N-methyl mesoporphyrin IX (NMM) as electrochemical probe for detection of guanine quadruplexes.

G-quadruplexes (G4) are stable alternative secondary structures of nucleic acids. With increasing understanding of their roles in biological processes and their application in bio- and nanotechnology, the exploration of novel methods for the analysis of these structures is becoming important. In this work, N-methyl mesoporphyrin IX (NMM) was used as a voltammetric probe for an easy electrochemical detection of G4s. Cyclic voltammetry on a hanging mercury drop electrode (HMDE) was used to detect NMM with a limit of detection (LOD) of 40 nM. Characteristic reduction signal of NMM was found to be substantially higher in the presence of G4 oligodeoxynucleotides (ODNs) than in the presence of single- or double-stranded ODNs and even ODNs susceptible to form G4s but in their unfolded, single-stranded forms. Gradual transition from unstructured single strand to G4, induced by increasing concentrations of the G4 stabilizing K+ ions, was detected by an electrochemical method for the first time. All obtained results were supported by circular dichroism spectroscopy. This work expands on the concept of electrochemical probes utilization in DNA secondary structure recognition and offers a proof of principle that can be potentially employed in the development of novel electroanalytical methods for nucleic acid structure studies.

Hexahelicene DNA-binding: Minor groove selectivity, semi-intercalation and chiral recognition.

In this contribution, we focused on a fundamental study targeting the interaction of water-soluble [6]helicene derivative 1 (1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide) with double-stranded (ds) DNA. A synthetic 30-base pair duplex, plasmid, chromosomal calf thymus and salmon DNA were investigated using electrochemistry, electrophoresis and spectroscopic tools supported by molecular dynamics (MD) and quantum mechanical approaches. Both experimental and theoretical work revealed the minor groove binding of 1 to the dsDNA. Both the positively charged imidazole ring and hydrophobic part of the side chain contributed to the accommodation of 1 into the dsDNA structure. Neither intercalation into the duplex DNA nor the stable binding of 1 to single-stranded DNA were found in topoisomerase relaxation experiments with structural components of 1, i.e. [6]helicene (2) and 1-butyl-3-methylimidazolium bromide (3), nor by theoretical calculations. Finally, the binding of optically pure enantiomers (P)-1 and (M)-1 was studied using circular dichroism spectroscopy, isothermal titration calorimetry and UV Resonance Raman (UVRR) methods. Using MD and quantum mechanical methods, minor groove and semi-intercalation were proposed for compound 1 as the predominant binding modes. From the UVRR findings, we also can conclude that 1 tends to preferentially interact with adenine and guanine residues in the structure of dsDNA.

Variability of Inverted Repeats in All Available Genomes of Bacteria.

Noncanonical secondary structures in nucleic acids have been studied intensively in recent years. Important biological roles of cruciform structures formed by inverted repeats (IRs) have been demonstrated in diverse organisms, including humans. Using Palindrome analyser, we analyzed IRs in all accessible bacterial genome sequences to determine their frequencies, lengths, and localizations. IR sequences were identified in all species, but their frequencies differed significantly across various evolutionary groups. We detected 242,373,717 IRs in all 1,565 bacterial genomes. The highest mean IR frequency was detected in the Tenericutes (61.89 IRs/kbp) and the lowest mean frequency was found in the Alphaproteobacteria (27.08 IRs/kbp). IRs were abundant near genes and around regulatory, tRNA, transfer-messenger RNA (tmRNA), and rRNA regions, pointing to the importance of IRs in such basic cellular processes as genome maintenance, DNA replication, and transcription. Moreover, we found that organisms with high IR frequencies were more likely to be endosymbiotic, antibiotic producing, or pathogenic. On the other hand, those with low IR frequencies were far more likely to be thermophilic. This first comprehensive analysis of IRs in all available bacterial genomes demonstrates their genomic ubiquity, nonrandom distribution, and enrichment in genomic regulatory regions. IMPORTANCE Our manuscript reports for the first time a complete analysis of inverted repeats in all fully sequenced bacterial genomes. Thanks to the availability of unique computational resources, we were able to statistically evaluate the presence and localization of these important regulatory sequences in bacterial genomes. This work revealed a strong abundance of these sequences in regulatory regions and provides researchers with a valuable tool for their manipulation.

G-quadruplexes in helminth parasites.

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

Electrochemistry of Cobalta Bis(dicarbollide) Ions Substituted at Carbon Atoms with Hydrophilic Alkylhydroxy and Carboxy Groups.

In this study we explore the effect on the electrochemical signals in aqueous buffers of the presence of hydrophilic alkylhydroxy and carboxy groups on the carbon atoms of cobalta bis(dicarbollide) ions. The oxygen-containing exo-skeletal substituents of cobalta bis(dicarbollide) ions belong to the perspective building blocks that are considered for bioconjugation. Carbon substitution provides wider versatility and applicability in terms of the flexibility of possible chemical pathways. However, until recently, the electrochemistry of compounds substituted only on boron atoms could be studied, due to the unavailability of carbon-substituted congeners. In the present study, electrochemistry in aqueous phosphate buffers is considered along with the dependence of electrochemical response on pH and concentration. The compounds used show electrochemical signals around -1.3 and +1.1 V of similar or slightly higher intensities than in the parent cobalta bis(dicarbollide) ion. The signals at positive electrochemical potential correspond to irreversible oxidation of the boron cage (the C2B9 building block) and at negative potential correspond to the reversible redox process of (CoIII/CoII) at the central atom. Although the first signal is typically sharp and its potential can be altered by a number of substituents, the second signal is complex and is composed of three overlapping peaks. This signal shows sigmoidal character at higher concentrations and may be used as a diagnostic tool for aggregation in solution. Surprisingly enough, the observed effects of the site of substitution (boron or carbon) and between individual groups on the electrochemical response were insignificant. Therefore, the substitutions would preserve promising properties of the parent cage for redox labelling, but would not allow for the further tuning of signal position in the electrochemical window.

Tracing dsDNA Virus-Host Coevolution through Correlation of Their G-Quadruplex-Forming Sequences.

The importance of gene expression regulation in viruses based upon G-quadruplex may point to its potential utilization in therapeutic targeting. Here, we present analyses as to the occurrence of putative G-quadruplex-forming sequences (PQS) in all reference viral dsDNA genomes and evaluate their dependence on PQS occurrence in host organisms using the G4Hunter tool. PQS frequencies differ across host taxa without regard to GC content. The overlay of PQS with annotated regions reveals the localization of PQS in specific regions. While abundance in some, such as repeat regions, is shared by all groups, others are unique. There is abundance within introns of Eukaryota-infecting viruses, but depletion of PQS in introns of bacteria-infecting viruses. We reveal a significant positive correlation between PQS frequencies in dsDNA viruses and corresponding hosts from archaea, bacteria, and eukaryotes. A strong relationship between PQS in a virus and its host indicates their close coevolution and evolutionarily reciprocal mimicking of genome organization.

Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection.

G-quadruplexes contribute to the regulation of key molecular processes. Their utilization for antiviral therapy is an emerging field of contemporary research. Here we present comprehensive analyses of the presence and localization of putative G-quadruplex forming sequences (PQS) in all viral genomes currently available in the NCBI database (including subviral agents). The G4Hunter algorithm was applied to a pool of 11,000 accessible viral genomes representing 350 Mbp in total. PQS frequencies differ across evolutionary groups of viruses, and are enriched in repeats, replication origins, 5'UTRs and 3'UTRs. Importantly, PQS presence and localization is connected to viral lifecycles and corresponds to the type of viral infection rather than to nucleic acid type; while viruses routinely causing persistent infections in Metazoa hosts are enriched for PQS, viruses causing acute infections are significantly depleted for PQS. The unique localization of PQS identifies the importance of G-quadruplex-based regulation of viral replication and life cycle, providing a tool for potential therapeutic targeting.

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.

Letter to the Editor: Significant mutation enrichment in inverted repeat sites of new SARS-CoV-2 strains.

In a recently published paper, we have found that SARS-CoV-2 hot-spot mutations are significantly associated with inverted repeat loci and CG dinucleotides. However, fast-spreading strains with new mutations (so-called mink farm mutations, England mutations and Japan mutations) have been recently described. We used the new datasets to check the positioning of mutation sites in genomes of the new SARS-CoV-2 strains. Using an open-access Palindrome analyzer tool, we found mutations in these new strains to be significantly enriched in inverted repeat loci.

G-quadruplexes in H1N1 influenza genomes.

BACKGROUND: Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment. RESULTS: PQS were identified in all 77 genomes. The total number of PQS in G4-EA-H1N1 genomes was 571. Interestingly, the number of PQS per genome in individual close relative viruses varied from 4 to 12. PQS were not randomly distributed in the 8 segments of the G4-EA-H1N1 genome, the highest frequency of PQS being found in the NP segment (1.39 per 1000 nt), which is considered a potential target for antiviral therapy. In contrast, no PQS was found in the NS segment. Analyses of variability pointed the importance of some PQS; even if genome variation of influenza virus is extreme, the PQS with the highest G4Hunter score is the most conserved in all tested genomes. G-quadruplex formation in vitro was experimentally confirmed using spectroscopic methods. CONCLUSIONS: The results presented here hint several G-quadruplex-forming sequences in G4-EA-H1N1 genomes, that could provide good therapeutic targets.

SARS-CoV-2 hot-spot mutations are significantly enriched within inverted repeats and CpG island loci.

SARS-CoV-2 is an intensively investigated virus from the order Nidovirales (Coronaviridae family) that causes COVID-19 disease in humans. Through enormous scientific effort, thousands of viral strains have been sequenced to date, thereby creating a strong background for deep bioinformatics studies of the SARS-CoV-2 genome. In this study, we inspected high-frequency mutations of SARS-CoV-2 and carried out systematic analyses of their overlay with inverted repeat (IR) loci and CpG islands. The main conclusion of our study is that SARS-CoV-2 hot-spot mutations are significantly enriched within both IRs and CpG island loci. This points to their role in genomic instability and may predict further mutational drive of the SARS-CoV-2 genome. Moreover, CpG islands are strongly enriched upstream from viral ORFs and thus could play important roles in transcription and the viral life cycle. We hypothesize that hypermethylation of these loci will decrease the transcription of viral ORFs and could therefore limit the progression of the disease.

Electrochemical Reduction and Oxidation of Eight Unnatural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode.

Recently we showed the reduction and oxidation of six natural 2'-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2'-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.

G-Quadruplexes in the Archaea Domain.

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.

Global analysis of inverted repeat sequences in human gene promoters reveals their non-random distribution and association with specific biological pathways.

Inverted repeats (IR) play important roles in specific DNA-dependent processes in simple prokaryotes to complex eukaryotes. They are recognized by a variety of proteins including restriction enzymes, helicases and transcription factors. We evaluate the presence and localization of IRs in all validated human promoter sequences within 1000 bp upstream and downstream of the transcription start site (TSS). The occurrence of 7 bp and longer IRs is located non-randomly in promoter regions, with enrichment within 200 bp upstream of the TSS. The highest frequency of IRs is just before TSS for repeats of 8 bp or longer. A comparison of promoters divided according to the occurrence of five individual promoter motifs shows unique location patterns of IRs. Principal component analyses and hierarchical clustering of IRs abundance demonstrated that they are depleted and/or not enriched in the promoters of stably expressed genes, but show significant enrichments for specific dynamically regulated biological pathways.

Structures and stability of simple DNA repeats from bacteria.

DNA is a fundamentally important molecule for all cellular organisms due to its biological role as the store of hereditary, genetic information. On the one hand, genomic DNA is very stable, both in chemical and biological contexts, and this assists its genetic functions. On the other hand, it is also a dynamic molecule, and constant changes in its structure and sequence drive many biological processes, including adaptation and evolution of organisms. DNA genomes contain significant amounts of repetitive sequences, which have divergent functions in the complex processes that involve DNA, including replication, recombination, repair, and transcription. Through their involvement in these processes, repetitive DNA sequences influence the genetic instability and evolution of DNA molecules and they are located non-randomly in all genomes. Mechanisms that influence such genetic instability have been studied in many organisms, including within human genomes where they are linked to various human diseases. Here, we review our understanding of short, simple DNA repeats across a diverse range of bacteria, comparing the prevalence of repetitive DNA sequences in different genomes. We describe the range of DNA structures that have been observed in such repeats, focusing on their propensity to form local, non-B-DNA structures. Finally, we discuss the biological significance of such unusual DNA structures and relate this to studies where the impacts of DNA metabolism on genetic stability are linked to human diseases. Overall, we show that simple DNA repeats in bacteria serve as excellent and tractable experimental models for biochemical studies of their cellular functions and influences.

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.

Electrodeposited silver amalgam particles on pyrolytic graphite in (spectro)electrochemical detection of 4-nitrophenol, DNA and green fluorescent protein.

Catalytic properties and high adsorption affinity of nucleic acids and proteins to silver amalgam electrode surface make this kind of electrified interface perspective for bioanalytical and biomedical applications. For the first time, a basal-plane pyrolytic graphite electrode (bPGE) has been used as a substrate for electrodeposition of silver amalgam particles (AgAPs). Optimization of the resulting composition, surface morphology and electrochemical properties of the AgAPs was done by scanning electron microscopy with energy disperse X-ray spectroscopy, image processing software and voltammetric detection of electrochemically reducible model organic nitro compound, 4-nitrophenol. Spectro-electrochemical applicability of bPGE-AgAP has been demonstrated by electrolysis of 4-nitrophenol. Simultaneous UV-Vis-chronoamperometry provided information on the number of exchange electrons and the reduction rate constants. Preferential adsorption of the fluorescently labelled calf thymus DNA and the green fluorescent protein (GFP) on the surface of AgAPs was observed by fluorescence microscopy. In contrast to previously studied indium-tin oxide and vapour-deposited gold decorated by AgAPs, herein the presented bPGE-AgAP has provided sufficiently wide negative potential window allowing direct electroanalysis of non-labelled DNA and GFP using intrinsic electrochemical signals independently of the fluorescent labelling. The bPGE-AgAP can thus be expected to find application opportunities in protein electrochemistry, (bio)sensor development or in-situ spectro-electrochemical studies.

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 Influence of Quadruplex Structure in Proximity to P53 Target Sequences on the Transactivation Potential of P53 Alpha Isoforms.

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.