) at a molar ratio of 1:1.05. For CB7 intervention assay, the duplex DNA construct (100 ng) was incubated with varied amounts of CB7 in 1 × ThermoPol™ reaction buffer for 20 min at room temperature, followed by the addition of dNTP mix (100 µM each) and Bst DNA pol (0.5 U). As a control without any CB7, the same volume (0.5 µL) of water was added to the solution instead. The reaction mixture was incubated at 37 °C for 1 h. Reactions were stopped by addition of a 4.5-fold excess of quenching solution (95% formamide, 25 mM EDTA at pH 8.0), and samples were analyzed on a denaturing 20% polyacrylamide gel (400 V, 4 h). After electrophoresis, the DNA oligomers in the gel were visualized using a Pharos FX Molecular imager (Bio-Rad, USA) in the fluorescence mode (λex = 488 nm). The starting duplex without any treatment is used as the control. For AM intervention assay, the sample was prepared in 1 × ThermoPol™ reaction buffer containing duplex DNA and CB7, followed by the addition of increasing concentrations of AM. The preparation was heated at 80 °C for 2 min, and then cooled to room temperature for 5 min. The following procedure is similar to the above one.

Synthesis and purification of ds templates. The cytosine or modified base (cytosine, 5mC, 5hmC, 5fC or 5caC) were added into the indicated positions of starting strand by PCR, using dCTP, 5-mdCTP, 5-hmdCTP, 5-fdCTP or 5cadCTP and Hot Start Taq DNA Pol. PCR products were separated on a native 20% polyacrylamide gel (400 V, 2 h). After electrophoresis, the specific DNA (band) was readily recovered from the gel by excision and extraction using the QIAquick® Gel Extraction Kit. qPCR assay. The qPCR assay was performed using a CFX96™ Real-Time System (Bio-Rad, USA). For CB7 intervention assay, the ds DNA template with or without SARP treatment was diluted to 0.01 ng/µL, and 0.5 µL was used in triplicate 20-µL qPCR reactions containing 1 × SYBR® Premix Ex Taq™ II Master (Takara, Japan), 0.5 µM forward and reverse primers, various concentrations of CB7 and nuclease-free water. To verify specificity, the total concentration of mixed templates was kept at 0.025 ng/µL. The cycling conditions are as following: initial denaturation at 95 °C for 15 s; 40 cycles of 95 °C for 5 s, 60 °C for 10 s. The fold-change of amplifiable DNA content was calculated as 2^-∆Ct, where ∆Ct = Ct (CB treated) – Ct (untreated). For AM intervention assay, the starting sample was prepared in 1 × SYBR® Premix Ex Taq™ II Master containing the ds DNA template (0.005 ng), 0.5 µM forward and reverse primers, 100 µM CB7 and nuclease-free water. The following procedure is similar to the above one wherein AM is added sequentially to the starting preparation prior to the start of PCR. UV melting studies. The UV melting experiments were carried out using a Jasco-810 spectropolarimeter (Jasco, Easton, MD, USA) equipped with a water bath temperature-control accessory. The detection is performed with a quartz cell (optical path length at 1 mm). DNA duplex (10 µM) was incubated in 10 mM Tris-HCl buffer (pH 7.0) containing 100 mM NaCl. The UV melting profiles were recorded by using a heating rate of 0.2 °C/min and the absorbance values were collected every 1 °C. The melting point (Tm) corresponds to the midtransition temperature, which was determined by the maximum of the first derivative of the absorbance as a function of temperature. Statistical analysis. Statistical analysis was performed using ORIGIN 8.5 software. The differences were considered to be significant for P < 0.05.

ASSOCIATED CONTENT Supporting Information. General methods; sequences of the used oligomers; more experimental data and more experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION CORRESPONDING AUTHOR * Profs. Dr. Xiang Zhou, Profs. Dr.Tian Tian



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College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Hubei, China. E-mail: [email protected], [email protected]

AUTHOR CONTRIBUTIONS ‡These authors contributed equally. NOTES The authors declare no competing financial interest.

ACKNOWLEDGMENT Zhou, Tian and Wang thank the National Science Foundation of China (No. 21432008, 21722803, 21721005, 81373256, 21372182 and 21672165).

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uril-driven host-guest chemistry for reversible intervention of 5

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