Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing

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.

Article Cite This: Acc. Chem. Res. 2019, 52, 1730−1737

pubs.acs.org/accounts

Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing, Cross-linking, and Modulation of Protein−DNA Binding and Transcription Michal Hocek*

Downloaded via 95.181.177.31 on July 26, 2019 at 12:51:57 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, 16610 Prague 6, Czech Republic Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic CONSPECTUS: Protein−DNA interactions are important in replication, transcription, repair, as well as epigenetic modifications of DNA, which involve methylation and demethylation of DNA resulting in regulation of gene expression. Understanding of these processes and chemical tools for studying and perhaps even modulating them could be of great relevance and importance not only in chemical biology but also in real diagnostics and treatment of diseases. In the past decade, we have been working on development of synthesis of base-modified 2′-deoxyribo- or ribonucleoside triphosphates (dNTPs or NTPs) and their use in enzymatic synthesis of modified nucleic acids using DNA or RNA polymerases. These synthetic and enzymatic methods are briefly summarized with focus on recent development and outlining of scope, limitations, and further challenges. The main focus of this Account is on applications of base-modified nucleic acids in sensing of protein−DNA interactions, in covalent cross-linking to DNA-binding proteins ,and in modulation of protein−DNA binding and transcription. Several environment-sensitive fluorescent nucleotides were incorporated to DNA probes which responded to protein binding by light-up, changing of color, or lifetime of fluorescence. Using a cyclodextrinpeptide transporter, fluorescent nucleotides can be transported through the cell membrane and incorporated to genomic DNA. Several dNTPs bearing reactive groups (i.e., vinylsulfonamide or chloroacetamide) were used for polymerase synthesis of DNA reactive probes which cross-link to Cys, His, or Lys in peptides or proteins. An attractive challenge is to use DNA modifications and bioorthogonal reactions in the major groove of DNA for modulation and switching of protein−DNA interactions. We have systematically explored the influence of major-groove modifications on recognition and cleavage of DNA by restriction endonucleases and constructed simple chemical switches of DNA cleavage. Systematic study of the influence of major-groove modifications on transcription with bacterial RNA polymerases revealed not only that some modified bases are tolerated, but also that the presence of 5-hydroxymethyluracil or -cytosine can even enhance the transcription (350 or 250% compared to native DNA). Based on these results, we have constructed the first chemical switch of transcription based on photocaging of hydroxymethylpyrimidines in DNA by 2-nitrobenzyl protection (transcription off), photochemical deprotection of the DNA (transcription on), and enzymatic phosphorylation (only for 5-hydroxymethyluracil, transcription off). Although it has been so far demonstrated only in vitro, it is the proof-of-principle first step toward chemical epigenetics.

■

INTRODUCTION Protein−DNA interactions are of paramount importance in many biological processes, i.e., DNA packaging, replication, transcription, epigenetic modifications, and repair. Sequencespecific recognition of DNA by proteins is mostly achieved1 through H-bonding interactions in the major groove of DNA, but also interactions in the minor groove, recognition of shape, and electrostatic interactions play important role. Particularly important is the sequence-specific binding of transcription factors (TFs) to promoter sequences upstream of the genes to either enhance or silence the gene expression. There are over 1600 human TFs identified, but the detailed role and binding motifs of many of them are still unknown.2 Although, there is a © 2019 American Chemical Society

number of methods for studying of protein−DNA interactions and for identification of DNA-binding proteins,3 there is still an urgent need of other alternative methods, in particular for weakly binding proteins. In Nature, the DNA methylation and demethylation processes are involved in regulation of gene expression and turning genes on or off during the cells differentiation.4 The most important epigenetic DNA modifications are 5methylcytosine (5mC) and its oxidized derivatives, i.e,. 5hydroxymethyl- (5hmC) or 5-formylcytosine (5fC).5 While Received: April 17, 2019 Published: June 5, 2019 1730

DOI: 10.1021/acs.accounts.9b00195 Acc. Chem. Res. 2019, 52, 1730−1737

Article

Accounts of Chemical Research

Figure 1. Synthesis of base-modified dNTPs (A), reactions useful for modification of dNTPs (B), and enzymatic methods (C) for construction of base-modified DNA.

RNA polymerases. The field has been reviewed several times in the recent years,14,16,17 so only a brief overview will be given here with focus on newest development. In the past decade, several straightforward methods for direct modifications of (d)NTPs have been developed in our and other groups (Figure 1).14 5-Iodopyrimidine or 7-iodo-7deazapurine dNTPs can be modified directly through Pdcatalyzed aqueous cross-coupling reactions (Figure 1A, Approach I).14 The Suzuki reaction with boronic acids or trifluoroborates can be used for introduction of aryl or alkenyl groups, the Sonogashira reaction with terminal alkynes for attachment of alkynyl-linked substituents (Figure 1B). These reactions have been thoroughly discusses in previous review.14 Meanwhile, also Stille18 and Heck19 coupling were reported for attachment of some aryl and alkenyl groups, respectively. So far, no direct coupling method is available for attachment of flexible alkyl groups (all relevant organometallics for those couplings, i.e., alkylmagnesium halides, -zinc halides, or cuprates, are not compatible with water). However, recently, we have reported the synthesis of flexible alkylsulfanylethyllinked dNTPs through the thiol−ene addition20 of thiols to 7vinyl-7-deazapurine or 5-vinylpyrimidine dNTPs and the synthesis of alkyl linked dNTPs through catalytic hydrogenation of the corresponding alkynyl-dNTPs. 21 Both reactions proceeded in methanol at ambient temperature, where the dNTPs were relatively stable toward hydrolysis. Certainly, some modified dNTPs can be also prepared in a more classical way by triphosphorylations of modified nucleosides (Approach II), but any reactive, in particular

5mC downregulates transcription through inhibition of binding of TFs and RNA polymerases to genomic DNA,6 5hmC is not only an intermediate in active demethylation of DNA,7 but it was also found to recruit some proteins and upregulate expression of certain genes.8 Also, 5-hydroxymethyluracil (5hmU) was identified in some genomes,9,10 but its role still remains elusive.11 Further research is needed to fully understand12,13 the epigenetic regulation and processes which certainly will have implications in diagnostics and therapy of cancer and other diseases. The importance of protein−DNA interactions in biology offers organic chemists an attractive and challenging opportunity to introduce non-natural chemical modifications into the major groove of DNA or even perform some bioorthogonal reactions on DNA. This Account will summarize our recent efforts in enzymatic synthesis of basemodified DNA for applications (i) in sensing of protein−DNA interactions, (ii) in cross-linking DNA-binding proteins, as well as (iii) in modulation or switching of protein−DNA interactions and transcription.

■

POLYMERASE SYNTHESIS OF BASE-MODIFIED NUCLEIC ACIDS Base-modified nucleic acids are useful tools in diagnostics and chemical biology. Their enzymatic synthesis14 using functionalized 2′-deoxyribonucleoside triphosphates (dNTPs) and DNA polymerase is a useful alternative to classical chemical synthesis on solid support. Similarly, modified RNA can be produced15 using ribonucleoside triphosphates (NTPs) and 1731


Article


DNA interactions (Figure 2). Several fluorophores including dimethylaminobenzylidene cyanoacetamide41 or tryptophan-

nucleophilic, functional groups must be protected. A simple and cheap method for attachment of some alkoxymethyl groups to uracil nucleosides is the bromination of thymine followed by ether formation with alkoxides (and triphoshorylation). This approach has been used for photocaging of 5hmU22 and 5hmC,23 as well as for tethering of reactive terminal acetylenes.24 The 5-substituted pyrimidine or 7-substituted 7-deazapurine dNTPs are usually good substrates for DNA polymerases and can be used for enzymatic synthesis of modified oligonucleotides (ONs) or DNA (Figure 1C).14 Some 7-aryl-, 7-alkenylor 7-alkynyl-7-deazapurine dNTPs (and in lesser extent also some 5-arylpyrimidine dNTPs) were even found25−27 to be better substrates for some DNA polymerases than natural dNTPs due to higher affinity (caused by increased cation-π stacking) to the active site of the ternary complex of polymerase, template and primer. From many commercially available DNA polymerases, thermostable DNA polymerases, i.e. KOD XL, Vent (exo-) and Pwo are typically tolerant to major-groove modification of dNTPs and still give high yields and fidelity of synthesis of modified DNA. Primer extension (PEX) with biotinylated template followed by magnetoseparation can be used28 for the synthesis of modified single strand oligonucleotides (ssONs), whereas PCR is used for synthesis of long DNA sequences modified in both strands.29 Recently, we have developed the synthesis of short modified ONs by nicking enzyme amplification reaction (NEAR, using Vent (exo-) DNA polymerase in the presence of a nicking endonuclease, i.e., Nt.BstNBI),30 as well as the synthesis of ONs containing a single modification by single-nucleotide extension (SNE) followed by PEX.31 Terminal deoxynucletidyl transferase (TdT) is used for 3′-tail-labeling.32 Rolling-cycle amplification (RCA) can be used for construction of long DNA with repetitive sequence.33 A major limitation of all of these methods is difficult scale up (they are typically performed in pico- or nanomole scale). Polymerase synthesis can be also used34 for minor-groove modification of DNA using 2-vinyl- or 2-ethynyl-dATP and thiol−ene or CuAAC click reactions of the vinyl- or ethynylmodified DNA. Interestingly, the corresponding 2-allylaminoor 2-propargylamino-dATP derivatives were very poor substrates for most polymerases, but Therminator polymerase was able to incorporate just one modified nucleotide and it could only continue by synthesis of nonmodified DNA using natural nucleotides.35 This finding has been used for sitespecific single modification in minor groove of DNA. Much less has been reported on enzymatic synthesis of modified RNA (typically using T7 RNA polymerase and one modified NTP only),15,36,37 but we have recently reported38 a systematic study of all four modified dNTPs showing that also the RNA polymerases can incorporate large portfolio of base-modified ribonucleotides.

Figure 2. Examples of environment-sensitive fluorescent modifications of DNA for sensing protein−DNA interactions.

based imidazolinone42 (analogue to fluorophore from cyan fluorescent protein) were incorporated to DNA probes which showed light-up response (2−4 fold increase of fluorescence intensity) upon binding of p53 (to duplex) or single-strand binding protein (SSB, to ssON). Push−pull substituted fluorene was used as solvatochromic label which changed color when p53 interacted with the corresponding DNA probe.43 A phenyl-bodipy molecular rotor was used44 as a viscosity probe which responded to protein binding by significant increase of lifetime (from 0.2 to 2.5 ns) not only in test tube but also in the cell (using fluorescence-lifetimeimaging microscopy). Conversely, the hexamethylated phenylbodipy derivative (where the rotation is blocked) was used as nonsensitive but very bright fluorophore and the corresponding modified dCTP was transfected to living cells using a cyclodextrin-peptide transporter45 resulting in staining of the genomic DNA by incorporation of the fluorescent nucleotide.46 Neither the transporter nor the fluorescent nucleotide were toxic to the cells which underwent mitosis observable under fluorescent microscope. Redox labeling of DNA can also be achieved through polymerase incorporation of modified nucleotides and many applications in electrochemical analysis,47 redox coding,48 and genosensing49 have been reported. For the detection of

■

APPLICATIONS OF BASE-MODIFIED DNA FOR SENSING PROTEIN−DNA INTERACTIONS Fluorescent labeling of DNA is a common technique useful for many applications in bioanalysis and imaging.39 Environment sensitive fluorophores can be used for sensing interactions of DNA with other (bio)molecules through changing of some measurable properties of the fluorescence (intensity, wavelength or lifetime).40 In our lab, we designed and synthesized dNTPs bearing environment-sensitive fluorophores and enzymatically constructed DNA probes for sensing protein− 1732


Article

Accounts of Chemical Research protein−DNA interactions, we have developed50 an approach based on construction of azido-labeled DNA probes (giving electrochemical reduction at −0.9 V) and its CuAAC click reaction with nitrophenylacetylene which transforms the azido group to nitrophenyltriazole (reduced at −0.4 V). When the click reaction is performed in the presence of DNA-binding protein which covers and shields part of the DNA probe, the transformation proceeds only at unshielded azido groups and then, after denaturation, we can determine how much of the DNA was covered by protein from the ratio of the two reduction peaks (Figure 3). Another recently reported

Figure 4. Examples of reactive groups in DNA cross-linking to proteins.

diols) and used the aldehyde-lined DNA for cross-linking with Lys in peptides by reductive amination. Since this method needs an additional reducing agent (e.g., NaBH3CN), we are currently testing other amine-specific reactive groups for reagent-free cross-linking with Lys. Based on some known56 covalent inhibitors of serine-proteases, we designed and synthesized trifluoroacetophenone-linked DNA which, however, did not give covalent cross-linking with the Ser mutant of p53.57 The reason could be that nonactivated Ser has a much higher pKa than serine does in active site of proteases. Other reactive groups targeting Tyr, Arg, and Trp are also currently investigated in our laboratories.

Figure 3. Clickable redox labeling for detection of protein−DNA binding through electrochemical analysis.

approach, which can even distinguish specific and nonspecific binding, is based on dual redox labeling of DNA probes and detection of binding protein through immunoprecipitation using a monoclonal antibody followed by electrochemical detection.51

■

APPLICATIONS OF BASE-MODIFIED DNA FOR CROSS-LINKING DNA-BINDING PROTEINS Unlike the chemical phosphoramidite synthesis, the polymerase synthesis of modified DNA can be used even for incorporation of even highly reactive functional groups. Examples of enzymatic incorporation of some clickable groups were reported24,52,53 and used for bioconjugations and bioorthogonal labeling; those modifications would not react with native proteins. However, cross-linking with amino acid side chains of native proteins is attractive for applications in DNA-proteomics, structural biology of protein−DNA complexes, as well as for potential construction of covalent inhibitors of some transcription factors. We have synthesized reactive vinylsulfonamide-linked dNTPs, enzymatically incorporated them to DNA probes which formed covalent crosslinks with Cys in recognition site of p53 protein (Figure 4).54 Similarly, we synthesized nucleotides and DNA probes bearing chloroacetamide, which cross-linked to Cys or His.55 In all those cases, the proximity effect was crucial for efficient crosslinking. More recently, we prepared21 aliphatic aldehydemodified dNTPs and DNA (through oxidative cleavage of

■

APPLICATIONS OF BASE-MODIFIED DNA FOR MODULATION AND SWITCHING OF PROTEIN−DNA BINDING A very attractive and hitherto unexplored possibility is to use non-natural modification of DNA (either in major or in minor groove) and bioorthogonal reactions to modulate or switch interactions of proteins with DNA. It would be an artificial biomimetic analogy of epigenetic regulation of transcription. In the first phase of this challenging project, we have chosen type II restriction endonucleases (REs) as simple models for sequence-specific DNA-binders with clear response (cleavage of DNA when the recognition and binding is successful). We have synthesized large set of dsDNA bearing diverse modified bases in the recognition sites of several REs and studied whether or not the REs will be able to tolerate the presence of the modification in the major groove of DNA and cleave the DNA. We found that several REs tolerated 5-substituted uracil58 or 7-substituted 7-deazaadenine59 bearing small modifications, whereas any bulky modifications or modified 1733


Article

Accounts of Chemical Research cytosines or guanines60 were not tolerated and fully inhibited the RE cleavage (Figure 5). Based on this knowledge, we have

Figure 6. Influence major-groove modifications on in vitro transcription with bacterial RNA polymerase (inhibition or increase of transcription).

to natural DNA). Then we studied the influence of 5-hmU and 5hmC (rare natural DNA modifications with unknown biological role) and found64 very surprising results showing that the presence of each of these bases in DNA can significantly enhance the transcription (350% with 5hmU and 250% with 5hmC compared to native DNA). The final goal was to apply the above-mentioned results and approaches for switching of transcription. In order to irreversibly turn off transcription, we constructed DNA template containing 5-ethynyluracil (which gave transcription at ca. 40% compared to natural DNA), and its CuAAC click reaction with 2,3-dihydroxypropylazide led to bulkier dihydroxypropyl-triazole modifed DNA which did not give any transcription.65 Far more interesting was an option of switching on transcription using light (Figure 7). To this end, we have synthesized by PCR DNA templates containing bulky 2-nitrobenzyl-photocaged 5hmU or 5hmC which did not give any transcription in vitro. By irradiation with visible light (400 nm) the nitrobenzyl groups were removed and the resulting hydroxymethylated DNA (containing either 5hmU or 5hmC) gave enhanced transcription (350 or 250%, see above).66 Treatment of the 5hmU-containing DNA with 5HMUD kinase led to phosphorylation of the hydroxymethyl groups which resulted in switching off the transcription again. This kinase is specific in phosphorylation of 5hmU, so when we treated the 5hmC-containing DNA with this enzyme and ATP, the transcription remained unchanged. Although the 5hmU and 5hmC have not (yet) been reported in bacterial genomes, they were found in bacteriophages,67 and our results suggest that their presence gives the bacteriophage DNA an advantage in transcription in bacteria and that perhaps the

Figure 5. Influence of major-groove modifications on cleavage of DNA by REs and switching of RE cleavage.

constructed two simplified switches for the RE cleavage. We used silyl-protected61 or nitrobenzyl22,23,62 photocaged DNA (where the protecting groups prevented binding of REs) and the deprotection (treatment with NH3 for desilylaltion or irradiation by UV or visible light for photochemical deprotection) led to unmasking of small modifications which were tolerated by the restriction enzymes and the DNA was cleaved. This gave us promising evidence that it is possible to regulate protein−DNA binding through chemical reactions on DNA. Encouraged by the results in modulation of RE cleavage of DNA, we proceeded to study of regulation of transcription. We have chosen a model of bacterial transcription using Pveg promoter from Bacillus subtilis which is well-recognized by the Escherichia coli or B. subtilis polymerase containing the primary sigma factor (Figure 6). At first, we prepared by PCR a library of DNA templates fully modified at one nucleobase with either 5-substituted U or C or with 7-substituted 7-deazaA or 7deazaG and studied the transcription with the bacterial RNA polymerases.63 As expected, some bulkier modifications in the major groove of DNA completely inhibited transcription from those templates. Also the replacement of T bu uracil completely blocked transcription. On the other hand, 7deazapurine- and 7-methyl-7-deazapurine-containing templates gave strong transcription comparable to natural DNA and also some pyrimidine modifications, e.g. 5-vinylcytosine or 5ethynyluracil gave significant transcription (20−40% compared 1734


Article


development of enzymatic methods for synthesis of modified DNA or RNA. In DNA, we have suitable methods for synthesis of both double- or single-stranded DNA containing one, several or many modifications. The potential of those methodologies should be further explored in selection of aptamers68 or DNAzymes.69 The remaining challenge is the enzymatic synthesis of larger quantities of modified DNA and in applications of DNA labeling in living cells and organisms. In RNA, the main remaining challenge is to synthesize sequences not compatible with specific promoter for T7 RNA polymerase. Major breakthrough can be expected when using engineered polymerases. We have shown that polymerase synthesis of DNA probes containing environment-sensitive fluorophores or redox labels has good potential in studying protein−DNA interactions. Using the (d)NTP transporter,45 we can perform labeling of DNA or RNA in living cells. The tolerance of polymerases to reactive groups enabled enzymatic synthesis of DNA probes bearing functional groups for selective cross-linking with several nucleophilic amino acid side-chains when proximity effect applied. The challenge is now to target less reactive amino acids (Ser, Tyr, Trp, etc.) in order to create a portfolio of reactive modifications for DNA proteomics or covalent inhibitors of oncogenic TFs. Systematic study of the influence of major-groove modifications on cleavage of DNA by REs revealed some unexpected tolerance of these enzymes to some A or T modifications. Bacterial RNA polymerases can also tolerate some DNA modifications, and, surprisingly, the presence of 5hydroxymethylpyrimidines in DNA template can significantly enhance transcription. Finally, we have constructed an in vitro transcription switch (turn on and off) based on photocaging, photochemical release, and phosphorylation of 5hmU (or 5hmC, where the phosphorylation does not proceed). Further research will be needed to fully understand the biological significance of these findings and to apply those approaches for regulation of gene expression in living cells. Nevertheless, it is the proof of principle and the first step toward the artificial chemical epigenetics, and our research also continues by testing other bioorthogonal reactions on DNA.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Michal Hocek: 0000-0002-1113-2047 Notes

The author declares no competing financial interest. Biography Figure 7. Switching of in vitro transcription through photocaging, photorelease, and phosphorylation of 5-hydroxymethylpyrimidines in DNA.

Michal Hocek received his M. S. degree from Prague Institute of Chemical Technology and Ph.D. from the Czech Academy of Sciences, and he did his postdoc at Universite Catholique de Louvain. He is now a group leader at IOCB and full professor of organic chemistry at the Charles University.

phosphorylation of 5hmU might be the bacterial defense against the virus. However, further experiments will be needed to confirm these hypotheses.

■

■

ACKNOWLEDGMENTS This work was supported by the Czech Academy of Sciences (RVO: 61388963 and the Praemium Academiae), by the Czech Science Foundation (17-03419S and 18-03305S), and

SUMMARY AND OUTLOOK In the past decade, great progress has been made in the synthesis of base-modified nucleotides and (d)NTPs and in 1735


Article


(20) Slavickova, M.; Pohl, R.; Hocek, M. Additions of Thiols to 7vinyl-7-deazaadenine Nucleosides and Nucleotides. Synthesis of Hydrophobic Derivatives of 2′-deoxyadenosine, dATP and DNA. J. Org. Chem. 2016, 81, 11115−11125. (21) Krömer, M.; Bártová, K.; Raindlová, V.; Hocek, M. Synthesis of Dihydroxyalkynyl and Dihydroxyalkyl Nucleotides as Building Blocks or Precursors for Introduction of Diol or Aldehyde Groups to DNA for Bioconjugations. Chem. - Eur. J. 2018, 24, 11890−11894. (22) Vaníková, Z.; Hocek, M. Polymerase Synthesis of Photocaged DNA Resistant Against Cleavage by Restriction Endonucleases. Angew. Chem., Int. Ed. 2014, 53, 6734−6737. (23) Bohácǒ vá, S.; Vaníková, Z.; Poštová Slavětínská, L.; Hocek, M. Protected 2′-deoxyribonucleoside Triphosphate Building Blocks for the Photocaging of Epigenetic 5-(hydroxymethyl)cytosine in DNA. Org. Biomol. Chem. 2018, 16, 5427−5432. (24) Panattoni, A.; Pohl, R.; Hocek, M. Flexible Alkyne-Linked Thymidine Phosphoramidites and Triphosphates for Chemical or Polymerase Synthesis and Fast Postsynthetic DNA Functionalization through Copper-Catalyzed Alkyne-Azide 1,3-Dipolar Cycloaddition. Org. Lett. 2018, 20, 3962−3965. (25) Kielkowski, P.; Fanfrlík, J.; Hocek, M. 7-Aryl-7-deazaadenine 2′-deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases Than dATP in Competitive Incorporations. Angew. Chem., Int. Ed. 2014, 53, 7552−7555. (26) Cahová, H.; Panattoni, A.; Kielkowski, P.; Fanfrlík, J.; Hocek, M. 5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs. ACS Chem. Biol. 2016, 11, 3165− 3171. (27) Hottin, A.; Betz, K.; Diederichs, K.; Marx, A. Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of AlkeneVersus Alkyne-Modified Nucleotides. Chem. - Eur. J. 2017, 23, 2109− 2118. (28) Brázdilová, P.; Vrábel, M.; Pohl, R.; Pivonková, H.; Havran, L.; Hocek, M.; Fojta, M. Ferrocenylethynyl Derivatives of Nucleoside Triphosphates: Synthesis, Incorporation, Electrochemistry, and Bioanalytical Applications. Chem. - Eur. J. 2007, 13, 9527−9533. (29) Jäger, S.; Rasched, G.; Kornreich-Leshem, H.; Engeser, M.; Thum, O.; Famulok, M. A Versatile Toolbox for Variable DNA Functionalization at High Density. J. Am. Chem. Soc. 2005, 127, 15071−15082. (30) Ménová, P.; Raindlová, V.; Hocek, M. Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Basemodified Oligonucleotides and Primers for PCR. Bioconjugate Chem. 2013, 24, 1081−1093. (31) Ménová, P.; Cahová, H.; Plucnara, M.; Havran, L.; Fojta, M.; Hocek, M. Polymerase Synthesis of Oligonucleotides Containing a Single Chemically Modified Nucleobase for Site-specific Redox Labelling. Chem. Commun. 2013, 49, 4652−4654. (32) Sarac, I.; Hollenstein, M. Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids. ChemBioChem 2019, 20, 860−871. (33) Hollenstein, M. Generation of Long, Fully Modified, and Serum-resistant Oligonucleotides by Rolling Circle Amplification. Org. Biomol. Chem. 2015, 13, 9820−9824. (34) Matyašovský, J.; Perlíková, P.; Malnuit, V.; Pohl, R.; Hocek, M. 2-Substituted dATP Derivatives as Building Blocks for PolymeraseCatalyzed Synthesis of DNA Modified in the Minor Groove. Angew. Chem., Int. Ed. 2016, 55, 15856−15859. (35) Matyašo vský , J.; Pohl, R.; Hocek, M. 2-Allyl- and Propargylamino-dATPs for Site-Specific Enzymatic Introduction of a Single Modification in the Minor Groove of DNA. Chem. - Eur. J. 2018, 24, 14938−14941. (36) Sawant, A. A.; Tanpure, A. A.; Mukherjee, P. P.; Athavale, S.; Kelkar, A.; Galande, S.; Srivatsan, S. G. A Versatile Toolbox for Posttranscriptional Chemical Labeling and Imaging of RNA. Nucleic Acids Res. 2016, 44, e16.

by the European Regional Development Fund; OP RDE (No. CZ.02.1.01/0.0/0.0/16_019/0000729).

■

REFERENCES

(1) Rohs, R.; Jin, X.; West, S. M.; Joshi, R.; Honig, B.; Mann, R. S. Origins of Specificity in protein-DNA Recognition. Annu. Rev. Biochem. 2010, 79, 233−269. (2) Lambert, S. A.; Jolma, A.; Campitelli, L. F.; Das, P. K.; Yin, Y.; Albu, M.; Chen, X.; Taipale, J.; Hughes, T. R.; Weirauch, M. T. The Human Transcription Factors. Cell 2018, 172, 650−665. (3) Furey, T. S. ChIP-seq and Beyond: New and Improved Methodologies to Detect and Characterize protein-DNA Interactions. Nat. Rev. Genet. 2012, 13, 840−852. (4) Chen, K.; Zhao, B. S.; He, C. Nucleic Acid Modifications in Regulation of Gene Expression. Cell Chem. Biol. 2016, 23, 74−85. (5) Carell, T.; Kurz, M. Q.; Müller, M.; Rossa, M.; Spada, F. Noncanonical Bases in the Genome: The Regulatory Information Layer in DNA. Angew. Chem., Int. Ed. 2018, 57, 4296−4312. (6) Jeltsch, A. Beyond Watson and Crick: DNA Methylation and Molecular Enzymology of DNA Methyltransferases. ChemBioChem 2002, 3, 274−293. (7) Wu, X.; Zhang, Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat. Rev. Genet. 2017, 18, 517− 534. (8) Perera, A.; Eisen, D.; Wagner, M.; Laube, S. K.; Künzel, A. F.; Koch, S.; Steinbacher, J.; Schulze, E.; Splith, V.; Mittermeier, N.; Muller, M.; Biel, M.; Carell, T.; Michalakis, S. ET3 Is Recruited by REST for Context-specific Hydroxymethylation and Induction of Gene Expression. Cell Rep. 2015, 11, 283−294. (9) Kawasaki, F.; Beraldi, D.; Hardisty, R. E.; McInroy, G. R.; van Delft, P.; Balasubramanian, S. Genome-wide Mapping of 5hydroxymethyluracil in the Eukaryote Parasite Leishmania. Genome Biol. 2017, 18, 23. (10) Pfaffeneder, T.; Spada, F.; Wagner, M.; Brandmayr, C.; Laube, S. K.; Eisen, D.; Truss, M.; Steinbacher, J.; Hackner, B.; Kotljarova, O.; Schuermann, D.; Michalakis, S.; Kosmatchev, O.; Schiesser, S.; Steigenberger, B.; Raddaoui, N.; Kashiwazaki, G.; Müller, U.; Spruijt, C. G.; Vermeulen, M.; Leonhardt, H.; Schär, P.; Müller, M.; Carell, T. Tet Oxidizes Thymine to 5-hydroxymethyluracil in Mouse Embryonic Stem Cell DNA. Nat. Chem. Biol. 2014, 10, 574−581. (11) Olinski, R.; Starczak, M.; Gackowski, D. Enigmatic 5hydroxymethyluracil: Oxidatively Modified Base, Epigenetic Mark or Both? Mutat. Res., Rev. Mutat. Res. 2016, 767, 59−66. (12) Raiber, E.-A.; Hardisty, R.; van Delft, P.; Balasubramanian, S. Mapping and elucidating the function of modified bases in DNA. Nat. Rev. Chem. 2017, 1, 0069. (13) Hofer, A.; Liu, Z. J.; Balasubramanian, S. Detection, Structure and Function of Modified DNA Bases. J. Am. Chem. Soc. 2019, 141, 6420. (14) Hocek, M. 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. 2014, 79, 9914−9921. (15) Flamme, M.; McKenzie, L. K.; Sarac, I.; Hollenstein, M. Chemical methods for the modification of RNA. Methods 2019, DOI: 10.1016/j.ymeth.2019.03.018. (16) Hollenstein, M. Nucleoside Triphosphates–building Blocks for the Modification of Nucleic Acids. Molecules 2012, 17, 13569−13591. (17) Hottin, A.; Marx, A. Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases. Acc. Chem. Res. 2016, 49, 418−427. (18) Krause, A.; Hertl, A.; Muttach, F.; Jäschke, A. Phosphine-free Stille-Migita Chemistry for the Mild and Orthogonal Modification of DNA and RNA. Chem. - Eur. J. 2014, 20, 16613−16619. (19) Dadová, J.; Vidláková, P.; Pohl, R.; Havran, L.; Fojta, M.; Hocek, M. Aqueous Heck Cross-coupling Preparation of Acrylatemodified Nucleotides and Nucleoside Triphosphates for Polymerase Synthesis of Acrylate-labeled DNA. J. Org. Chem. 2013, 78, 9627− 9637. 1736


Article

Accounts of Chemical Research (37) George, J. T.; Srivatsan, S. G. Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA. Bioconjugate Chem. 2017, 28, 1529−1536. (38) Milisavljevič, N.; Perlíková, P.; Pohl, R.; Hocek, M. Enzymatic Synthesis of Base-modified RNA by T7 RNA Polymerase. A Systematic Study and Comparison of 5-substituted Pyrimidine and 7-substituted 7-deazapurine Nucleoside Triphosphates as Substrates. Org. Biomol. Chem. 2018, 16, 5800−5807. (39) Xu, W.; Chan, K. M.; Kool, E. T. Fluorescent Nucleobases as Tools for Studying DNA and RNA. Nat. Chem. 2017, 9, 1043−1055. (40) Tanpure, A. A.; Pawar, M. G.; Srivatsan, S. G. Fluorescent Nucleoside Analogs: Probes for Investigating Nucleic Acid Structure and Function. Isr. J. Chem. 2013, 53, 366−378. (41) Dziuba, D.; Pohl, R.; Hocek, M. Polymerase Synthesis of DNA Labelled with Benzylidene Cyanoacetamide-based Fluorescent Molecular Rotors: Fluorescent Light-up Probes for DNA-binding Proteins. Chem. Commun. 2015, 51, 4880−4882. (42) Kuba, M.; Pohl, R.; Hocek, M. Synthesis of 2′-Deoxycytidine and Its Triphosphate Bearing Tryptophan-based Imidazolinone Fluorophore for Environment Sensitive Fluorescent Labelling of DNA. Tetrahedron 2018, 74, 6621−6629. (43) Dziuba, D.; Pospisil, P.; Matyasovsky, J.; Brynda, J.; Nachtigallova, D.; Rulisek, L.; Pohl, R.; Hof, M.; Hocek, M. Solvatochromic Fluorene-Linked Nucleoside and DNA as ColorChanging Fluorescent Probes for Sensing Interactions. Chem. Sci. 2016, 7, 5775−5785. (44) Dziuba, D.; Jurkiewicz, P.; Cebecauer, M.; Hof, M.; Hocek, M. A Rotational BODIPY Nucleotide: An Environment-Sensitive Fluorescence-Lifetime Probe for DNA Interactions and Applications in Live-Cell Microscopy. Angew. Chem., Int. Ed. 2016, 55, 174−178. (45) Zawada, Z.; Tatar, A.; Mocilac, P.; Buděsí̌ nský, M.; Kraus, T. Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters. Angew. Chem., Int. Ed. 2018, 57, 9891−9895. (46) Güixens-Gallardo, P.; Zawada, Z.; Matyašovský, J.; Dziuba, D.; Pohl, R.; Kraus, T.; Hocek, M. Brightly Fluorescent 2′-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry. Bioconjugate Chem. 2018, 29, 3906−3912. (47) Paleček, E.; Bartošík, M. Electrochemistry of Nucleic Acids. Chem. Rev. 2012, 112, 3427−3481. (48) Balintová, J.; Plucnara, M.; Vidláková, P.; Pohl, R.; Havran, L.; Fojta, M.; Hocek, M. Benzofurazane as a New Redox Label for Electrochemical Detection of DNA: Towards Multipotential Redox Coding of DNA Bases. Chem. - Eur. J. 2013, 19, 12720−12731. (49) Magriña,́ I.; Toldrà, A.; Campàs, M.; Ortiz, M.; Simonova, A.; Katakis, I.; Hocek, M.; O’Sullivan, C. K. Electrochemical Genosensor for the Direct Detection of Tailed PCR Amplicons Incorporating Ferrocene Labelled dATP. Biosens. Bioelectron. 2019, 134, 76−82. (50) Balintová, J.; Š paček, J.; Pohl, R.; Brázdová, M.; Havran, L.; Fojta, M.; Hocek, M. Azidophenyl as a Click-transformable Redox Label of DNA Suitable for Electrochemical Detection of DNA− protein Interactions. Chem. Sci. 2015, 6, 575−587. (51) Hermanová, M.; Orság, P.; Balintová, J.; Hocek, M.; Fojta, M. Dual Redox Labeling of DNA as a Tool for Electrochemical Detection of P53 protein-DNA Interactions. Anal. Chim. Acta 2019, 1050, 123− 131. (52) Reisacher, U.; Ploschik, D.; Rönicke, F.; Cserép, G. B.; Kele, P.; Wagenknecht, H.-A. Copper-free Dual Labeling of DNA by Triazines and Cyclopropenes as Minimal Orthogonal and Bioorthogonal Functions. Chem. Sci. 2019, 10, 4032−4037. (53) Ren, X.; El-Sagheer, A. H.; Brown, T. Efficient Enzymatic Synthesis and Dual-colour Fluorescent Labelling of DNA Probes Using Long Chain azido-dUTP and BCN Dyes. Nucleic Acids Res. 2016, 44, e79. (54) Dadová, J.; Orság, P.; Pohl, R.; Brázdová, M.; Fojta, M.; Hocek, M. Vinylsulfonamide and Acrylamide Modification of DNA for Crosslinking with Proteins. Angew. Chem., Int. Ed. 2013, 52, 10515−10518.

(55) Olszewska, A.; Pohl, R.; Brázdová, M.; Fojta, M.; Hocek, M. Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins. Bioconjugate Chem. 2016, 27, 2089−2094. (56) Lin, J.; Westler, W. M.; Cleland, W. W.; Markley, J. L.; Frey, P. A. Fractionation Factors and Activation Energies for Exchange of the Low Barrier Hydrogen Bonding Proton in Peptidyl Trifluoromethyl Ketone Complexes of Chymotrypsin. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 14664−14668. (57) Olszewska, A.; Pohl, R.; Hocek, M. TrifluoroacetophenoneLinked Nucleotides and DNA for Studying of DNA-Protein Interactions by 19F NMR Spectroscopy. J. Org. Chem. 2017, 82, 11431−11439. (58) Macíčková-Cahová, H.; Pohl, R.; Hocek, M. Cleavage of Functionalized DNA Containing 5-modified Pyrimidines by Type II Restriction Endonucleases. ChemBioChem 2011, 12, 431−438. (59) Macíčková-Cahová, H.; Hocek, M. Cleavage of Adeninemodified Functionalized DNA by Type II Restriction Endonucleases. Nucleic Acids Res. 2009, 37, 7612−7622. (60) Mačková, M.; Bohácǒ vá, S.; Perlíková, P.; Poštová Slavětínská, L.; Hocek, M. Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7-Substituted 7-Deazaguanine Nucleobases. ChemBioChem 2015, 16, 2225−2236. (61) Kielkowski, P.; Macíčková-Cahová, H.; Pohl, R.; Hocek, M. Transient and Switchable (triethylsilyl)ethynyl Protection of DNA Against Cleavage by Restriction Endonucleases. Angew. Chem., Int. Ed. 2011, 50, 8727−8730. (62) Bohácǒ vá, S.; Vaníková, Z.; Poštová Slavětínská, L.; Hocek, M. Protected 2′-deoxyribonucleoside Triphosphate Building Blocks for the Photocaging of Epigenetic 5-(hydroxymethyl)cytosine in DNA. Org. Biomol. Chem. 2018, 16, 5427−5432. (63) Raindlová, V.; Janoušková, M.; Slavíčková, M.; Perlíková, P.; Bohácǒ vá, S.; Milisavljevič, N.; Š anderová, H.; Benda, M.; Barvík, I.; Krásný, L.; Hocek, M. Influence of Major-groove Chemical Modifications of DNA on Transcription by Bacterial RNA Polymerases. Nucleic Acids Res. 2016, 44, 3000−3012. (64) Janoušková, M.; Vaníková, Z.; Nici, F.; Bohácǒ vá, S.; Vítovská, D.; Š anderová, H.; Hocek, M.; Krásný, L. 5-(Hydroxymethyl)uracil and -cytosine as Potential Epigenetic Marks Enhancing or Inhibiting Transcription with Bacterial RNA Polymerase. Chem. Commun. 2017, 53, 13253−13255. (65) Slavı ́čková, M.; Janoušková, M.; Š imonová, A.; Cahová, H.; Kambová, M.; Š anderová, H.; Krásný, L.; Hocek, M. Turning Off Transcription with Bacterial RNA Polymerase through CuAAC Click Reactions of DNA Containing 5-Ethynyluracil. Chem. Eur. J. 2018, 24, 8311−8314. (66) Vanı ́ková, Z.; Janoušková, M.; Kambová, M.; Krásný, L.; Hocek, M. Switching Transcription with Bacterial RNA Polymerase through Photocaging, Photorelease and Phosphorylation Reactions in the Major Groove of DNA. Chem. Sci. 2019, 10, 3937−3942. (67) Weigele, P.; Raleigh, E. A. Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses. Chem. Rev. 2016, 116, 12655−12687. (68) Diafa, S.; Hollenstein, M. Generation of Aptamers with an Expanded Chemical Repertoire. Molecules 2015, 20, 16643−16671. (69) Silverman, S. K. DNA as a Versatile Chemical Component for Catalysis, Encoding, and Stereocontrol. Angew. Chem., Int. Ed. 2010, 49, 7180−7201.

1737


Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing

Recommend Documents