Sequence-Specific Generation of 1,N6-Ethenoadenine and 3,N4

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Article 6

Sequence-Specific Generation of 1,N-Ethenoadenine and 3,N-Ethenocytosine in Single-Stranded Unmodified DNA 4

David Egloff, Igor A. Oleinich, and Eva Freisinger ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500497p • Publication Date (Web): 17 Nov 2014 Downloaded from http://pubs.acs.org on November 18, 2014

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ACS Chemical Biology

Sequence-Specific Generation of 1,N6-Ethenoadenine and 3,N4-Ethenocytosine in Single-Stranded Unmodified DNA David Egloff, Igor A. Oleinich, and Eva Freisinger*

Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. *e-mail: [email protected]

ABSTRACT: DNA lesions such as 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC) are ubiquitously present in genomes of different organisms and show increasing levels upon exposure to mutagenic substances or under conditions of chronic inflammations and infections. To facilitate investigations of the mutagenic properties and repair mechanisms of etheno-base adducts, access to oligonucleotides bearing these lesions at defined positions is of great advantage. In this study we report a new synthetic strategy to sequence-specifically generate etheno-adducts in a single-stranded unmodified DNA sequence making use of a DNAtemplated approach that positions the alkylating agent close in space to the respective target base. In contrast to solid-phase synthesis of modified oligonucleotides such DNA-templated methods can be applied to single-stranded nucleic acids of unrestricted lengths. The modular nature of the system allows straightforward adaptation to different sequences.

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INTRODUCTION The formal addition of an etheno-bridge between the exocyclic amino group and one ring nitrogen atom of the nucleobases cytosine, adenine, and guanine produces the exocyclic DNA adducts 3,N4-ethenodeoxycytidine (εdC), 1,N6-ethenodeoxyadenosine (εdA), and N2,3ethenodeoxyguanosine (εdG), respectively. In vivo, these adducts are generated by reactive metabolites after exposure to e.g. vinyl chloride, but also endogenous formation in the context of lipid peroxydation and oxidative stress is reported.1-3 Etheno base adducts can be excised by specific DNA glycosylases or directly repaired by the action of iron- and α-ketoglutaratedependent dioxygenases, i.e. Escherichia coli AlkB or its mammalian homologues.4-7 During DNA replication, etheno adducts can lead to mispairing and hence are potentially mutagenic.8,9 Unlike A, 1,N6-ethenoadenine (εA) exhibits fluorescent properties and is used as a fluorescent nucleobase analogue, e.g. for functional studies of various enzymes.10-14 Due to its small size, incorporation of εA into DNA and RNA causes usually only little local distortions of the nucleic acid structure.15,16 As the position of εA is rather fixed within a duplex structure the heterogeneity of the fluorescence signal is rather low compared to out of chain fluorescence labels, which are usually attached to the nucleic acid using relatively long and often flexible linkers. A drawback of fluorescent base analogues can be quenching of the signal and hence significant reduction of the quantum yield due to stacking within the nucleic acid strand. Another disadvantage is the blockage of the Watson-Crick hydrogen bonding face by the etheno-bridge, which can hamper investigations relying on molecular recognition processes via this site. So far, εdA and εdC containing nucleic acids were mainly used for the investigation of biochemical responses to this lesion, e.g. to evaluate the replication fidelity and processivity of numerous polymerases8,9,17-19 and the action of specific glycosylases from the base excision 2 ACS Paragon Plus Environment

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repair pathway.4,5,20,21 Shorter oligonucleotides for in vitro studies can be prepared by solidphase synthesis using the corresponding commercially available phosphoramidite building blocks of the lesion for incorporation at a defined position.22,23 However, the preparation of longer strands with solid-phase synthesis is more costly and gives lower yields. Plasmid DNA or viral vectors for in vivo studies are either pre-treated with the lesion forming agent, e.g. chloroacetaldehyde, thereby generating multiple lesions at rather undefined locations, or the lesion is specifically incorporated at a defined position by ligation with a synthetically produced short oligonucleotide.24 In this work, we describe a new modular DNA-templated approach for the sequencespecific generation of εdA or εdC modifications in DNA single strands (Fig. 1a): A target DNA strand of any source or sequence is incubated with a complementary strand denoted the reactive strand. This reactive strand comprises the recognition sequence, i.e. the complementary part, and a specially designed chemically reactive group. DNA duplex formation will place the reactive group in very close proximity to the target A or C base in the target DNA strand (Fig. 1a, step 1). After specific activation of the reactive group the target base is converted into its corresponding ε-base (step 2) and the two strands can be separated (step 3), e.g. by denaturating polyacrylamide gel electrophoresis (PAGE). The idea of using nucleic acid templates to facilitate and direct chemical reactions is not new as such and was already applied, e.g., in DNA-templated organic synthesis25,26 or for the formation of interstrand cross-links, strand breaks or other modifications within nucleic acids.27-31 The beauty of the modular approach described here is that the system can be easily adapted to different sequence contexts thanks to its modularity. No solid-phase oligonucleotide synthesis using special phosphoramidites is required as the reactive group can be coupled to commercially available 3'-phosphorylated oligonucleotide sequences (Scheme



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1b). In addition, the reactive group used for the introduction of the etheno-bridge generates unsubstituted εA and εC in contrast to a previously described approach.27

RESULTS AND DISCUSSION Design and synthesis of the reactive strand. The formation of etheno-adducts of A and C derivatives upon incubation with chloroacetaldehyde (1, Fig. 1b) under slightly acidic conditions was discovered more than 40 years ago and is still the most frequently used method for this purpose.32 Thus, a suitable reactive group for the newly designed system should possess comparable chemical properties as chloroacetaldehyde, i.e. an aldehyde functionality as well as an electron-withdrawing group. This group must in addition offer the possibility to attach a linker for coupling to the recognition sequence. These properties are combined in the aryl sulfonate group (2e, Fig. 1b, Scheme 1a). In addition to its electronwithdrawing properties, the sulfonate group is a highly reactive leaving group due to resonance stabilization of the resulting structure. The phenyl ring structure can be used for the linkage to the recognition sequence as described in the Supplementary Information. To prevent premature reaction of the aldehyde group with A and C before strand annealing a 1,2diol group was inserted instead. The vicinal diol functionality can be regarded as a protected form of an aldehyde group, to which it can be converted by oxidative cleavage with NaIO4 in situ. While NaIO4 is completely biocompatible with DNA, it slowly degrades εA and hence the excess of oxidizing agent was quenched with ethylene glycol after 15 min, the time required for complete conversion of the vicinal diol into the aldehyde group. To test the efficiency of the newly designed reactive group, compound 2e′ was reacted with 9ethyladenine (3a) as shown in Fig. 1c and the progress of 9-ethyl-ε-adenine (4a) formation followed with fluorescence spectroscopy, LC-MS, and high pressure liquid chromatography (HPLC) using a reverse-phase (RP) C18 column (Supplementary Fig. S3-S5). Yields of the reaction are roughly 75% after 1 day and 95% after 6 days. 4 ACS Paragon Plus Environment

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For coupling to the recognition sequence a reactive group with a carboxy group in place of the toluene methyl group was synthesized and the carboxy group activated by formation of an N-hydroxysuccinimide (NHS) ester (2e, Scheme 1a). Coupling of this ester to the aminomodified recognition sequence, prepared from the 3'-phosphorylated oligonucleotides as described,33 results in a stable peptide bond with a yield of approximately 90 % (Scheme 1b).

Formation and detection of εA. For the sequence-specific formation of εA within the DNA template, reactive strand 7c was incubated with the two complementary target strands 8 and 9 as well as with the non-complementary strand 10 (Fig. 2a). Reactive and target strands were allowed to anneal for 15 min at RT prior to addition of NaIO4 and subsequent quenching with ethylene glycol, and the reaction was left to proceed for 5 days. Spectrophotometric data show an increasing fluorescence emission characteristic for εA formation (Fig. 2b) for the reaction with target strand 9, reaching 75% of the maximum intensity after 1, 90% after 2, and 95% after 3 days of incubation, which was chosen as standard reaction time for all further experiments. No increase of the fluorescence emission was observed with target strand 8, however, stacking interactions of the target adenine base with the neighboring guanine can lead to fluorescence quenching of εA, while the thymine base in 9 has a significantly smaller influence.34 For the quantification of εA formation the reaction mixture was digested with DNase I and phosphodiesterase I followed by treatment with calf intestinal alkaline phosphatase, and the resulting nucleosides were separated with RP-HPLC (Fig. 2c). Peaks were assigned by comparison with commercial standards (dA, dT, dG, dC) or with εdA that was prepared by reaction of dA with chloroacetaldehyde.23,35 εdA was additionally identified with ESI-MS/MS measurements (Supplementary Fig. S16). εdA formation was only detected in the reactions with target strands 8 and 9 but not with the non-complementary strand 10. Modification yields were determined by comparing the peak area of εdA in the chromatogram 5 ACS Paragon Plus Environment


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of the digested reaction mixture with the peak area in the chromatogram obtained after injection of a defined amount of synthetic εdA. For both target strands 8 and 9 the yield of modification was about 30%. RP-HPLC and ESI-MS/MS analysis also reveal small amounts of εdC in the nucleoside mixture after digestion, which is formed by reaction of the reactive sequence with itself, probably attacking the C moiety next to the reactive group (Supplementary Fig. S10 and S11). This shows the importance of using a protected aldehyde and of pre-annealing of primer-template DNA before activation with NaIO4 to prevent premature reactions with other nucleobases than the target base as well as self-reaction of the reactive sequence as far as possible. Nevertheless, commonly used annealing procedures, which include heating of the sample, should not be used as this accelerates the perpetual cleavage at the sulfonate ester moiety present also at lower temperatures (Figure S6) and hence leads to the loss of the 1,2-diol protected aldehyde functionality. For example, heating of reactive strand 7c to 90 °C for 2 min leads to approximately 50% degradation (Figure S9). Accordingly, RT was used as reaction temperature for all experiments.

Sequence-specificity of εA formation. To evaluate the sequence- as well as the sitespecificity of εA formation polymerase stop assays (PSAs) were performed using Thermus aquaticus (Taq) DNA polymerase, which lacks proofreading 3' to 5' exonuclease activity and is efficiently stopped by a single εA or εC in the template strand.36,37 For the PSA experiments reactive strand 7c (Fig. 2a) was incubated with the longer target strands 11-16 (Fig. 3a) to test εA generation at positions n+1 to n+9. Subsequently, PSAs were performed using the 5'-32Plabeled primer 23 (Fig. 3b). The elongation product mixtures were separated with denaturing PAGE, and products imaged using a phosphorimager. The PAGE gels show two major bands, one for the fully extended and hence unmodified template and a second one at lower strand length corresponding to the fraction of templates that were modified (Fig. 3b). Comparison of 6 ACS Paragon Plus Environment

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the intensities of the two bands with each other allows determining the percentage of εA generation. These yields are in good agreement with the results obtained from the enzymatic digestion experiments (Fig. 2c, Table 1). The highest yield of εA formation, i.e. approximately 30%, is detected for A in position n+1 and yields decrease steadily for target bases located further away from the reactive group. For positions n+6 and higher, yields become negligible. Other residues, e.g. A in position n-3, are not modified according to the PSA experiments. These results show that the system exhibits selectivity for the positions closest to the reactive group. The system was also tested with considerably longer sequences, i.e. reactive strand 7c was also incubated with a 123 nt long single strand, and in addition also the influence of mismatches in the recognition sequence on the modification yield was evaluated (Supplementary Information and Figs. S19 and S20). Results show again a modification yield of approximately 30% for A in the n+1 position. Mismatches with the 5’-end of reactive strand 7c have a minor influence on εA formation in the n+1 position with yields of 25-30%, while two mismatches in the (n-1) and (n) positions just 3’ of the target A base result in a significantly decreased modification yield of approximately 17%.

εC formation and selectivity in competition reactions. As εC is formed under similar conditions as εA, reactive strand 7c was also tested for its ability to modify C residues at different positions (target strands 17-22, Fig. 3a). The modification yields were again analyzed with PSAs (Fig. 3b, Table 1). Also here, yields for εC formation decrease with increasing distance between the target C and the reactive group in the complementary strand. The yield of εC formation in position n+1 is slightly lower compared toεA, i.e. around 26% compared to 32%, and yields become negligible already with C in position n+3. To further evaluate the site-specificity of the system, reactive strand 7c was incubated with the mixed target strands 24-35, which contain combinations of multiple A and C bases in 7 ACS Paragon Plus Environment


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positions n+1 to n+4 (Fig. 3c,d, Table 1). In most reactions, the target base at or closest to position n+1 is converted most efficiently into its etheno analog (25-28, 30, 31, 33-35). Nevertheless, the specificity for the n+1 position drops in some cases considerably (28, 29) or is even inverted (24, 32) if another target base is positioned just next to it in n+2, the exception being target 34. If A is located in position n+1 and C is close-by (32, 33), the yield for εA is dramatically decreased compared to sequences without C. In the reverse situation with C in position n+1 the presence of A has no negative influence but rather improves the formation of εC (34, 35). Rather unexpected and surprising is the high yield of εC formation in position n+2 observed with target 32, which is much higher that yields observed when only a single C is located in position n+1 or n+2 (17, 18). Accordingly, to efficiently target an A base at a specific position no other A and C bases should be located in direct vicinity as a significant decrease of εA formation at the target site will occur. For the modification of C in a specific position only a neighboring C base lowers the yield (29), while the presence of A even seems to increase it.

CONCLUSIONS In summary, the method presented here is a viable alternative to the presently used procedures for the preparation of single-stranded DNA sequences containing etheno bases at a specific position. The conversion of individual A and C residues to their corresponding εbases succeeds at moderate to good yields, probably facilitated by the close proximity of the reaction partners in the annealed strands, which also increases the apparent concentration and hence the yield of the reaction.26 Nevertheless, the presence of other nucleobases in the vicinity that are susceptible to ε-bridge formation can have an influence on the modification yield as well as on the degree of specificity. The purity of the modified target strands obtained with the presented modular system is lower than what is obtained with standard solid-phase 8 ACS Paragon Plus Environment

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synthesis due to the difficulties of separating modified from unmodified strands. In addition, the highest yields obtained are in the range of 30%, which is mainly based on the compromise between the good leaving group properties of the tosylate moiety and its associated proneness to hydrolysis. However, for selected applications, e.g. for longer DNA strands, this new strategy can be a good alternative. Further applications might also include the modification of native DNA or even RNA strands including double stranded structures, although the applicability and especially the modification efficiency still needs to be evaluated. For the modification of double stranded structures strand separation and hybridization with a homologous single strand was reported to be efficiently catalyzed by the Escherichia coli recombinase RecA.38,39 This approach might also be feasible for the here presented modular system, which will be tested in future experiments. With this work we introduced a new way to generate sequence-specifically εA and εC in a single-stranded target DNA molecule. In addition, our modular system offers a general way of targeting individual bases in DNA strands for modification. By selecting the corresponding recognition sequence for the reactive strand and by designing a suitable reactive group, virtually any base in any sequence context can be targeted and modified as desired.

EXPERIMENTAL PROCEDURES

Formation of 9-ethyl-εεA. For the model reaction 2e' (8 mM), 3a (8 mM), and NaIO4 (120 mM) were stirred at RT for 15 min in 1 mL 100 mM sodium acetate (pH=5.0). NaIO4 was quenched by the addition of 20 µL ethylene glycol (360 µmol) and the reaction was continued under the same conditions. Samples were taken at different time points and analyzed by fluorescence spectroscopy, RP-HPLC and LC-MS (Supplementary figures S3-S5).



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Synthesis of reactive strand 7c. Preparation of 7c was performed according to a modified literature procedure.33 The corresponding 3’-phosphorylated oligonucleotide (25 µL, 4 mM) was precipitated with 6 µL 8% (w/v) aqueous cetyltrimethylammoniumbromide (CTAB), dried and redissolved in 50 µL dimethyl sulfoxide (DMSO). After the addition of 2,2’dithiodipyridine (PDS) in DMSO (50 µL, 1.6 mM), 4-dimethylaminopyridine (DMAP) in DMSO (50 µL, 1.2 M) and triphenylphosphine (PPh3) in dimethyl formamide (DMF) (50 µL, 1.2 M), the reaction mixture was incubated at 25°C for 25 min. 8 µL (approx. 1000 equiv.) ethylene diamine was added and incubation was continued for 30 min. The resulting aminomodified oligonucleotide was precipitated with 2% (w/v) LiClO4 in acetone and washed with acetone. For the next step, the dry oligonucleotide (approx. 90 nmol) was dissolved in 90 µL 100 mM potassium phosphate buffer (pH=7.5) and 18 µL of the NHS-ester 2e of the reactive group (for synthesis see Supporting information) in DMSO (100 mM) was added. Incubation at RT for 4 h and precipitation with NaCl and EtOH yielded reactive strand 7c in approx. 90 % yield. 7c was characterized by RP-HPLC and MALDI-TOF (Supplementary figures S6, S8). Sequence-specific formation of etheno-bases. Reactive strand 7c (5 µL, 120 µM) and the respected target strand (5 µL, 60 µM) were combined with 60 µL 1 M NaCl and 50 µL 20 mM sodium acetate (pH=5.5) and incubated for 15 min. After the addition of 10 mM NaIO4 in 30 µL 20 mM sodium acetate (pH=5.5) the mixture was incubated in a Thermomixer at 25°C and 500 rpm for 90 min. NaIO4 was quenched with 15 mM ethylene glycol in 30 µL 20 mM sodium acetate (pH=5.5) and incubation was continued under the same conditions. The reaction was stopped after 3 days and purified by NAP-5 columns (GE Healthcare) for enzymatic digestion or by ZipTip C18 (Millipore) for PSA. Enzymatic digestion of target strands (Fig. 2c). Reaction mixtures, containing typically 3 nmol of the respective target strand (8-10) purified with a NAP-5 column, were incubated for 10 ACS Paragon Plus Environment

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15 h at 37 °C with bovine DNase I (Thermo Scientific AG, 25 µL, 25 U) and snake venom phosphodiesterase I (USBiological, 2.5 µL, 0.5 U) in 5 µL buffered solution containing 500 mM Tris-HCl, 100 mM MgCl2, 1 mM CaCl2 at pH 6.8. The final volume was adjusted to 42.5 µL with H2O. The mixture was then incubated for 30 min at 30°C with calf intestinal alkaline phosphatase (Promega, 2.5 µL, 2.5 U), diluted with H2O (55 µL) and directly injected into the HPLC system For enzymatic digestions of reaction mixtures with the long target sequences 11-26 volumes were increased by a factor of 1.6. The final volume before injection into the HPLC system was adjusted to 100 µL. Polymerase stop assays (PSA). After incubation with reactive strand 7c the target strands were purified with ZipTip C18 and annealed with the 32P-labeled primer 23 by incubating at 90°C for 2 min and slowly cooling down. After addition of dNTPs and Taq DNA polymerase (Promega), the mixtures were incubated at 55°C for 30 min. Final reaction mixtures with a total volume of 20 µL contained target strand (10 nM), 32P-labeled primer 23 (10 nM), dNTPs (0.25 mM) and Taq DNA polymerase (0.01 U/µL) in 5x Taq reaction buffer (1:5 dilution). The reactions were quenched with stop solution (82% formamide, 0.16% xylene cynol, 0.16% bromophenol blue) and incubated at 90°C for 5 min prior to analysis by 12% denaturing PAGE. Visualization and quantification was conducted on a Typhoon Scanner FLA 9500 with ImageQuant TL 1D (GE Healthcare).

ASSOCIATED CONTENT Supporting Information Experimental procedures including syntheses and analyses of compounds (Figures S1, S2, S6S8), investigation of model and side reactions (Figures S3-S5, S9-S11, S17, S18, Schemes S1, S2), as well as procedures for and evaluation of ε-base formation in the target strands (Figures



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S12-16, S19, S20, Scheme S3). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. M. Zhao for his assistance with gel imaging. Financial support from the Swiss National Science Foundation (E.F), the Forschungskredit of the University of Zurich (D.E.), and the COST CM1105 Action is gratefully acknowledged.

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(19) Spengler, S., and Singer, B. (1981) Transcriptional errors and ambiguity resulting from the presence of 1,N6-ethenoadenosine or 3,N4-ethenocytidine in polyribonucleotides. Nucl. Acids Res. 9, 365-373. (20) Sauvaigo, S., Guerniou, V., Rapin, D., Gasparutto, D., Caillat, S., and Favier, A. (2004) An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage. Anal. Biochem. 333, 182-192. (21) Singer, B., Antoccia, A., Basu, A. K., Dosanjh, M. K., Fraenkel-Conrat, H., Gallagher, P. E., Kusmierek, J. T., Qiu, Z. H., and Rydberg, B. (1992) Both purified human 1,N6ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine. Proc. Natl. Acad. Sci. U.S.A. 89, 9386-9390. (22) Srivastava, S. C., Raza, S. K., and Misra, R. (1994) 1,N6-etheno deoxy and ribo adenosine and 3,N4-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules. Nucl. Acids Res. 22, 1296-1304. (23) Zhang, W. F., Rieger, R., Iden, C., and Johnson, F. (1995) Synthesis of 3,N4-etheno, 3,N4-ethano, and 3-(2-hydroxyethyl) derivatives of 2'-deoxycytidine and their incorporation into oligomeric DNA. Chem. Res. Toxicol. 8, 148-156. (24) Shrivastav, N., Li, D., and Essigmann, J. M. (2010) Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation. Carcinogenesis 31, 59-70. (25) Brunner, J., Mokhir, A., and Kraemer, R. (2003) DNA-templated metal catalysis. J. Am. Chem. Soc. 125, 12410-12411. (26) Li, X. Y., and Liu, D. R. (2004) DNA-templated organic synthesis: Nature's strategy for controlling chemical reactivity applied to synthetic molecules. Angew. Chem., Int. Ed. 43, 4848-4870.



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(27) Kobori, A., Morita, J., Ikeda, M., Yamayoshi, A., and Murakami, A. (2009) Sequence selective formation of 1,N6-ethenoadenine in DNA by furan-conjugated probe. Bioorg Med. Chem. Lett. 19, 3657-3660. (28) Nakatani, K., Hagihara, S., Sando, S., Miyazaki, H., Tanabe, K., and Saito, I. (2000) Site selective formation of thymine glycol-containing oligodeoxynucleotides by oxidation with osmium tetroxide and bipyridine-tethered oligonucleotide. J. Am. Chem. Soc. 122, 63096310. (29) Onizuka, K., Taniguchi, Y., and Sasaki, S. (2010) A new usage of functionalized oligodeoxynucleotide probe for site-specific modification of a guanine base within RNA. Nucl. Acids Res. 38, 1760-1766. (30) Pieck, J. C., Kuch, D., Grolle, F., Linne, U., Haas, C., and Carell, T. (2006) PNAbased reagents for the direct and site-specific synthesis of thymine dimer lesions in genomic DNA. J. Am. Chem. Soc. 128, 1404-1405. (31) Stevens, K., Claeys, D. D., Catak, S., Figaroli, S., Hocek, M., Tromp, J. M., Schurch, S., Van Speybroeck, V., and Madder, A. (2011) Furan-oxidation-triggered inducible DNA cross-linking: Acyclic versus cyclic furan-containing building blocks - On the benefit of restoring the cyclic sugar backbone. Chem.—Eur. J. 17, 6940-6953. (32) Kochetkov, N. K., Shibaev, V. N., and Kost, A. A. (1971) New reaction of adenine and cytosine derivatives, potentially useful for nucleic acids modification. Tetrahedron Lett. 12, 1993-1996. (33) Grimm, G. N., Boutorine, A. S., and Helene, C. (2000) Rapid routes of synthesis of oligonucleotide conjugates from non-protected oligonucleotides and ligands possessing different nucleophilic or electrophilic functional groups. Nucleosides, Nucleotides Nucleic Acids 19, 1943-1965.


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(34) Tolman, G. L., Barrio, J. R., and Leonard, N. J. (1974) Chloroacetaldehyde-modified dinucleoside phosphates. Dynamic fluorescence quenching and quenching due to intramolecular complexation. Biochemistry 13, 4869-4878. (35) Sugiyama, T., Schweinberger, E., Kazimierczuk, Z., Ramzaeva, N., Rosemeyer, H., and Seela, F. (2000) 2-aza-2'-deoxyadenosine: Synthesis, base-pairing selectivity, and stacking properties of oligonucleotides. Chem. Eur. J. 6, 369-378. (36) Choi, J.-H., and Pfeifer, G. P. (2004) DNA damage and mutations produced by chloroacetaldehyde in a CpG-methylated target gene. Mutat. Res.-Fund. Mol. M. 568, 245256. (37) Patel, P. H., Kawate, H., Adman, E., Ashbach, M., and Loeb, L. K. (2001) A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity. J. Biol. Chem. 276, 5044-5051. (38) Belousov, E. S., Afonina, I. A., Podyminogin, M. A., Gamper, H. B., Reed, M. W., Wydro, R. M., and Meyer, R. B. (1997) Sequence-specific targeting and covalent modification of human genomic DNA. Nucl. Acids Res. 25, 3440-3444. (39) Podyminogin, M. A., Meyer, R. B., and Gamper, H. B. (1995) Sequence-specific covalent modification of DNA by cross-linking oligonucleotides. Catalysis by RecA and implication for the mechanism of synaptic joint formation. Biochemistry 34, 13098-13108.



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FIGURE LEGENDS Figure 1. Strategy for sequence-specific generation of εdA and εdC: a) An unmodified target strand (black, target base marked gray) is mixed with a reactive strand consisting of a short complementary recognition sequence (orange) and a reactive group (green). After strand annealing (step 1) and activation of the reactive group with NaIO4 an ε-bridge at the target A (3, Fig. 1c) or C (5) base is formed (step 2). Strand separation yields a single stranded sequence-specifically modified DNA (step 3). b) The reactive group (2e), an aryl sulfonate derivative with a 1,2-diol masked aldehyde functionality, is designed to be a functional analogue of chloroacetaldehyde (1). The model reaction was performed with 2e′, while the Nhydroxysuccinimide (NHS or -OSu) ester 2e was used for the coupling reaction to form reactive strand 7 (Fig. 2a). c) Model reaction of reactive group 2e′ with 9-ethyl-A (3a) yields the ε-derivative 4a, while incubation of strands with a target A (3b) or C (5b) with reactive strand 7c induces the generation of the corresponding ε-bases (4b, 6b).

Figure 2. Detection of εA formation within DNA target strands: a) Sequences of reactive (7c) and target (8-10) strands used to detect εdA formation in short oligonucleotides (X in 7c denotes the position of the reactive group). Strands 8 and 9 bear the target A in position n+1 and n+2 relative to the position of the cytosine base carrying the reactive group in strand 7c, respectively. Strand 10 is non-complementary (nc) to the reactive strand 7c. b) Fluorescence spectra of the reaction of 7c with 9 recorded upon excitation at 275 nm show an increase of fluorescence emission over time, i.e. 0 to 5 days, indicative for εA formation (results combined from two measurements). c) RP-HPLC trace of the reaction monitored in b) after digestion to the single nucleosides. In addition to the four unmodified nucleosides also εdA (rt = 28.8 min) is detected. The chromatogram recorded at 330 nm (red) clearly distinguishes the signal for εdA from the other nucleosides, which do not show absorption at this wavelength. 18 ACS Paragon Plus Environment

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Figure 3. Site-specificity of εA and εC formation in target DNA strands. a) Sequences of 40mer target strands with A (11-16) and C (17-22) in positions n+1 to n+9 relative to the position of the cytosine base carrying the reactive group in 7c. b) Denaturing PAGE of PSA reactions performed with the 5’-32P-labeled primer 23 and the different target strands 11-22, which were modified with the reactive strand 7c. The gels reveal fully-extended as well as truncated products that are indicative for the presence of εA or εC lesions in position n+x. Lane P marks the position of primer 23 and U the position of the fully-extended unmodified target strand 11. The origin of the double band for the fully extended unmodified strand 19 (C in position n+3) remains enigmatic (Supplementary figures S17, S18). c) Sequences of mixed target strands 24-35 with multiple A and C bases in positions n+1 to n+4. d) Denaturing PAGE of PSA reactions using target strands 24-35. The high resolution of the left gel reveals in addition a mechanistic detail of the PSA assay, i.e. that the Taq polymerase is able to add a single base opposite the ε-base and only thereafter the extension reaction is stopped. As for strand 19 in part b), also with strand 30 (C in positions n+1 and n+3) a double band for the fully extended product is observed.



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Scheme 1. a) Synthesis of reactive group 2e and b) coupling reaction to the 3’-phosphorylated recognition strand 7a.*

*

Abbreviations used: 3-Clba, 3-chlorosulfonyl benzoic acid; IPG, D,L-

isopropylideneglycerol; NEt3, triethyl amine; THF, tetrahydrofurane


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TABLES Table 1. Yields including standard deviation of ε-modification using reactive strand 7c and target strands (TS) 11-22 and 24-35 as determined by PSA or by enzymatic digestion following separation of products by HPLC. TS 11 12 13 14 15 16 17 18 19 20 21 22

HPLC (%) 30 27 19 14 6 3 -

PSA n+x (%) 32 ± 0.5 25 ± 2.0 17 ± 0.3 11 ± 2.0 5 ± 0.3 3 ± 0.6 26 ± 6.2 14 ± 3.8 5 ± 3.5 1 ± 0.9 6 ± 1.9 1 ± 1.0

TS 24 25 26 27 28 29 30 31 32 33 34 35

PSA n+1 (%) 22 ± 1.4 33 ± 3.3 37 ± 2.7 22 ± 2.0 11 ± 3.1 34 ± 5.9 21 ± 7.3 9 ± 0.9 14 ± 2.5 31 ± 3.1 29 ± 1.4

PSA n+2 (%) 30 ± 1.3 28 ± 4.5 18 ± 1.4 10 ± 2.2 40 ± 1.9 4 ± 1.1 -


PSA n+3 (%) 15 ± 2.6 16 ± 2.5 16 ± 1.3 3 ± 1.0 -

PSA n+4 (%) 6 ± 1.7 4 ± 0.9 3 ± 1.0 1 ± 0.2


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SYNOPSIS TOC 1,N6-Ethenoadenine (εA) and 3,N4-ethenocytosine (εC) are naturally occurring base adducts. In this study a new synthetic strategy to sequence-specifically generate etheno-adducts in an unmodified single-stranded nucleic acid is reported making use of a DNA-templated approach that positions the required alkylating agent close in space to the respective target base. The length of the target strand should not restrict the applicability of the presented method.


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Scheme 1. a) Synthesis of reactive group 2e and b) coupling reaction to the 3’-phosphorylated recognition strand 7a. 186x200mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 1. Strategy for sequence-specific generation of εdA and εdC: a) An unmodified target strand (black, target base marked gray) is mixed with a reactive strand consisting of a short complementary recognition sequence (orange) and a reactive group (green). After strand annealing (step 1) and activation of the reactive group with NaIO4 an ε-bridge at the target A (3, Fig. 1c) or C (5) base is formed (step 2). Strand separation yields a single stranded sequence-specifically modified DNA (step 3). b) The reactive group (2e), an aryl sulfonate derivative with a 1,2-diol masked aldehyde functionality, is designed to be a functional analogue of chloroacetaldehyde (1). The model reaction was performed with 2e′, while the Nhydroxysuccinimide (NHS or -OSu) ester 2e was used for the coupling reaction to form reactive strand 7 (Fig. 2a). c) Model reaction of reactive group 2e′ with 9-ethyl-A (3a) yields the ε-derivative 4a, while incubation of strands with a target A (3b) or C (5b) with reactive strand 7c induces the generation of the corresponding ε-bases (4b, 6b). 151x138mm (300 x 300 DPI)


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Figure 2. Detection of εA formation within DNA target strands: a) Sequences of reactive (7c) and target (810) strands used to detect εdA formation in short oligonucleotides (X in 7c denotes the position of the reactive group). Strands 8 and 9 bear the target A in position n+1 and n+2 relative to the position of the reactive group in strand 7c, respectively. Strand 10 is non-complementary (nc) to the reactive strand 7c. b) Fluorescence spectra of the reaction of 7c with 9 recorded upon excitation at 275 nm show an increase of fluorescence emission over time, i.e. 0 to 5 days, indicative for εA formation (results combined from two measurements). c) RP-HPLC trace of the reaction monitored in b) after digestion to the single nucleosides. In addition to the four unmodified nucleosides also εdA (rt = 28.8 min) is detected. The chromatogram recorded at 330 nm (red) clearly distinguishes the signal for εdA from the other nucleosides, which do not show absorption at this wavelength. 286x519mm (300 x 300 DPI)



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Figure 3. Site-specificity of εA and εC formation in target DNA strands. a) Sequences of 40mer target strands with A (11-16) and C (17-22) in positions n+1 to n+9 relative to the position of the reactive group in 7c. b) Denaturing PAGE of PSA reactions performed with the 5’-32P-labeled primer 23 and the different target strands 11-22, which were modified with the reactive strand 7c. The gels reveal fully-extended as well as truncated products that are indicative for the presence of εA or εC lesions in position n+x. Lane P marks the position of primer 23 and U the position of the fully-extended unmodified target strand 11. The origin of the double band for the fully extended unmodified strand 19 (C in position n+3) remains enigmatic (Supplementary figures S17, S18). c) Sequences of mixed target strands 24-35 with multiple A and C bases in positions n+1 to n+4. d) Denaturing PAGE of PSA reactions using target strands 24-35. The high resolution of the left gel reveals in addition a mechanistic detail of the PSA assay, i.e. that the Taq polymerase is able to add a single base opposite the ε-base and only thereafter the extension reaction is stopped. As for strand 19 in part b), also with strand 30 (C in positions n+1 and n+3) a double band for the fully extended product is observed.


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207x340mm (300 x 300 DPI)



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Figure for Table of Contents 53x44mm (300 x 300 DPI)


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Sequence-Specific Generation of 1,N6-Ethenoadenine and 3,N4

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