5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Articles pubs.acs.org/acschemicalbiology

5‑Substituted Pyrimidine and 7‑Substituted 7‑Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs Hana Cahová,† Alessandro Panattoni,† Pavel Kielkowski,† Jindřich Fanfrlík,† and Michal Hocek*,†,‡ †

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo namesti 2, CZ-16610 Prague 6, Czech Republic ‡ Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague-2 12843, Czech Republic S Supporting Information *

ABSTRACT: A complete series of 5-substituted uracil or cytosine, as well as 7-substituted 7-deazaadenine and 7deazaguanine 2′-deoxyribonucleoside triphosphates (dNTPs) bearing substituents of increasing bulkiness (H, Me, vinyl, ethynyl, and phenyl) were systematically studied in competitive primer extension in the presence of their natural counterparts (nonmodified dNTPs), and their kinetic data were determined. The results show that modified dNTPs bearing π-electroncontaining substituents (vinyl, ethynyl, Ph) are typically excellent substrates for DNA polymerases comparable to or better than natural dNTPs. The kinetic studies revealed that these modified dNTPs have higher affinity to the active site of the enzyme−primer−template complex, and the calculations (semiempirical quantum mechanical scoring function) suggest that it is due to the cation−π interaction of the modified dNTP with Arg629 in the active site of Bst DNA polymerase.

B

corresponding nucleosides), no systematic quantitative studies of competitive incorporations of modified nucleotides in the presence of natural dNTPs has been reported, and the modified dNTPs were generally believed to be worse substrates for DNA polymerases than their natural counterparts. Recently, we reported a preliminary communication38 on competitive incorporation of several 7-substituted-7-deazaadenine and 5substituted cytosine dNTPs and found that 7-aryl-7-deazadATP analogues (dARTPs) are significantly better substrates for DNA polymerases than dATP, whereas the corresponding 5-modified dCRTP analogues were comparable or slightly worse substrates than dCTP. Here, we report the results of the extended comprehensive and systematic study of competitive incorporations of all four modified nucleotides by various polymerases, kinetic studies, and molecular modeling in order to explain the previous surprising results (Scheme 1).

ase-functionalized DNA or oligonucleotides have attracted growing interest and find diverse applications.1 Apart from chemical synthesis on solid support, they can be efficiently prepared by enzymatic synthesis using DNA polymerases and base-modified 2′-deoxyribonucleoside triphosphates (dNTPs) as substrates.2−4 Number of methods based on the polymerase incorporations of modified nucleotides have been developed,2 and the applications include fluorescent,5 spin,6 or redox7 labeling; introduction of functional groups for DNAzymes or aptamers;8−13 reactive groups for cross-linking14 or even polymers,15 oligonucleotides,16 and protein17 molecules; or protection of DNA against cleavage by restriction endonucleases18−22 or regulation of transcription.23 Mechanistic and structural studies showed3,24−29 that there is a significant space in the major groove direction on the complex of polymerase, primer and template which enables efficient incorporation of dNTPs bearing even bulky substituents if they are linked to position 5 of pyrimidines or position 7 of 7-deazapurines. In most of these enzymatic methods, the polymerase reactions using a modified dNTP are performed in the absence of its natural counterpart (unmodified dNTP). Although some PCR reactions30−32 or primer extension (PEX)24−29 studies were reported with mixtures of natural and modified dNTPs and metabolic labeling for quantification of DNA synthesis is based on competitive incorporations of bromouracil,33 or more recently of ethynyl-34−36 or azido-substituted37 nucleotides (generated in vivo by intracellular phosphorylation of the © XXXX American Chemical Society

■

RESULTS AND DISCUSSION In our previous works,18−22 we studied the influence of majorgroove modifications on cleavage of DNA by Type II restriction endonucleases (REs) and found that some of these enzymes can tolerate small substituents, whereas the presence of bulkier Received: August 17, 2016 Accepted: September 26, 2016

A

DOI: 10.1021/acschembio.6b00714 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology

Chart 1. Chemical Structures of Base-Modified dNRTPs Used in This Study

Scheme 1. Principle of the Competition Assay

Table 1. Incorporation of Functionalized dNRTPs with Bst DNA Polymerase dNRTP

competitiona

5′-...G/AATTCC...-3′ 3′-...CTTAA/GG...-5′ dGTP dG7DTP dGMeTP dGViTP dGETP dGPhTP

groups in the major groove typically blocks the enzymatic cleavage. This knowledge has helped us to develop an assay38 for the competitive incorporations of modified nucleotides in the presence of their natural counterparts. The assay (Scheme 1) was based on the PEX reaction using a FAM-labeled primer, template (containing a recognition sequence for a RE), polymerase, and a mixture of a natural dNTP and the corresponding modified dNRTP (in the presence of the three complementary natural dNTPs). The mixture of PEX products was then incubated with a RE which cleaves the nonmodified DNA, whereas the DNA containing a modified recognition sequence remains intact. The PAGE analysis is used for quantification of the ratio of modified versus natural nucleotides in the particular site. In addition to previously studied38 ethynyl and phenyl 7substituted 7-deazaadenine dARTPs (dAETP and dAPhTP)18 and 5-substituted dCRTPs (dCETP and dCPhTP),19 we also included 7-methyl and 7-vinyl-7-deaza-dATPs (dAMeTP23 and dAViTP20), vinyl-substituted dCTP (dC ViTP),20 and a complete series of four 7-substituted 7-deazaguanine dGRTPs (dGMeTP, dGViTP, dGETP, and dGPhTP),21 unsubstituted 7deazaguanine dG7DTP, and three 5-substituted dUTPs (dUViTP,20 dUETP,19 and dUPhTP19) as analogues of TTP (Chart 1). The substituents in the major groove were chosen to show the effect of the bulkiness of the substituent (increasing bulkiness from H to phenyl in deazapurines), as well as the possible influence of the π-electronic systems bearing functional groups of varying bulkiness. For each nucleobase, we had to design a specific sequence of a primer and template leading to a formation of PEX product which would have exactly one modified nucleobase in the

33 42 39 58 67

5′-...G/AATTCC...-3′ 3′-...CTTAA/GG...-5′ TTP dUViTP dUETP dUPhTP

41 28 57

5′-...G/AATTCC...-3′ 3′-...CTTAA/GG...-5′ dATP dAMeTP dAViTP dAETP dAPhTP

35 70 72 76

5′-...AGC/GCT...-3′ 3′-...TCG/CGA...-5′ dCTP dCViTP dCETP dCPhTP a

49 49 52

Percentage of incorporation of the modified nucleotide.

recognition site for the particular RE (see Table 1 and Table S1 in Supporting Information). For the study of all A, G, and U B


Articles


Table 2. Competitive Incorporation of Selected Modified 7Deazaguanine and Uracil Nucleotides Using 4 DNA polymerasesa Bst

a

KOD XL

Pwo

Vent (exo-)

R

dN TP

1:1

1:10

1:1

1:10

1:1

1:10

1:1

1:10

dGETP dGPhTP dUETP dUPhTP

58 66 28 57

81 90 63 80

51 58 37 17

88 89 88 57

37 43 26 6

61 80 53 57

24 41 20 3

70 84 60 34

Percentage of incorporation of the modified nucleotide.

except for dUETP), whereas KOD XL polymerase preferred only deazapurine dNRTPs. Pwo and Vent (exo-) preferred natural dNTPs, and in particular, U-modified nucleotides were poorly incorporated in competition with natural TTP (Table 2, Figure S2). When using an excess of the modified dNRTP (10:1), we almost always observed a large proportion of the modified DNA (57−90%) except for dUPhTP with Vent (exo-). Previously, we have shown38 that the competitive incorporations (with dAPhTP and dCPhTP) were sequence-dependent. Therefore, we have now also performed competitive experiments with dGPhTP or dUPhTP using four different sequences where the incoming modified nucleotide follows a different nucleobase in the primer. The results (Table S3 in the SI) showed that the competitive incorporation of dUPh nucleotide is almost the same in all tested sequences (54−57%), whereas the incorporation of dGPh nucleotide varies between 43 and 67% in different sequences. In order to further prove the higher affinity of polymerase Bst large fragment toward 7-phenyl-7-deazapurine dNTPs in comparison with other modified nucleotides, we performed a direct competition PEX experiment with a mixture of dGPhTP and unsubstituted deazapurine dG7DTP followed by cleavage by PvuII (which is known21 to cleave DNA containing 7deazaG but not 7-Ph-7-deazaG). In accord with our expectations, this experiment yielded 86% of phenylated DNA (Figure 2). To investigate the effect of the substrate activity of modified dNRTPs in a more complex process, we performed a competitive PCR experiment with various ratios of natural dNTPs and dUPhTP and dGPhTP. As Bst large fragment is not a thermally stable DNA polymerase, we used KOD XL polymerase in the PCR experiments instead. We designed a 339-bp-long PCR product with a cleavage site for EcoRI and performed cleavage of the PCR products with this RE. It is important to note that the recognition site for EcoRI contains four thymines and two guanines (Table 1) and replacement of any one of them by the dUPh or dGPh would cause resistance of the sequence toward cleavage. Thus, the ratio between cleaved and noncleaved DNA does not represent the percentage of modification at each site (as it is in the PEX), but the percentages of the presence of at least one (or more) modified nucleotide(s) in the whole recognition sequence and the PCR products are a statistical mixture of all possible combinations of modified and nonmodified sequences. The dUPhTP was found to be a good substrate in PCR (Figure 3). At a ratio of 1:1 with natural TTP, we observed a 74% presence of uncleaved DNA, whereas even at a ratio of 1:2 (2-fold excess of TTP), there was a 50% uncleaved DNA. Although we cannot fully quantify the competition ratio at each site, the experiments show that there is a significant incorporation of the dUPh (statistically at least 18% in the ratio 1:1) even in the very complex PCR process.

Figure 1. Page analyses of PEX experiments with Bst DNA polymerase and cleavage products with dG7DTP and dGPhTP. Lane 1, N: product of PEX with natural dGTP. Lanes 2 and 3, 1:1 and 1:10: product of PEX with three natural dNTPs and corresponding ratio of dGTP/ dGRTP. Lane 4, N: product of cleavage of unmodified DNA by EcoRI. Lanes 5 and 6, 1:1 and 1:10: products of cleavage of modified DNAs by RE.

modifications, we used EcoRI RE, which is known to be blocked by all major-groove modifications.18−22 In order to study C modifications, we have chosen AfeI, which contains a suitable cytosine in close proximity to the cleavage site (Table 1). We also retested some of the previously studied dARTP and dCRTP analogues,38 and since the competition is also sequence dependent38 and we used different conditions, their data slightly differ from previously published values but generally follow the same trends.38 At first, we studied the competitive incorporations of each of the above-mentioned modified nucleotides in a ratio of 1:1 with their natural counterparts in the presence of the Bst (large fragment) polymerase (Figure 1, Table 1, Figure S1). From the modified dG R TPs, the unsubstituted 7-deazaguanine (dG7DTP) as well as the 7-methyl- (dGMeTP) and 7-vinyl(dGViTP) derivatives were slightly worse substrates than dGTP, giving 33−42% of incorporation of the modified nucleotide. On the other hand, the 7-ethynyl (dGETP) and 7-phenyl- (dGPhTP) derivatives were better substrates than the natural nucleotide (58% and 67% of incorporation, respectively). From 5-modified dURTPs, the vinyl and ethynyl derivatives (dUViTP and dUETP) were worse substrates (41% and 28% incorporation), while only the phenyl derivative (dUPhTP) was a slightly better substrate (57%) than TTP. From the modified 7-deaza-dATP analogues (dARTPs), the 7methyl derivative (dAMeTP) was a worse substrate (35%) than dATP (similarly to previously published38 unsubstituted 7deaza-dATP), whereas the vinyl (dAViTP), ethynyl (dAETP), and phenyl (dAPhTP) derivatives were all significantly better substrates (70−76% incorporation) than dATP. In the modified cytosine nucleotides, all three modified derivatives (dCViTP, dCETP and dCPhTP) were comparable to dCTP (49−52% incorporation). Then, we studied selected modified deazaguanine and uracil triphosphates (dGETP, dGPhTP, dUETP, and dUPhTP) in competitive PEX experiments in the presence of different DNA polymerases (Bst large fragment, KOD XL, Pwo, Vent(exo-)) in two different ratios of functionalized dNRTP and natural dNTP (1:1 and 10:1). In accord with the previously reported similar experiments with dAPhTP,38 when using equimolar mixtures of natural and modified dNTPs, Bst polymerase was found to give the highest preference to modified dNRTPs (all C


Articles


Table 3. Incorporation of Functionalized dNRTPs with Bst DNA Polymerase 5′-...NCCC···-3′

Figure 2. Page analyses of PEX experiments with Bst DNA polymerase with dG7DTP and dGPhTP and RE cleavage. Lane 1, N: product of PEX with dG7DTP. Lanes 2 and 3, 1:1 and 1:10: product of PEX with two natural dNTPs dG7DTP and a corresponding ratio of dG7DTP /dGPhTP. Lane 4, N: product of cleavage of unmodified DNA by PvuII. Lanes 5 and 6, 1:1 and 1:10: products of cleavage of modified DNAs by RE.

3′-...XGGG···-5′

KMa

kcatb

kcat/KM

ratioc

dGTP dG7DTP dGMeTP dGViTP dGETP dGPhTP TTP dUViTP dUETP dUPhTP dATP dAMeTP dAViTP dAETP dAPhTP dCTP dCViTP dCETP dCPhTP

5.3 12.5 11.1 9.5 7.5 4.1 3.2 13.1 6.3 7.9 8.7 6.1 10.9 7.3 5.6 6.7 14.1 15.1 6

13.1 12.1 12.7 13.8 17.5 13.7 6.6 17.7 4.6 9.3 8.0 5.5 7.1 6.3 13.5 6.1 15.4 10.4 7.5

2.47 0.97 1.14 1.45 2.33 3.34 2.06 1.35 0.73 1.18 0.99 0.9 0.65 0.88 2.41 0.91 1.1 0.69 1.25

1 0.4 0.5 0.6 0.9 1.4 1 0.7 0.4 0.6 1 0.9 0.7 0.9 2.4 1 1.2 0.8 1.4

The units for KM are μM. bthe units for kcat are s−1. cRatio is defined as (kcat/KM)Modif/(kcat/KM)natural, and the units are s−1 μM−1.

a

the unsubstituted or methylated 7-deazapurine dNTPs. All modified dURTPs and dCRTPs had higher KM values than TTP or dCTP, except for dCPhTP, which was comparable to the natural counterpart. In accord with the previous report,38 the ethynyl and phenyl-modified 7-deaza-dATPs, and surprisingly also methylated dAMeTP, had lower KM values than natural dATP. The kcat values for the modified versus nonmodified dNTPs did not show very significant differences, but the kcat/ KM ratios were the highest for the 7-phenyl-substituted 7deazapurine nucleotides (dGPhTP and dAPhTP), which also performed the best in competitive experiments and indicates their higher affinity for the active site of the complex of polymerase with primer and template. The phenylated deazapurine nucleotides (dGPhTP and dAPhTP) also showed the highest discrimination rate, calculated as (kcat/KM)Modif/ (kcat/KM)natural. Also, two of the cytosine nucleotides (dCPhTP and dCViTP) gave a discrimination rate > 1, which does not fully correspond to their performance in the competition assay (comparable to natural dCTP), but the sequences and experimental setup were not identical, which doesn’t allow for expressing such minute differences. To explain the fact that phenyl-substituted 7-deazapurine nucleotides (dAPhTP and dGPhTP), but not phenylpyrimidine nucleotides (dCPhTP and dUPhTP), were significantly better substrates for DNA polymerase than natural dNTPs, we performed a molecular modeling study. We modeled tertiary complexes of Bst polymerase with a template, primer, and a dNTP (docked to the published crystal structure42) using semiempirical quantum mechanical scoring function43,44 (Figure 4). The 7-phenyl-7-deazapurine nucleotides (dGPhTP and dAPhTP) had larger affinity (more negative score) than the native dGTP or dATP (by 20.5 and 10.5 kcal/mol, respectively, Table 4). The score of dCPhTP was only slightly more negative than that of dCTP (by 1.8 kcal/mol), whereas the modified dUPhTP was computed to have a lower affinity than the natural TTP (by 8.7 kcal/mol) or than unsubstituted dUTP (by 10.6

Figure 3. Agarose gel analysis of competitive PCR with dUPhTP using KOD XL followed by cleavage by EcoRI. Lane 1, 100 bp ladder; lane 2, product of PCR with natural dNTPs cleaved by EcoRI; lanes 3−9, products of PCR with different ratios of TTP/dUPhTP after cleavage by EcoRI.

On the other hand, the PCR amplification with dGPhTP was very poor (in accord with our previous work,21 see Figure S3 in the SI). Since the dGPhTP is a superior substrate for the polymerase, the low efficiency of the PCR must be due to a difficult reading of the enzyme through the dGPh-modified template in the PCR cycles (similar effects we often observed for other bulkier modifications2). Then, we studied the kinetics of single nucleotide incorporations38−41 of each of the modified dNRTPs with Bst polymerase and compared them to those of their natural counterparts (Table 3 and Supporting Information Table S4). The KM values of most of the modified dGRTPs were somewhat higher (7.5−12.5) than that of natural dGTP (5.3), except for dGPhTP (4.1), which was the best substrate also in the competition experiments. They were generally lower for dGRTPs bearing π-electron-containing substituents than for D


Articles


varying bulkiness in the presence of their natural counterparts (unmodified dNTPs). Most of these modified dNRTP’s were good to excellent substrates for Bst and KOD XL polymerases and still moderate to good substrates for Pwo and Vent(exo-) polymerases. 7-Deazapurine dNTPs bearing π-electron-containing substituents (ethynyl and phenyl, as well as 7-vinyl-7deazaadenine) are generally better substrates of Bst polymerase than natural dATP or dGTP, respectively. The corresponding 5-substituted cytosine dNTP’s (dCRTP) are comparable to dCTP, whereas the 5-substituted uracil dURTPs are generally worse substrates than TTP. The measured kinetic parameters (KM) follow the same trend and confirm that 7-phenyl-7deazapurine dNTPs have higher affinity to the active site of the polymerase than their natural counterparts. The calculations also confirmed the higher affinity of the phenylated deazapurine nucleotides to the active site and revealed that the most significant contributing factor is the cation−π interaction of the modified dNPhTP with Arg629. These results not only confirm and extend our previous observations38 of the superior substrate activities of 7-aryl-7deaza-dATP analogues to the corresponding 7-deazaguanine nucleotides but also shed more light onto the mechanism of polymerase incorporation of base-modified nucleotides. The very good competition of 7-substituted 7-deazapurine dNTPs, and still reasonably good activity of 5-substituted pyrimidine dNTPs, in the presence of their natural counterparts is very encouraging for further development of methods of polymerase synthesis of modified DNA and for possible in cellulo and even in vivo applications if satisfactory delivery45 of modified dNTPs will be solved. Studies in these directions are under way in our lab.

Figure 4. Molecular modeling data. (a) Modeled structure of dGPhTP in the active site of the complex of Bst polymerase, primer, and template. (b) Contribution of selected Bst polymerase amino acid residues to the dGTP (in black) and dGPhTP (in red) binding. The energy contribution of single amino acids to the binding obtained from the “virtual glycine scan.”

■

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00714. List of all ON and DNA sequences, additional tables with data, additional PAGE analyses of all experiments, and full experimental section with all materials and procedures (PDF)

Ph

Table 4. Scores of the Natural dNTPs and dN TPs dNTP

score

dGTP dGPhTP dUTP TTP dUPhTP dATP dAPhTP dCTP dCPhTP

−108.5 −129.0 −72.4 −70.5 −61.8 −86.9 −97.4 −68.4 −70.2

ASSOCIATED CONTENT

relative score −20.5

■

10.5 (to UTP), 8.7 (to TTP) −10.5

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

−1.8

Author Contributions

M.H., H.C., and P.K. designed the study. H.C. and A.P. performed the experiments. J.F. performed the calculations. M.H. and H.C. wrote the paper with contributions of all other authors. All authors have given approval to the final version of the manuscript.

kcal/mol). The modeled structure of dGPhTP in the active site shows that the phenyl ring occupied the space between the aliphatic chain of Arg629 and the phenyl of Phe710. The virtual glycine scan showed that the increase in the binding is largely due to the cation−π interaction of Arg629 with the phenyl group of dGPhTP. Thus, the results of this molecular modeling study reflect the discrimination rate calculated from kinetic data for phenylated derivatives (deazapurines versus pyrimidines) and are in accord with the previously reported mechanistic considerations.29

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Academy of Sciences of the Czech Republic (RVO: 61388963 and Praemium Academiae to M.H.), the Czech Science Foundation (14-04289S to M.H.), by Marie Sklodowska-Curie Innovative Training Network (ITN) Click Gene (H2020-MSCA-ITN-2014-642023 to A.P.), and by Gilead Sciences, Inc. (Foster City, CA, U.S.A.).

■

CONCLUSIONS In conclusion, we performed a systematic study of competition PEX experiments with a series of 7-substituted 7-deazapurine and 5-substituted pyrimidine dNTPs bearing substituents of E


Articles


■

(18) Macíčková-Cahová, H., and Hocek, M. (2009) Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases. Nucleic Acids Res. 37, 7612−7622. (19) Macíčková-Cahová, H., Pohl, R., and Hocek, M. (2011) Cleavage of Functionalized DNA Containing 5-Modified Pyrimidines by Type II Restriction Endonucleases. ChemBioChem 12, 431−438. (20) Mačková, M., Pohl, R., and Hocek, M. (2014) Polymerase Synthesis of DNAs Bearing Vinyl Groups in the Major Groove and their Cleavage by Restriction Endonucleases. ChemBioChem 15, 2306− 2312. (21) Mačková, M., Bohácǒ vá, S., Perlíková, P., Poštová Slavětínská, L., and Hocek, M. (2015) Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7 Substituted 7-Deazaguanine Nucleobases. ChemBioChem 16, 2225−2236. (22) Kielkowski, P., Macíčková-Cahová, H., Pohl, R., and Hocek, M. (2011) Transient and switchable (triethylsilyl)ethynyl protection of DNA against cleavage by restriction endonucleases. Angew. Chem., Int. Ed. 50, 8727−8730. (23) Raindlová, V., Janoušková, M., Slavíčková, M., Perlíková, P., Bohácǒ vá, S., Milisavljevič, N., Šanderová, H., Benda, M., Barvík, I., Krásný, L., and Hocek, M. (2016) Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases. Nucleic Acids Res. 44, 3000−3012. (24) Obeid, S., Baccaro, A., Welte, W., Diederichs, K., and Marx, A. (2010) Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U. S. A. 107, 21327−21331. (25) Obeid, S., Bußkamp, H., Welte, W., Diederichs, K., and Marx, A. (2013) Snapshot of a DNA polymerase while incorporating two consecutive C5-modified nucleotides. J. Am. Chem. Soc. 135, 15667− 15669. (26) Bergen, K., Steck, A. L., Strütt, S., Baccaro, A., Welte, W., Diederichs, K., and Marx, A. (2012) Structures of KlenTaq DNA polymerase caught while incorporating C5-modified pyrimidine and C7-modified 7-deazapurine nucleoside triphosphates. J. Am. Chem. Soc. 134, 11840−11843. (27) Bergen, K., Betz, K., Welte, W., Diederichs, K., and Marx, A. (2013) Structures of KOD and 9°N DNA polymerases complexed with primer template duplex. ChemBioChem 14, 1058−1062. (28) Betz, K., Streckenbach, F., Schnur, A., Exner, T., Welte, W., Diederichs, K., and Marx, A. (2010) Structures of DNA polymerases caught processing size-augmented nucleotide probes. Angew. Chem., Int. Ed. 49, 5181−5184. (29) Obeid, S., Busskamp, H., Welte, W., Diederichs, K., and Marx, A. (2012) Interactions of non-polar and ‘‘Click-able’’ nucleotides in the confines of a DNA polymerase active site. Chem. Commun. 48, 8320− 8322. (30) Seela, F., and Röling, A. (1992) 7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7IdTP incorporation during PCRamplification and protection from endodeoxyribonuclease hydrolysis. Nucleic Acids Res. 20, 55−61. (31) Shoji, A., Hasegawa, T., Kuwahara, M., Ozaki, H., and Sawai, H. (2007) Chemico-enzymatic synthesis of a new fluorescent-labeled DNA by PCR with a thymidine nucleotide analogue bearing an acridone derivative. Bioorg. Med. Chem. Lett. 17, 776−779. (32) Ren, X., El-Sagheer, A. H., and Brown, T. (2016) Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes. Nucleic Acids Res. 44, e79. (33) Bessman, M. J., Lehman, I. R., Adler, J., Zimmerman, S. B., Simms, E. S., and Kornberg, A. (1958) Enzymatic synthesis of deoxyribonucleic acid. III. The incorporation of pyrimidine and purine analogues into deoxyribonucleic acid. Proc. Natl. Acad. Sci. U. S. A. 44, 633−640. (34) Guan, L., van der Heijden, G. W., Bortvin, A., and Greenberg, M. M. (2011) Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2′-deoxycytidine. ChemBioChem 12, 2184−2190.

ABBREVIATIONS RE, restriction endonuclease; PEX, primer extension; dNTP, 2′-deoxyribonucleoside triphosphate

■

REFERENCES

(1) Nakatani, K., and Tor, Y. Modified Nucleic Acids, Nucleic Acids and Molecular Biology Series; Springer, 2016; Vol. 31, pp 1−276. (2) Hocek, M. (2014) Synthesis of base-modified 2′-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology. J. Org. Chem. 79, 9914−9921. (3) Hottin, A., and Marx, A. (2016) Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases. Acc. Chem. Res. 49, 418−427. (4) Hollenstein, M. (2012) Nucleoside triphosphates–building blocks for the modification of nucleic acids. Molecules 17, 13569−13591. (5) Dziuba, D., Jurkiewicz, P., Cebecauer, M., Hof, M., and Hocek, M. (2016) A Rotational BODIPY Nucleotide: An EnvironmentSensitive Fluorescence-Lifetime Probe for DNA Interactions and Applications in Live-Cell Microscopy. Angew. Chem., Int. Ed. 55, 174− 178. (6) Obeid, S., Yulikov, M., Jeschke, G., and Marx, A. (2008) Enzymatic synthesis of multiple spin-labeled DNA. Angew. Chem., Int. Ed. 47, 6782−6785. (7) Balintová, J., Špaček, J., Pohl, R., Brázdová, M., Havran, L., Fojta, M., and Hocek, M. (2015) Azidophenyl as a click-transformable redox label of DNA suitable for electrochemical detection of DNA−protein interactions. Chem. Sci. 6, 575−587. (8) Hollenstein, M., Hipolito, C. J., Lam, C. H., and Perrin, D. M. (2009) A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+). Nucleic Acids Res. 37, 1638−1649. (9) Kuwahara, M., Nagashima, J., Hasegawa, M., Tamura, T., Kitagata, R., Hanawa, K., Hososhima, S., Kasamatsu, T., Ozaki, H., and Sawai, H. (2006) Systematic characterization of 2′-deoxynucleoside5′-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA. Nucleic Acids Res. 34, 5383−5394. (10) Gourlain, T., Sidorov, A., Mignet, N., Thorpe, S. J., Lee, S. E., Grasby, J. A., and Williams, D. M. (2001) Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR. Nucleic Acids Res. 29, 1898−1905. (11) Sawai, H., Ozaki, A. N., Satoh, F., Ohbayashi, T., Masud, M. M., and Ozaki, H. (2001) Expansion of structural and functional diversities of DNA using new 5-substituted deoxyuridine derivatives by PCR with superthermophilic KOD Dash DNA polymerase. Chem. Commun., 2604−2605. (12) Sakthivel, and Barbas, C. F., III (1998) Expanding the Potential of DNA for Binding and Catalysis: Highly Functionalized dUTP Derivatives That Are Substrates for Thermostable DNA Polymerases. Angew. Chem., Int. Ed. 37, 2872−2875. (13) Kajiyama, T., Kuwahara, M., Goto, M., and Kambara, H. (2011) Optimization of pyrosequencing reads by superior successive incorporation efficiency of improved 2′-deoxyadenosine-5′-triphosphate analogs. Anal. Biochem. 416, 8−17. (14) Dadová, J., Orság, P., Pohl, R., Brázdová, M., Fojta, M., and Hocek, M. (2013) Vinylsulfonamide and acrylamide modification of DNA for cross-linking with proteins. Angew. Chem., Int. Ed. 52, 10515−10518. (15) Baccaro, A., and Marx, A. (2010) Enzymatic synthesis of organic-polymer-grafted DNA. Chem. - Eur. J. 16, 218−226. (16) Baccaro, A., Steck, A. L., and Marx, A. (2012) Barcoded nucleotides. Angew. Chem., Int. Ed. 51, 254−257. (17) Welter, M., Verga, D., and Marx, A. (2016) Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases. Angew. Chem., Int. Ed. 55, 10131−10135. F


Articles

ACS Chemical Biology (35) Neef, A. B., and Luedtke, N. W. (2011) Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc. Natl. Acad. Sci. U. S. A. 108, 20404−20409. (36) Neef, A. B., Pernot, L., Schreier, V. N., Scapozza, L., and Luedtke, N. W. (2015) A Bioorthogonal Chemical Reporter of Viral Infection. Angew. Chem., Int. Ed. 54, 7911−7914. (37) Neef, A. B., and Luedtke, N. W. (2014) An azide-modified nucleoside for metabolic labeling of DNA. ChemBioChem 15, 789− 793. (38) Kielkowski, P., Fanfrlík, J., and Hocek, M. (2014) 7-Aryl-7deazaadenine 2′-Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations. Angew. Chem., Int. Ed. 53, 7552−7555. (39) Polesky, A. H., Steitz, T. A., Grindley, N. D., and Joyce, C. M. (1990) Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli. J. Biol. Chem. 265, 14579−14591. (40) Kong, H., Kucera, R. B., and Jack, W. E. (1993) Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J. Biol. Chem. 268, 1965−1975. (41) Patro, J. N., Urban, M., and Kuchta, R. D. (2009) Interaction of human DNA polymerase alpha and DNA polymerase I from Bacillus stearothermophilus with hypoxanthine and 8-oxoguanine nucleotides. Biochemistry 48, 8271−8278. (42) Kiefer, J. R., Mao, C., Braman, J. C., and Beese, L. S. (1998) Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature 391, 304−307. (43) Fanfrlik, J., Bronowska, A. K., Rezac, J., Prenosil, O., Konvalinka, J., and Hobza, P. (2010) A reliable docking/scoring scheme based on the semiempirical quantum mechanical PM6-DH2 method accurately covering dispersion and H-bonding: HIV-1 protease with 22 ligands. J. Phys. Chem. B 114, 12666−12678. (44) Lepsik, M., Rezac, J., Kolař, M., Pecina, A., Hobza, P., and Fanfrlik, J. (2013) The semiempirical quantum mechanical scoring function for in silico drug design. ChemPlusChem 78, 921−931. (45) Gollnest, T., Dinis de Oliveira, T., Rath, A., Hauber, I., Schols, D., Balzarini, J., and Meier, C. (2016) Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues. Angew. Chem., Int. Ed. 55, 5255−5258.

G


5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as

Recommend Documents