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We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure-activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.

DNA molecules containing 5-vinyluracil, 5-vinylcytosine, or 7-deaza-7-vinyladenine were prepared by polymerase incorporation of the corresponding vinyl-modified 2′-deoxyribonucleoside triphosphates, and the influence of the vinyl group in the major groove of DNA on the cleavage by diverse type II restriction endonucleases (REs) was studied. The presence of 5-vinyluracil was tolerated by most of the REs, whereas only some REs were able to cleave sequences containing 7-deaza-7-vinyladenine. The enzyme ScaI was found to cleave DNA containing 5-vinylcytosine efficiently but not DNA containing the related 5-ethynylcytosine. All other REs failed to cleave sequences containing any cytosine modifications.

 Functionalized nucleic acids have found applications in chemical biology, bioanalysis, catalysis and nanotechnology.  Modifications can be introduced into the phosphate backbone, sugar moiety and, most often, onto a nucleobase.  Base-modified DNA can be synthesized chemically or enzymatically.  Chemical synthesis is carried out on solid support and utilizes phosphorimidate building blocks.  Enzymatic synthesis of (base-modified) DNA is based on the incorporation of (functionalized) nucleoside triphosphates by a DNA polymerase.

4-Hydroxy-3-methoxyphenyl (MOP) and 2,3-dihydrobenzofuran-5-yl (DHB) groups were tested as potential new oxidizable labels for electrochemical detection of DNA. The corresponding 5-aryl-cytosine and 7-aryl-7-deazaadenine 2-deoxyribonucleoside triphosphates (dNTPs) were prepared by the Suzuki coupling of halogenated dNTPs or by triphosphorylation of modified nucleosides, and were incorporated enzymatically into DNA by polymerase. In parallel, the electrochemical behavior of the labeled nucleosides, nucleotides, and DNA was studied to show that most of them give analytically useful signals of oxidation on the carbon electrode. Particularly, MOP appears to be a useful oxidizable DNA label since it offers independent detection of the modified DNA.

The Suzuki-Miyaura cross-coupling reactions of 9-ben- zyl-6-chloropurine, 9- or 3-benzyl-8-bromoadenine and 2,6-diha- lopurines with boronic acids gave the corresponding 6-, 8- or 2-aryl- or -alkenylpurines in good yields. Anhydrous conditions in toluene were superior for coupling of electron-rich boronic acids, while aqueous DME was used for electron-poor arylboronic acids as well as for alkenylboronic acids. A good regioselectivity was observed for the coupling of 2,6-dihalopurines: 9-benzyl-2,6-dichloropurine reacted with one equivalent of phenyl boronic acid to give 9-benzyl- 2-chloro-6-phenylpurine, while an analogous reaction of 9-benzyl- 6-chloro-2-iodopurine gave selectively 9-benzyl-6-chloro-2-phe- nylpurine.

A series of fifteen 6-aryl-, 6-hetaryl- and 6-benzylpurine ribonucleosides has been prepared by Pd-catalyzed cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)- purine with arylboronic acids, hetarylzinc halides, hetarylstannanes or benzylzinc halides followed by deprotection. Structure-activity relationship study revealed that besides 6-(4-substituted phenyl)purine nucleosides, also some 6-hetaryl- and 6-benzylpurine ribonucleosides possess considerable cytostatic activity.

Three approaches to the synthesis of the title (purin-6-yl)methylphosphonates were investigated and compared. While, the Arbuzov reaction of 6-(iodomethyl)purines with triethyl phosphite did not work, Michaelis–Becker alkylation of the sodium salt of diethyl phosphonate with 6-(mesyloxymethyl)purines gave the desired products in good yields. The best method was based on Rh- or Pd-catalyzed cross-coupling reactions of 6-iodopurines with (diisopropoxyphosphorylmethyl)zinc bromide. In this way a small series of 6-(diisopropoxyphosphorylmethyl)purine bases and nucleosides was prepared in high yields.

Standard Pd-catalyzed cross-coupling reactions proceed chemoselectively at the position of the bromine, whereas nucleophilic substitutions are unselective.

The Suzuki-Miyaura reaction of protected 6-chloropurine and 2-amino-6-chloropurine bases and nucleosides with substituted phenylboronic acids led to the corresponding protected 6-(substituted phenyl)purine derivatives 6-9. Their deprotection yielded a series of substituted 6-phenylpurine bases and nucleosides 10-13. Significant cytostatic activity (IC(50) 0.25-20 micromol/L) in CCRF-CEM, HeLa, and L1210 cell lines was found for several 6-(4-X-substituted phenyl)purine ribonucleosides 12 (X = H, F, Cl, and OR), while the 6-phenylpurine and 2-amino-6-phenylpurine bases 10 and 11, as well as 2-amino-6-phenylpurine ribosides 13, were entirely inactive against these cell lines.

Cross-coupling reactions of 2-amino-6-chloro-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}purine (1) and 2-amino-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}-6-iodopurine (2) with diverse types of organometallic reagents have been studied. Arylboronic acids reacted with 1 to give the corresponding 2-amino-6-arylpurines 3a-3d in good yields. Analogously, trialkylaluminium reagents were used for the preparation of 6-alkyl-2-aminopurines 3k and 3l from 1. Hetarylzinc halides and hetarylstannanes required the use of 2-amino-6-iodopurine 2 to give the corresponding 2-amino-6-hetarylpurines 3e-3j in fair to good yields. A CuI/KF mediated coupling of perfluoroalkylsilanes with 2 afforded the 2-amino-6-perfluoroalkylpurines 3m and 3n in moderate yields. Cleavage of the esters 3 with bromo(trimethyl)silane gave the target free phosphonates 4 that were purified by ion- exchange chromatography. The title compounds were tested on antiviral and cytostatic activity.

Glycosylation and alkylation of 6-(trifluoromethyl)purine by several protected halogenoses or hydroxyalkyl chlorides afforded regio- and stereoselectively the 9-β-nucleosides or 9-alkylated purine derivatives in good yields. Deprotection of these intermediates gave a series of nucleoside (β-D-ribofuranosyl, 2-deoxy-β-D-ribofuranosyl and β-D-arabinofuranosyl) and acyclonucleoside (2,3-dihydroxypropyl and (2-hydroxyethyl)oxymethyl) derivatives of 6-(trifluoromethyl)purine. While the ribofuranosyl derivative 1 showed significant cytostatic activity, the other derivatives were inactive.

The Stille coupling of 9-2-[bis(2-propoxy)phosphonylmethoxy]ethyl-6-chloropurines with 1-(ethoxyvinyl)tributyltin afforded 6-(1-ethoxyvinyl)purine derivatives. Their acid hydrolysis gave 6-acetylpurine derivatives that after reductive amination using various primary and secondary amine hydrochlorides and sodium cyanoborohydride followed by deprotection afforded the title N-substituted 6-(1-aminoethyl)-9-(2-phosphonomethoxyethyl)purine derivatives.

A novel, efficient, two-step methodology is presented for construction of base-modified oligonucleotides or DNA, involving aqueous cross-coupling reactions of halogenated nucleoside triphosphates (dNTPs) with terminal acetylenes or arylboronic acids, followed by polymerase incorporation of the modified dNTPs either using primer extension (PEX) or polymerase chain reaction (PCR). Curr. Protoc. Chem Biol. 2:1-14. © 2010 by John Wiley & Sons, Inc. Keywords: nucleoside triphosphates; DNA polymerase; base-modified DNA; cross-coupling

A general method for Cu-catalyzed C–H sulfenylation of purines, 7-deaza- and 9-deazapurines with aryl- or alkyldisulfides has been developed. In purines, the reaction occurs at position 8, in 7-deazapurines at position 7 and in 9-deazapurines at position 9, leading to new interesting arylsulfanyl derivatives of purine or deazapurine bases. The resulting 8-arylsulfanylpurines undergo Liebesking–Srogl coupling with arylstannanes or boronic acids, whereas the (arylsulfanyl)deazapurines are not reactive under these conditions.

Benzofurazane has been attached to nucleosides and dNTPs, either directly or through an acetylene linker, as a new redox label for electrochemical analysis of nucleotide sequences. Primer extension incorporation of the benzofurazane-modified dNTPs by polymerases has been developed for the construction of labeled oligonucleotide probes. In combination with nitrophenyl and aminophenyl labels, we have successfully developed a three-potential coding of DNA bases and have explored the relevant electrochemical potentials. The combination of benzofurazane and nitrophenyl reducible labels has proved to be excellent for ratiometric analysis of nucleotide sequences and is suitable for bioanalytical applications.

Bioorthogonal covalent cross-linking of DNA-binding proteins (p53) to DNA was achieved through novel DNA probes bearing a reactive vinylsulfonamide (VS) group. The VS-modified dCTP served as building block for polymerase synthesis of modified DNA, which was readily conjugated with cysteine-containing peptides and proteins by Michael addition.

A series of sugar-modified derivatives of cytostatic 7-heteroaryl-7-deazaadenosines (2'-deoxy-2'-fluororibo- and 2'-deoxy-2',2'-difluororibonucleosides) bearing an aryl or heteroaryl group at position 7 was prepared and screened for biological activity. The difluororibonucleosides were prepared by non- stereoselective glycosidation of 6-chloro-7-deazapurine with benzoyl-protected 2-deoxy-2,2-difluoro-D-erythro-pentofuranosyl-1-mesylate, followed by amination and aqueous Suzuki cross-couplings with (het)arylboronic acids. The fluororibo derivatives were prepared by aqueous palladium-catalyzed cross-coupling reactions of the corresponding 7-iodo-7-deazaadenine 2'-deoxy-2'-fluororibonucleoside 20 with (het)arylboronic acids. The key intermediate 20 was prepared by a six-step sequence from the corresponding arabinonucleoside by selective protection of 3'- and 5'-hydroxy groups with acid-labile groups, followed by stereoselective SN 2 fluorination and deprotection. Some of the title nucleosides and 7-iodo-7-deazaadenine intermediates showed micromolar cytostatic or anti-HCV activity. The most active were 7-iodo and 7-ethynyl derivatives. The corresponding 2'-deoxy-2',2'-difluororibonucleoside 5'-O-triphosphates were found to be good substrates for bacterial DNA polymerases, but are inhibitors of human polymerase α.

The synthesis of a series of 9-(2-phosphonomethoxyalkyl) derivatives of 6-(aminomethyl)purine 11, 2-amino-6-(aminomethyl)purine 12 and purine-6-carboxamidine 14 is reported. The 6-cyanopurines 1 and 2 were selectively alkylated with 2-[bis(isopropyloxy)phosphonylmethoxy]alkyl synthons 3 and 4 at the 9-position. Catalytic hydrogenation of the obtained 9-{2-[bis(isopropyloxy)phosphonylmethoxy]alkyl}-6-cyanopurines 9 and 10 followed by treatment with bromotrimethylsilane afforded the title compounds 11 and 12. Analogous acyclic nucleotides derived from purine-6-carboxamidines 14 were prepared from the cyanopurines 9a and 10a by treatment with sodium methoxide and ammonium chloride followed by deprotection. Compounds 11 and 12 exhibited moderate activity (MIC50 = 3-50 μg/ml) against herpes simplex virus type 1, varicella-zoster virus and Moloney murine sarcoma virus in vitro.

A facile method of preparation of 6-cyanopurine bases from commercially available 6-chloropurines is reported. The 6-chloropurines were selectively protected by THP-functions and the protected derivatives reacted smoothly with tetraethylammonium cyanide and DABCO to give 6-cyanopurine derivatives that were easily deprotected to afford the title compounds.

Synthesis of a series of 2-(aminomethyl)-9-(2-phosphonomethoxyalkyl)adenines VI and hypoxanthines VII is reported. The protected 2-(aminomethyl)adenine I was selectively alkylated with [bis(2-propoxy)phosphonylmethoxy]alkyl chlorides or tosylates II and the obtained 9-[bis(2-propoxy)phosphonylmethoxy]alkyl-2-(benzyloxycarbonyla minomethyl)adenines IV were oxodeaminated to give the corresponding hypoxanthine derivatives V. The intermediates IV and V were completely deprotected by treatment with iodotrimethylsilane under formation of the title compounds VI and VII.

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

An efficient two-step procedure allows the synthesis of 2,4-diarylpyrimidines via phosphonium-mediated Suzuki coupling and Liebeskind—Srogl reaction.

A general strategy allows to synthesize a broad spectrum of title compounds via nucleophilic displacement of 2,4-dichloropyrimidine nucleoside substrates as is demonstrated by some selected examples in the scheme.

1,5-Diones Ib - Ie were converted to spirocyclic 1,4-dihydropyridines IIb - IIe by treatment with methanolic methylamine. The starting diketones Ib - Ie were obtained from corresponding acetophenones 4-X-C6H4COMe and fluorenone with sodium amide except of If where only ketone III and 1,4-dione IV were isolated. Molecular spectra (UV-VIS, NMR, IR and MS) of compounds Ib - Ie, IIa - IIe, III, and IV were assigned and interpreted.

Molecular geometries of 1,4-dihydropyridines Ia, Ib and their possible photoisomers IIa - Va and IIb - Vb were optimized by the PM3 method. The electronic absorption spectra were calculated by the CNDO/S-CI procedure and compared with the experimental spectroscopic data. A probable mechanism of the photocolouration of Ia, Ib, caused by sunlight or UV illumination, is proposed, and structures IIIa, IIIb are assigned to violet or green coloured species.

The design of four new fluorinated biaryl fluorescent labels and their attachment to nucleosides and nucleoside triphosphates (dNTPs) by the aqueous cross-coupling reactions of biarylboronates is reported. The modified dNTPs were good substrates for KOD XL polymerase and were enzymatically incorporated into DNA probes. The photophysical properties of the biaryl-modified nucleosides, dNTPs, and DNA were studied systematically. The different substitution pattern of the biaryls was used for tuning of emission maxima in the broad range of 366-565 nm. Using methods of computational chemistry the emission maxima were reproduced with a satisfactory degree of accuracy, and it was shown that the large solvatochromic shifts observed for the studied probes are proportional to the differences in dipole moments of the ground (S(0)) and excited (S(1)) states that add on top of smaller shifts predicted already for these systems in vacuo. Thus, we present a set of compounds that may serve as multipurpose base-discriminating fluorophores for sensing of hairpins, deletions, and mismatches by the change of emission maxima and intensities of fluorescence and that can be also conviently studied by (19)F NMR spectroscopy. In addition, aminobenzoxazolyl-fluorophenyl-labeled nucleotides and DNA also exert dual pH-sensitive and solvatochromic fluorescence, which may imply diverse applications.

A series of novel sugar-modified derivatives of cytostatic 6-hetaryl-7-deazapinine ribonucleoside is prepared via Pd-catalyzed cross coupling reactions of different 7-deazapurines with arylboronic acids, aryl stannanes or trimethylaluminum.

Iridium(I)-catalyzed allylation of the enantiopure monoprotected copper(I) alkoxide, generated from (S)-5a, with the enantiopure allylic carbonates (R)-9a,b has been developed as the key step in a new approach to C-nucleoside analogues. The anomeric center was thus constructed via a stereocontrolled formation of the C-O rather than C-C bond with retention of configuration. The resulting bisallyl ethers 15a,b (≥90% de and99% ee) were converted into C-ribosides 29a,b via the Ru-catalyzed ring-closing metathesis, followed by a diastereoselective dihydroxylation catalyzed by OsO(4) or RuO(4) and deprotection. Variation of the absolute configuration of the starting segments 5a and 9a,b allowed a stereocontrolled synthesis of all four α/β-D/L-combinations.

A range of purine derivatives modified at position 6 of the basic purine skeleton exhibit a variety of biological activities. Several derivatives are used or tested nowadays for pharmacological treatments. The present work aims to analyze the effects of substituents on the electron distribution in the purine core as reflected by NMR chemical shifts. We collected a comprehensive set of experimental NMR data for a variety of 6-substituted purines (-NH(2), -NHMe, -NMe(2), -OMe, -Me, -CCH, and -CN) and determined the molecular and crystal structures of three derivatives (-NHMe, -CCH, and -CN) by X-ray diffraction. The density-functional methods calibrated in our recent study (Phys. Chem. Chem. Phys., 2010, 12, 5126) have been employed to enable understanding of the substituent-induced changes in the NMR chemical shifts of the atoms in the purine skeleton. Analyses of the nuclear shielding using localized molecular orbitals (LMOs), specifically the natural LMOs (NLMOs) and Pipek-Mezey LMOs, were used to break down the values of the isotropic (13)C and (15)N NMR chemical shifts and the chemical shift tensors into the contributions of the individual LMOs. The experimental and calculated trends in the chemical shift of the N-3 atom correlate nicely with the Hammett constants (σ(para)) and the calculated natural charges on N-3, whereas the contributions of the LMOs to the N-1 and C-6 chemical shifts are found to be more complex.

Accurate replication of genomic DNA is critical for cellular survival and proliferation. Despite the complexity of the cellular DNA replication process, the overall error rates are remarkably low. Three discrete processes contribute to the accuracy of replication. First, replicative DNA polymerases typically exhibit relatively low error frequencies of 10−3 to 10−6 errors per dNTP inserted (1). Second, misincorporation events decrease the rate of elongation significantly, thereby allowing 3′-5′ exonucleolytic proofreading to occur and decreasing the error rate by 100-fold or so. Finally, postreplicative repair enzymes reduce the overall error frequency to roughly 10−9 errors per nucleotide inserted (2). The mechanisms by which various polymerases efficiently distinguish between correct and incorrect dNTP substrates during polymerization are not yet fully understood. Furthermore, different polymerases appear to utilize significantly different mechanisms (3-4). Some low-fidelity enzymes, such as human primase, herpes primase, and Y-family DNA polymerases, appear to largely utilize the Watson-Crick hydrogen bonding groups on the incoming (d)NTPs and the templating base to help identify correct and incorrect incorporation events (5-10). On the other hand, various studies have shown that A and B family DNA polymerases do not require Watson-Crick hydrogen bond formation for dNTP incorporation (11-12). B family polymerases use specific functional groups on the base of an incoming purine dNTP to prevent misincorporation and to enhance correct incorporation (4, 13-16). The methods used by A family polymerases are less well understood; while some studies indicate that shape selectivity may be critical for correct dNTP incorporation, others imply that shape is not important (16-26). For example, Kool and coworkers showed that some A family polymerases efficiently incorporate 2,4-dihalotoluene dNTPs in a manner consistent with the enzyme using shape as a primary determinant (27). However, these enzymes also readily incorporate purine dNTP opposite a templating T and dITP opposite a templating C (19, 28), even though the shapes of purine and hypoxanthine significantly vary from the shapes of adenine and guanine, respectively. DNA polymerase α (pol α1) is a key polymerase (along with primase, pol δ, and pol e) required during nuclear DNA replication (29). Pol α is a B-family polymerase that typically exhibits low processivity, polymerizing ~12 nucleotides before dissociating, and moderate fidelity, making 10−3-10−5 errors per dNTP polymerized (30-31). Biologically, pol α is responsible for the initial extension of primase-synthesized RNA primers in all new DNA strands. Pol α lacks 3′-5′ exonuclease activity; therefore, the incorporation of an incorrect dNTP results in the dissociation of pol α and subsequent association of a processive replicative polymerase with exonuclease activity (pol δ or pol e) (29, 32). Pol α requires neither the formation of Watson-Crick hydrogen bonds nor a correctly shaped base pair for the rapid incorporation of a dNTP (16, 33-34). Instead, recent work has shown that during incorporation of dATP and related purine analogues, pol α employs a combination of positive and negative selectivity to ensure accuracy of replication. N-1 and N-3 serve as negative selectors and help prevent misincorporation, while N-1 and N6 act as positive selectors and enhance correct incorporation (4). Thus, a combination of positive and negative selectivity provides accuracy for pol α during dNTP incorporation, rather than shape or hydrogen bonding patterns. The HSV-1 polymerase, another B-family DNA polymerase, uses the same general mechanisms as pol α, although the precise roles of N-1 and N6 vary between the two enzymes (14). While the chemical features of purine bases have been examined with respect to their roles in correct incorporation and preventing misincorporation by pol α and herpes DNA polymerase, the contributions of the different functional groups on pyrimidines have not been examined. Accordingly, we examined the role of O2, N-3, and N4/O4 of pyrimidine dNTPs and template bases for incorporation and fidelity with these two enzymes.

A general modular and practical methodology for preparation of diverse 5-substituted pyridin-2-yl and 6-substituted pyridin-3-yl C-ribonucleosides was developed. Regioselective lithiation of 2,5-dibromopyridine proceeded at position 5 or 2 depending on the solvent, and the resulting bromopyridyl lithium species underwent additions to TBS-protected ribonolactone and follow-up transformations to corresponding acetylated hemiketal intermediates 7 and 10 that were diastereoselectively reduced to give either 5-bromopyridin-2-yl or 6-bromopyridin-3-yl silyl-protected C-ribonucleosides 8 or 11 in 68% and 77% overall yields as pure β-anomers. These bromopyridyl C-nucleoside intermediates were then subjected to a series of palladium-catalyzed cross-coupling reactions, aminations, aminocarbonylations, and hydroxylations to give a series of protected 1β-(5-alkyl-, 5-aryl-, 5-amino-, 5-carbamoyl-, and 5-hydroxypyridin-2-yl)-C-ribonucleosides 13a-i and β-(6-alkyl-, 6-aryl-, 6-amino-, 6-carbamoyl-, and 6-hydroxypyridin-3-yl)-C-ribonucleosides 15a-i. Deprotection of silylated nucleosides by Et(3)N·3HF, TBAF, or TFA gave a series of free C-nucleosides 14a-i and 16a-i.

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines were prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.

Basic aspects of DNA electrochemistry with a strong focus on the use of modified nucleobases as redox probes for electrochemical bioanalysis are reviewed. Intrinsic electrochemical properties of nucleobases in combination with artificial redox-active nucleobase modifications are frequently applied in this field. Synthetic approaches (both chemical and enzymatic) to base-modified nucleic acids are briefly summarized and their applications in redox labelling are discussed. Finally, analytical applications including DNA hybridization, primer extension, PCR, SNP typing, DNA damage and DNA–protein interaction analysis are presented (critical review, 91 references).