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Templated DNA ligation with thiol chemistry† Cite this: Org. Biomol. Chem., 2014, 12, 8823

Dadong Li,‡ Xiaojian Wang,‡ Fubo Shi,‡ Ruojie Sha, Nadrian C. Seeman* and James W. Canary*

Received 23rd July 2014, Accepted 24th September 2014 DOI: 10.1039/c4ob01552e www.rsc.org/obc

We describe two DNA-templated ligation strategies: native chemical ligation (NCL), and thiol-disulfide exchange. Both systems result in successful ligation in the presence of a DNA template. The stability of the product from the NCL reaction relies on exogenous thiol, while the thiol-disulfide reaction proceeds in a catalyst-free manner.

Template-directed DNA ligation reactions are of great interest. For example, such reactions are essential in self-replicating systems, where a template initiates a process and released products serve as templates for subsequent reactions.1 In nature, DNA replication is one of the fundamental processes of any living system. Not surprisingly, systems utilizing DNA as templates have drawn great attention for decades. Various reactions have been reported to ligate two DNA strands, including phosphodiester formation activated by carbodiimide2 or N-cyanoimidazole,3 imine formation,4 photoinduced [2 + 2] cycloaddition,5 azide–alkyne [3 + 2] cycloaddition,6 and the Wittig reaction.7 The key challenge has always been to achieve high yields with simple reaction conditions. Our groups have reported DNA-templated ligations utilizing amide formation with excellent coupling yields. However, catalysis was required to activate the carboxylates.8 Here we report two DNA-templated systems utilizing thiol chemistry, including native chemical ligation (NCL) and disulfide bond formation (DSF) (Scheme 1). The concept of NCL was first introduced in 1990s by the Kent group,9 and it has been shown to be the most robust and practical of the available ligation chemistries for covalently conjugating two unprotected peptide segments.10 Not limited to protein ligations, NCL has been applied to the conjugation of a variety of molecules, including peptide-oligonucleotide,11 small molecule-oligonucleotide,12 protein-liposome,13 and

Department of Chemistry, New York University, 100 Washington Sq. East, New York, NY 10003, USA. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4ob01552e ‡ These authors contributed equally to this work.

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Scheme 1 Templated DNA ligation using thiol-based chemistry. (A) General ligation scheme; (B) native chemical ligation (NCL); (C) disulphide exchange (DSF). The template strand was omitted in both (B) and (C). RSH: thiophenol or 4-mercaptophenylacetic acid (MPAA). TCEP: tris (2-carboxyethyl) phosphine.

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templated PNA ligations.14 Nevertheless, it has not been reported that NCL can be applied to DNA–DNA ligation. Our initial plan to achieve DNA–DNA ligation followed the principle of NCL: an exogenous thiol activates the thioester and catalyzes the ligation, transthioesterification with the side chain thiol of an N-terminal cysteine creates a thioester-linked intermediate, and the thioester-linked intermediate undergoes intramolecular nucleophilic rearrangement to form an amide bond at the ligation site.15 In addition, the presence of a template strand should provide substantial acceleration of the reaction. As shown in Fig. 1, when the template strand (S0, Table 1) was absent, the coupling product (S1_1–S2) was not observed. When the template was present, the NCL reaction proceeded smoothly. The conjugation proceeded rapidly upon initiation by reducing the disulfide bond, yielding substantial conjugated product almost immediately, regardless of whether exogenous thiol catalysts were present or not. The conjugation yield was between 60% to 90% based on quantitation of electrophoretograms.16 This finding clearly demonstrates that templation accelerated the coupling so effectively that the catalytic effect from exogenous thiol could not be observed. Indeed, the exogenous thiol showed a surprising protective effect on the conjugate, especially at higher temperature. When the reaction mixture was incubated for a longer time at 37 °C, much more conjugate decomposed in the thiol-free condition compared to the mixture containing an exogenous thiol. About 50% of the conjugate decomposed after 8 h of incubation, and less than 10% conjugate was left after 50 h of incubation. Since two strands were conjugated through linkers, the T spacing on the template was tested. With slight differences from 0 T to 5 T,

Fig. 1 Templated DNA native chemical ligation reaction. (A) Schematic of the designed strands: top, template strand, S0; bottom left, strand containing thioester, S1-thioester (S1_1); bottom right, strand containing cysteine, S2-Cys-S-tBu (S2). (B) Time course of templated DNA NCL: denaturing gels developed with Stains-All (Sigma) show the NCL conjugate product. Gels contained 15% polyacrylamide, and were run in 1X TBE buffer at 55 °C. The reaction solution was sampled after incubation at 37 °C for the following times (left to right) 5 min, 1 h, 3 h, 8 h, 29 h, 50 h, and 74 h. Within each time slot, from left to right, the lanes include a 10 nucleotide ladder, reaction with both a 43-mer template (S0–43mer) and 1% v/v thiophenol, reaction with S0–43-mer but no thiophenol, and reaction with 1% v/v thiophenol but no template. The reaction was initialized by adding 20 mM TCEP.

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Organic & Biomolecular Chemistry Table 1

Strand sequences

Code

Oligonucleotide sequences

S0

5′-TTTTGTAGGACAGCTCTCTG(T)n GAGTTACTGGACAAGATTTT-3′ 5′-CAGAGAGCTGTCCTAC-3′ 5′-CAGAGAGC-3′ 5′-TCTTGTCCAGTAACTC-3′ 5′-AGTAACTC-3′ 5′-CAGAGAGCTGTCCTAC∼TCTTGTCCAGTAACTC-3′ 5′-CAGAGAGC∼AGTAACTC-3′ 5′-CAGAGAGC∼TCTTGTCCAGTAACTC-3′ 5′-CAGAGAGC∼AGTAACTC-3′

S1a S1′ a S2b S2′ b S1_1–S2 S1′_1–S2′ S1′_2–S2 S1′_2–S2′

a S1_1 and S1′_1 were modified with thioester at 5′ end. S1_2 and S1′_2 were modified with MPAA at 5′ end. b S2 and S2′ were modified with tBu-S-Cys at 3′ end. Detailed structures of the modifications were shown in Scheme 1. “∼” denotes the variant linkage as shown in Scheme 1.

maximum at 3 T, the yields dropped significantly with longer spacers, which imposed extra distance and thus decreased the templating effect (Fig. 2). MALDI-TOF MS showed that the conjugated product obtained from the reaction with 1% thiophenol (PhSH) present contained an extra thiophenol motif (Fig. S3A,† S1_1–S2–PhSH), and this extra thiophenol was cleaved by treatment with dithiothreitol (DTT) (Fig. S3B,† S1_1–S2), which indicated that a disulfide bond was formed between the cysteine side chain thiol and thiophenol. Systems with shorter strands (S1′_1, S2′) and two different exogenous thiols, thiophenol (Fig. 3) or MPAA (ESI, Fig. S4†) groups were also tested. MALDI-TOF spectra with better resolution were obtained (about 20 Da at 5 kDa), and similar results were observed. This indicated that an N–S acyl shift might occur with an uncapped cysteine thiol group and lead to the decomposition of the conjugated product (ESI, Fig. S5†).13,17 We also noticed that the decomposition was slower at lower temperature (Fig. 4). To explore further the role of exogenous thiols, experiments were carried out under TCEP-free conditions. As expected,

Fig. 2 Templated NCL reaction with differently spaced templates. The system consists of a (40 + n) mer template S0, 16mer S1-thioester (S1_1), 16mer S2-Cys-S-tBu (S2), 1% v/v thiophenol, and incubated at 37 °C for 72 h. Different spaced templates are used, and n indicates the number of T spacing used for each template. The marker lane on the left is a 10 nucleotide ladder. The gel contains 15% polyacrylamide, run in 1X TBE buffer at 55 °C, stained with ethidium bromide (EB).

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Fig. 3 MALDI-TOF MS of conjugation product from shorter strands. (A) Before DTT treatment: m/z calculated (S1’_1–S2’–PhSH, M − H+): 5490.2, found: 5490. (B) After DTT treatment: m/z calculated (S1’_1–S2’, M − H+): 5380.1, found: 5384. The reaction mixture was incubated with 1% v/v thiophenol at room temperature for 21 h.

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Fig. 5 Denaturing gel stained with EB showing the conjugate product with thiol-disulfide exchange deprotection. From left to right, lanes include a 10 nucleotide ladder, 3 h reaction with S0, S1_1, S2 and 1% v/v thiophenol, 1 h reaction with S0, S1_1, S2 and 1% v/v thiophenol, 3 h reaction with S0, S1_1, S2 and 200 mM MPAA, 1 h reaction with S0, S1_1, S2 and 200 mM MPAA, 3 h reaction with S0, S1_1 and S2 and (no exogenous thiol), and 1 h reaction with S0, S1_1 and S2 and (no exogenous thiol).

when there was no exogenous thiol, the capped cysteine strand did not react with the thioester strand. This is consistent with the accepted mechanism of NCL, wherein the transthioesterification step is critical for NCL to proceed.15 Interestingly, with exogenous thiol present, coupling of the two strands proceeded smoothly (Fig. 5). A thiol-disulfide exchange18 was possibly the initial step that freed the cysteine side chain thiol for the NCL reaction. Inspired by successful deprotection via the thiol-disulfide exchange reaction, a DNA-templated conjugation system was developed based on disulfide chemistry. Instead of a thioester motif, the S1 strand was modified with MPAA (S1_2), which

Fig. 4 Time course of DNA-templated NCL with S0, S1_1 and S2 at 23 °C. They were all 15% acrylamide, run in 1X TBE buffer at 55 °C and stained with EB. The reaction solution was sampled at incubation time t = 0 (adding thiol), 5 min, 10 min, 20 min, 30 min, 1 h, 1.5 h, 3 h, 24 h, 48 h, 72 h. In each gel from left to right, the lanes show a 10 nucleotide ladder, t = 0, 5 min, 10 min, 20 min, 30 min, 1 h, 1.5 h, 3 h, 24 h, 48 h, 72 h. The thiols added in were (A) 2% v/v thiophenol, (B) 20 mM MPAA, (C) no exogenous thiol added.

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Fig. 6 Characterization of DNA-templated disulfide conjugation. (A) Denaturing gels stained with EB showed the DSF conjugate product. From left to right, lanes contain S0; S1_2; S2; 24 h reaction with S0, S1_2, and S2; a 10 nucleotide ladder; 24 h reaction with S0, S1_2, and S2’; S2’. (B) MALDI-TOF spectrum for the crude reaction mixture of S0, S1_2, and S2. Inset: enlarged view of the mass range between 5 to 5.3 kDa. Peaks are assigned as follows: S0 m/z calculated (M − H+): 12 347, found: 12 343; S1_2–S2 m/z calculated (M − H+): 10 283, found: 10 280; S1_2 m/z calculated (M − H+): 5195.6, found: 5199; S2 m/z calculated (M − H+): 5177.7, found: 5179. (C) MALDI-TOF spectrum for the crude reaction mixture of S0, S1_2, and S2’. Peaks are assigned as follows: S0 m/z calculated (M − H+): 12 347, found: 12 349; S1_2–S2’ m/z calculated (M − H+): 7869.5, found: 7871; S1_2 m/z calculated (M − H+): 5195.6, found: 5199; S2’ m/z calculated (M − H+): 2764.1, found: 2765.

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Acknowledgements This research was supported by NSF grant CMMI-1120890 to JWC and NCS and by the following grants to NCS: GM-29554 from NIGMS, CCF-1117210 from the NSF, MURI W911NF-11-10024 from ARO, grants N000141110729 and N000140911118 from ONR. We acknowledge the support of DE3C0007991 from DOE for DNA purchase and synthesis expenses.

Fig. 7 Denaturing gels stained with Stains-all showed the DSF conjugate product. From left to right, lanes include 24 h reaction with 16mer S1_2 and 16mer S2; 24 h reaction with S1_2 and 8mer S2’; 24 h reaction with S0, S1_2, and S2; 24 h reaction with S0, S1_2, and S2’; a 10 nucleotide ladder; template strand S0; S1_2; 16mer S2; 24 h reaction with S0, S1_2, and S2’; S2’.

reacted with a tert-butyl mercaptan-capped cysteine motif (tBuS-Cys) in an S2 strand (S2), yielding cross-linked products (Fig. 6). Initial attempts with 16-mer S1 and 16-mer S2 were promising, as MALDI-TOF MS of the reaction crude products showed the target mass peak corresponding to the S1_2–S2 conjugate. However, the gel was hard to interpret because the S1 strand dimerized with itself, yielding a 32-mer S1_2–S2 conjugate which had similar mobility to the 32mer S1_2–S2. To confirm the conjugation reaction, an 8-mer S2′ was prepared and tested. Both MALDI-TOF MS and the gel showed clearly that the S1_2–S2′ conjugate was formed. The conjugation yield was about 90%. Similar to the NCL reaction, the template strand was essential for the coupling reaction, without which no observable conjugated product formed (Fig. 7). While this manuscript was in preparation, another report of a sulfur exchange-based DNA coupling reaction in forming 5′-3′ disulfide linkages appeared. Although similar chemistry was studied, a rather complicated protocol was required to prepare the solid support for the synthesis of oligonucleotide, while our system utilized only commercially available materials.19

Conclusions In conclusion, two DNA-templated conjugation systems were developed based on the chemistry of thiols. One of them followed native chemical ligation, and interestingly a protective, instead of catalytic, role of exogenous thiols was observed. The other system utilized a thiol-disulfide exchange reaction and the desired cross-linked product formed without the need for any additional reagents but the template DNA. Ultimately, these two systems provide new methods to ligate together two oligonucleotides via enzyme-free processes. There is considerable potential for developing new DNA nano-materials by applying these chemistries, such as constructing catenanes;20 or producing asymmetric three-way branched oligonucleotides,21 which can be processed further to assemble more complex nanostructures.

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Notes and references 1 (a) L. E. Orgel, Nature, 1992, 358, 203–209; (b) A. Robertson, A. J. Sinclair and D. Philp, Chem. Soc. Rev., 2000, 29, 141–152; (c) S. Liao and N. C. Seeman, Science, 2004, 306, 2072–2074; (d) T. Wang, R. Sha, R. Dreyfus, M. E. Leunissen, C. Maass, D. J. Pine, P. M. Chaikin and N. C. Seeman, Nature, 2011, 478, 225– 228. 2 (a) G. von Kiedrowski, Angew. Chem., Int. Ed. Engl., 1986, 25, 932–935; (b) R. Naylor and P. T. Gilham, Biochemistry, 1966, 5, 2722–2728. 3 T. Li and K. C. Nicolaou, Nature, 1994, 369, 218–221. 4 J. T. Goodwin and D. G. Lynn, J. Am. Chem. Soc., 1992, 114, 9197–9198. 5 K. Fujimoto, S. Matsuda, N. Takahashi and I. Saito, J. Am. Chem. Soc., 2000, 122, 5646–5647. 6 (a) A. H. El-Sagheer and T. Brown, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 15329–15334; (b) M. Shelbourne, X. Chen, T. Brown and A. H. El-Sagheer, Chem. Commun., 2011, 47, 6257–6259. 7 M. L. McKee, P. J. Milnes, J. Bath, E. Stulz, R. K. O’Reilly and A. J. Turberfield, J. Am. Chem. Soc., 2012, 134, 1446– 1449. 8 (a) Y. Liu, A. Kuzuya, R. Sha, J. Guillaume, R. Wang, J. W. Canary and N. C. Seeman, J. Am. Chem. Soc., 2008, 130, 10882–10883; (b) M. Ye, J. Guillaume, Y. Liu, R. Sha, R. Wang, N. C. Seeman and J. W. Canary, Chem. Sci., 2013, 4, 1319–1329. 9 P. E. Dawson, T. W. Muir, I. Clarklewis and S. B. H. Kent, Science, 1994, 266, 776–779. 10 S. B. H. Kent, Chem. Soc. Rev., 2009, 38, 338–351. 11 D. A. Stetsenko and M. J. Gait, J. Org. Chem., 2000, 65, 4900–4908. 12 J. Michaelis, A. Maruyama and O. Seitz, Chem. Commun., 2013, 49, 618–620. 13 S. W. A. Reulen, W. W. T. Brusselaars, S. Langereis, W. J. M. Mulder, M. Breurken and M. Merkx, Bioconjugate Chem., 2007, 18, 590–596. 14 (a) A. Mattes and O. Seitz, Chem. Commun., 2001, 2050– 2051; (b) S. Ficht, A. Mattes and O. Seitz, J. Am. Chem. Soc., 2004, 126, 9970–9981; (c) A. Roloff and O. Seitz, Chem. Sci., 2013, 4, 432–436. 15 E. C. B. Johnson and S. B. H. Kent, J. Am. Chem. Soc., 2006, 128, 6640–6646.

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19 V. Patzke, J. S. McCaskill and G. von Kiedrowski, Angew. Chem., Int. Ed., 2014, 53, 4222–4226. 20 R. Kumar, A. El-Sagheer, J. Tumpane, P. Lincoln, L. M. Wilhelmsson and T. Brown, J. Am. Chem. Soc., 2007, 129, 6859–6864. 21 M. F. Jacobsen, J. B. Ravnsbaek and K. V. Gothelf, Org. Biomol. Chem., 2010, 8, 50–52.

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16 X. Li, Z. J. Gartner, B. N. Tse and D. R. Liu, J. Am. Chem. Soc., 2004, 126, 5090–5092. 17 J. Kang and D. Macmillan, Org. Biomol. Chem., 2010, 8, 1993–2002. 18 (a) P. A. Fernandes and M. J. Ramos, Chem. – Eur. J., 2004, 10, 257–266; (b) A. Fava, A. Iliceto and E. Camera, J. Am. Chem. Soc., 1957, 79, 833–838.

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Org. Biomol. Chem., 2014, 12, 8823–8827 | 8827

Templated DNA ligation with thiol chemistry.

We describe two DNA-templated ligation strategies: native chemical ligation (NCL), and thiol-disulfide exchange. Both systems result in successful lig...
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