Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases.

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) derived from 5-substituted pyrimidines and 7-substituted 7-deazapurines bearing anionic substituents (carboxylate, sulfonate, phosphonate, and phosphate). The anion-linked dNTPs were used for enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase containing one, two, three, or four modified nucleotides. The polymerase was able to synthesize even long sequences of >100 modified nucleotides in a row by primer extension (PEX). We also successfully combined two anionic and two hydrophobic dNTPs bearing phenyl and indole moieties. In PCR, the combinations of one or two modified dNTPs gave exponential amplification, while most of the combinations of three or four modified dNTPs gave only linear amplification in asymmetric PCR. The hypermodified ONs were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies including hybridization, denaturation, CD spectroscopy and molecular modelling and dynamics suggest that the presence of anionic modifications in one strand decreases the stability of duplexes while still preserving the B-DNA conformation, whilst the DNA hypermodified in both strands adopts a different secondary structure.

ABNOH-Linked Nucleotides and DNA for Bioconjugation and Cross-linking with Tryptophan-Containing Peptides and Proteins.

Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC reaction of ABNOH-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable tricyclic fused hexahydropyrrolo-indole conjugates. Similarly modified ABNOH-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNOH-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.

Normalized Multipotential Redox Coding of DNA Bases for Determination of Total Nucleotide Composition.

The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging.

A series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels-Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.

Polymerase Synthesis of DNA Containing Iodinated Pyrimidine or 7-Deazapurine Nucleobases and Their Post-synthetic Modifications through the Suzuki-Miyaura Cross-Coupling Reactions.

All four iodinated 2'-deoxyribonucleoside triphosphates (dNTPs) derived from 5-iodouracil, 5-iodocytosine, 7-iodo-7-deazaadenine and 7-iodo-7-deazaguanine were prepared and studied as substrates for KOD XL DNA polymerase. All of the nucleotides were readily incorporated by primer extension and by PCR amplification to form DNA containing iodinated nucleobases. Systematic study of the Suzuki-Miyaura cross-coupling reactions with two bulkier arylboronic acids revealed that the 5-iodopyrimidines were more reactive and gave cross-coupling products both in the terminal or internal position in single-stranded oligonucleotides (ssONs) and in the terminal position of double-stranded DNA (dsDNA), whereas the 7-iodo-7-deazapurines were less reactive and gave cross-coupling products only in the terminal position. None of the four iodinated bases reacted in an internal position of dsDNA. These findings are useful for the use of the iodinated nucleobases for post-synthetic modification of DNA with functional groups for various applications.

Glyoxal-Linked Nucleotides and DNA for Bioconjugations and Crosslinking with Arginine-Containing Peptides and Proteins.

Glyoxal-linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of 4-azidophenylglyoxal or a Sonogashira reaction of 4-bromophenylglyoxal with 5-ethynyl-dUMP or -dUTP. The triphosphates were used as substrates for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The glyoxal-linked nucleotides reacted with arginine-containing peptides to form stable imizadolone-linked conjugates. This reactive glyoxal modification in DNA was used for efficient bioconjugations and crosslinking with Arg-containing peptides or proteins (e. g., histones) and was found to be more reactive than previously reported 1,3-diketone-linked DNA probes.

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins.

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

Vicinal Diol-Tethered Nucleobases as Targets for DNA Redox Labeling with Osmate Complexes.

Six-valent osmium (osmate) complexes with nitrogenous ligands have previously been used for the modification and redox labeling of biomolecules involving vicinal diol moieties (typically, saccharides or RNA). In this work, aliphatic (3,4-dihydroxybutyl and 3,4-dihydroxybut-1-ynyl) or cyclic (6-oxo-6-(cis-3,4-dihydroxypyrrolidin-1-yl)hex-2-yn-1-yl, PDI) vicinal diols are attached to nucleobases to functionalize DNA for subsequent redox labeling with osmium(VI) complexes. The diol-linked 2'-deoxyribonucleoside triphosphates were used for the polymerase synthesis of diol-linked DNA, which, upon treatment with K2 OsO3 and bidentate nitrogen ligands, gave the desired Os-labeled DNA, which were characterized by means of the gel-shift assay and ESI-MS. Through ex situ square-wave voltammetry at a basal plane pyrolytic graphite electrode, the efficiency of modification/labeling of individual diols was evaluated. The results show that the cyclic cis-diol (PDI) was a better target for osmylation than that of the flexible aliphatic ones (alkyl- or alkynyl-linked). The osmate adduct-specific voltammetric signal obtained for OsVI -treated DNA decorated with PDI showed good proportionality to the number of PDI per DNA molecule. The OsVI reagents (unlike OsO4 ) do not attack nucleobases; thus offering specificity of modification on the introduced glycol targets.

Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA.

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.

The selection of a hydrophobic 7-phenylbutyl-7-deazaadenine-modified DNA aptamer with high binding affinity for the Heat Shock Protein 70.

Nucleic acids aptamers often fail to efficiently target some proteins because of the hydrophilic character of the natural nucleotides. Here we present hydrophobic 7-phenylbutyl-7-deaadenine-modified DNA aptamers against the Heat Shock Protein 70 that were selected via PEX and magnetic bead-based SELEX. After 9 rounds of selection, the pool was sequenced and a number of candidates were identified. Following initial screening, two modified aptamers were chemically synthesised in-house and their binding affinity analysed by two methods, bio-layer interferometry and fluorescent-plate-based binding assay. The binding affinities of the modified aptamers were compared with that of their natural counterparts. The resulting modified aptamers bound with higher affinity (low nanomolar range) to the Hsp70 than their natural sequence (>5 µM) and hence have potential for applications and further development towards Hsp70 diagnostics or even therapeutics.

Traceless enzymatic synthesis of monodispersed hypermodified oligodeoxyribonucleotide polymers from RNA templates.

We have developed a new alternative for enzymatic synthesis of single-stranded hypermodified oligodeoxyribonucleotides displaying four different hydrophobic groups based on reverse transcription from RNA templates catalyzed by DNA polymerases using a set of base-modified dNTPs followed by digestion of RNA by RNases. Using mixed oligodeoxyribonucleotide primers containing a ribonucleotide at the 3'-end, RNase AT1 simultaneously digested the template and cleaved off the primer to release a fully modified oligonucleotide that can be further 3'-labelled with a fluorescent nucleotide using TdT. The resulting hypermodified oligonucleotides could find applications in selection of aptamers or other functional macromolecules.

Correction: Traceless enzymatic synthesis of monodispersed hypermodified oligodeoxyribonucleotide polymers from RNA templates.

Correction for 'Traceless enzymatic synthesis of monodispersed hypermodified oligodeoxyribonucleotide polymers from RNA templates' by Marek Ondrus et al., Chem. Commun., 2022, 58, 11248-11251, https://doi.org/10.1039/D2CC03588J.

Epigenetic Pyrimidine Nucleotides in Competition with Natural dNTPs as Substrates for Diverse DNA Polymerases.

Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

Homologues of epigenetic pyrimidines: 5-alkyl-, 5-hydroxyalkyl and 5-acyluracil and -cytosine nucleotides: synthesis, enzymatic incorporation into DNA and effect on transcription with bacterial RNA polymerase.

Homologues of natural epigenetic pyrimidine nucleosides and nucleotides were designed and synthesized. They included 5-ethyl-, 5-propyl-, 5-(1-hydroxyethyl)-, 5-(1-hydroxypropyl)- and 5-acetyl- and 5-propionylcytosine and -uracil 2'-deoxyribonucleosides and their corresponding 5'-O-triphosphates (dNXTPs). The epimers of 5-(1-hydroxyethyl)- and 5-(1-hydroxypropyl)pyrimidine nucleosides were separated and their absolute configuration was determined by a combination of X-ray and NMR analysis. The modified dNXTPs were used as substrates for PCR synthesis of modified DNA templates used for the study of transcription with bacterial RNA polymerase. Fundamental differences in transcription efficiency were observed, depending on the various modifications. The most notable effects included pronounced stimulation of transcription from 5-ethyluracil-bearing templates (200% transcription yield compared to natural thymine) and an enhancing effect of 5-acetylcytosine versus inhibiting effect of 5-acetyluracil. In summary, these results reveal that RNA polymerase copes with dramatically altered DNA structure and suggest that these nucleobases could potentially play roles as artificial epigenetic DNA nucleobases.

Antiviral Activity of 7-Substituted 7-Deazapurine Ribonucleosides, Monophosphate Prodrugs, and Triphoshates against Emerging RNA Viruses.

A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.