Nucleobase Modifications Research Articles

Genomic Uracil and Aberrant Profile of Demethylation Intermediates in Epigenetics and Hematologic Malignancies.

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

Recognition of Target Site in Various Forms of DNA and RNA by Peptide Nucleic Acid (PNA): From Fundamentals to Practical Applications

Abstract Peptide nucleic acid (PNA) is a DNA analog, in which the sugar-phosphate backbone in DNA is replaced by poly[N-(2-aminoethyl)glycine]. Since its discovery in the early 1990s, PNA has been widely employed in chemistry, biochemistry, medicine, nanotechnology, and many other fields. This account surveys recent developments on the design of PNA derivatives and their applications. In the first part, PNAs for sequence-specific recognition of DNA and RNA (single-strands, double-strands, G-quadruplexes, i-motifs, and others) are comprehensively covered. Modifications of nucleobases and of the main chain effectively promote both the strength of binding and the selectivity of recognition. In the second half of this account, practical applications of PNA are presented. Structural restraints, induced by complex formation of PNA with DNA and RNA substrates, lead to selective transformation of target sites to desired structures. Applications to regulation of gene expression, gene editing, construction of sophisticated nanostructures, and others are also described. Advantages and disadvantages of PNAs, compared with other sequence-recognizing molecules hitherto reported, are discussed in terms of various physicochemical and biological features.

Combined use of chemically modified nucleobases and nanostructured DNA for enhanced immunostimulatory activity of CpG oligodeoxynucleotide

Oligodeoxynucleotide (ODN) containing a cytosine-phosphate-guanine (CpG) motif, or CpG ODN, is considered suitable for treating immune diseases, including allergies. Although the phosphorothioate modification is used to enhance the stability and immunostimulatory activity of CpG ODNs, it is associated with the risk of adverse effects. Construction of nanostructured DNA assemblies, such as tripod- and hexapod-like structured DNAs, tripodna and hexapodna, respectively, were also found to increase this activity. The chemical modification of nucleobases could be another approach for enhancing CpG ODN activity. Here, we examined whether chemically modified nucleobase substitutions can enhance CpG ODN activity by measuring tumor necrosis factor α (TNF-α) release after addition to murine macrophage-like RAW264.7 cells. First, the guanine at the 18th position of phosphodiester CpG 1668 was substituted with several chemically modified guanines, and then the various guanines were substituted. Among all tested substitutions, 15,18-thdG, in which two guanines outside the CpG motif were substituted with the 2-aminothieno[3,4-d]pyrimidine guanine mimic (thdG), was the most effective. Compared to 32P-CpG 1668, 32P-15,18-thdG was taken up more efficiently by the RAW264.7 cells. Then, 15,18-thdG was incorporated into tripodna and hexapodna. 15,18-thdG/tri- or hexapodna induced higher TNF-α release from the RAW264.7 cells than PO CpG 1668/tri- or hexapodna, respectively. These results indicate that the thdG substitution is a useful effective strategy for enhancing the immunostimulatory activity of CpG DNAs in both single stranded and DNA nanostructure forms.

A new Pb2+-specific DNAzyme by revisiting the catalytic core of 10–23 DNAzyme

10–23 DNAzyme is a catalytic DNA molecule from in vitro selection, the 15-mer catalytic core was investigated for more DNAzyme variants by block deletions. DNAzyme DZM01 was selected with metal ion dependence of Pb2+ ≫ Mn2+, with no activity in the presence of Mg2+ (20 mM), Ca2+ (20 mM), Zn2+ (20 mM, pH 6). The unique binding properties of Pb2+ with nucleic acids might be responsible for the formation of the catalytic core, which is different from that of other divalent metal ions. More DNAzyme variants are expected to be derived for specific metal ion dependence by various nucleobase sequences and modifications.

Mass Spectrometry-Based Analysis of DNA Modifications: Potential Applications in Basic Research and Clinic.

Stable-isotope-dilution tandem mass spectrometry is the most advanced technique used for quantitative determination of a wide spectrum of endogenously generated DNA nucleobase modifications. It is regarded as a gold standard for such analyses. Here, we consider the requirements for reliable identification and quantification of DNA adducts/modifications, whether endogenously derived or not, and discuss how their quantification can provide information on the mechanism of action and the biological relevance of individual nucleobase modifications. A clinical application of such measurements will only be possible after a full validation of the assay and once we have gained a better understanding of the exact role that these DNA modifications play in disease pathogenesis. Once these prerequisites are satisfied, DNA modification measurements may be helpful as clinical parameters for treatment monitoring, for risk group identification and for the development of prevention strategies.

Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach.

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

Molecular Features and Metal Ions That Influence 10-23 DNAzyme Activity.

Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.

Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels.

Fluorescence labeling and probing are fundamental techniques for nucleic acid analysis and quantification. However, new fluorescent probes and approaches are urgently needed in order to accurately determine structural and conformational dynamics of DNA and RNA at the level of single nucleobases/base pairs, and to probe the interactions between nucleic acids with proteins. This review describes the means by which to achieve these goals using nucleobase replacement or modification with advanced fluorescent dyes that respond by the changing of their fluorescence parameters to their local environment (altered polarity, hydration, flipping dynamics, and formation/breaking of hydrogen bonds).

Automated screening for oxidative or methylation-induced DNA damage in human cells.

The assessment of genotoxicity upon exposure to chemical and environmental agents plays an important role in basic research as well as in pharmaceutical, chemical, cosmetic and food industry. Low sensitivity and lack of inter-laboratory comparability are considered problematic issues in genotoxicity testing. Moreover, commonly used mutagenicity assays lack information about early and specific genotoxic events. Previously, we developed an automated version of the “Fluorimetric detection of Alkaline DNA Unwinding” (FADU) assay as a high-throughput screening method for the detection of DNA strand breaks in living cells. Here, we report an enzyme-modified version of the cell-based FADU assay (emFADU) for the determination of oxidative and methylation lesions in cellular DNA. Our method is based on the use of formamidopyrimidine DNA glycosylase or human alkylad­enine DNA glycosylase for the detection of chemically-induced nucleobase modifications in lysates of immortalized cell lines, growing in suspension or as adherent cells, and in peripheral blood mononuclear cells. We could show that upon treatment with sub-cytotoxic doses of known genotoxins, oxidative and methylation lesions are readily detectable. This fast, inexpensive, and convenient method could be useful as a high-content screening approach for the sensitive and specific assessment of genotoxicity in human cells. Thus, when implemented in the early compound development in an industrial setting, the emFADU assay could help reduce the number of animals used for toxicity testing. Furthermore, as we established the method for different cell types, this new assay may provide an opportunity for population studies and/or mechanistic research into DNA repair pathways.

A Benzophenone-Based Photocaging Strategy for the N7 Position of Guanosine.

Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring‐nitrogen methylation, such as m7G and m1A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α‐hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho‐nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site‐specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m7G and m1A with spatiotemporal control.

Chemical-Labeling-Assisted Detection of Nucleobase Modifications by Quantum-Tunneling-Based Single-Molecule Sensing.

Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling. We used 5-Formyl-2'-deoxyuridine as a model compound and demonstrated that chemical labeling dramatically alters its molecular conductance compared with that of canonical nucleotides; thus, facilitating statistical discrimination, which is impeded in the unlabeled state. This work introduces a chemical strategy that overcomes the intrinsic difficulty in quantum-tunneling-based modification analysis-the similarity of the molecular conductance of the nucleobases of interest.

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

AbstractA tandem enzymatic strategy to enhance the scope of C‐alkylation of small molecules via the in situ formation of S‐adenosyl methionine (SAM) cofactor analogues is described. A solvent‐exposed channel present in the SAM‐forming enzyme SalL tolerates 5′‐chloro‐5′‐deoxyadenosine (ClDA) analogues modified at the 2‐position of the adenine nucleobase. Coupling SalL‐catalyzed cofactor production with C‐(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C‐(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C‐alkylation provides the basis to develop a late‐stage enzymatic platform for the preparation of high value small molecules.

S-Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C-Alkylation.

A tandem enzymatic strategy to enhance the scope of C-alkylation of small molecules via the in situ formation of S-adenosyl methionine (SAM) cofactor analogues is described. A solvent-exposed channel present in the SAM-forming enzyme SalL tolerates 5'-chloro-5'-deoxyadenosine (ClDA) analogues modified at the 2-position of the adenine nucleobase. Coupling SalL-catalyzed cofactor production with C-(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C-(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C-alkylation provides the basis to develop a late-stage enzymatic platform for the preparation of high value small molecules.

6-Phenylpyrrolocytidine: An Intrinsically Fluorescent, Environmentally Responsive Nucleoside Analogue.

The detailed synthetic protocols for the preparation of phosphoramidite reagents compatible with standard, automated oligonucleotide synthesis for the 2'-deoxy- and ribo-6-phenylpyrrolocyitidine are reported. Each protocol starts with the parent nucleoside and prepares the 5'-O-dimethoxytrityl-N4 -benzoyl-5-iodocytosine derivative for the nucleobase modification chemistry. The key step is the direct formation of 6-phenylpyrrolocytosine aglycon via a sequential, one-pot Pd-catalyzed Sonogashira-type cross- coupling followed by a 5-endo-dig cyclization. Subsequent standard transformations provide the deoxy- and 2'-O-tert-butyldimethysilyl protected ribo- nucleoside phosphoramidite reagents. © 2019 by John Wiley & Sons, Inc.

Targeting Base Excision Repair Glycosylases with DNA Containing Transition State Mimics Prepared via Click Chemistry.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( Kd) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar Kd values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.

Genome, Epigenome, and Transcriptome Editing via Chemical Modification of Nucleobases in Living Cells.

Base editors are tools that chemically modify the nucleobases of DNA and RNA in a programmable manner, allowing for genome, epigenome, and transcriptome editing in live cells. These tools can be used to introduce specific base transitions in DNA or RNA, manipulate methylation patterns in the epigenome, and create genetically encoded libraries in target genes. These various functions can be used to modulate every aspect of the central dogma. The efficiency and precision of base editors makes them useful in both basic research and the development of new therapies. Here we describe currently available base editors and the ways that they can be used to better understand and manipulate different aspects of the central dogma.

RNAm5Cfinder: A Web-server for Predicting RNA 5-methylcytosine (m5C) Sites Based on Random Forest

5-methylcytosine (m5C) is a common nucleobase modification, and recent investigations have indicated its prevalence in cellular RNAs including mRNA, tRNA and rRNA. With the rapid accumulation of m5C sites data, it becomes not only feasible but also important to build an accurate model to predict m5C sites in silico. For this purpose, here, we developed a web-server named RNAm5Cfinder based on RNA sequence features and machine learning method to predict RNA m5C sites in eight tissue/cell types from mouse and human. We confirmed the accuracy and usefulness of RNAm5Cfinder by independent tests, and the results show that the comprehensive and cell-specific predictors could pinpoint the generic or tissue-specific m5C sites with the Area Under Curve (AUC) no less than 0.77 and 0.87, respectively. RNAm5Cfinder web-server is freely available at http://www.rnanut.net/rnam5cfinder.

A versatile method for the UVA-induced cross-linking of acetophenone- or benzophenone-functionalized DNA

Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3′-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3′-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.

Selectivity and Promiscuity in TET-Mediated Oxidation of 5-Methylcytosine in DNA and RNA.

Enzymes of the ten-eleven translocation (TET) family add diversity to the repertoire of nucleobase modifications by catalyzing the oxidation of 5-methylcytosine (5mC). TET enzymes were initially found to oxidize 5-methyl-2'-deoxycytidine in genomic DNA, yielding products that contribute to epigenetic regulation in mammalian cells, but have since been found to also oxidize 5-methylcytidine in RNA. Considering the different configurations of single-stranded (ss) and double-stranded (ds) DNA and RNA that coexist in a cell, defining the scope of TET's preferred activity and the mechanisms of substrate selectivity is critical to better understand the enzymes' biological functions. To this end, we have systematically examined the activity of human TET2 on DNA, RNA, and hybrid substrates in vitro. We found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA. Chimeric and hybrid substrates containing mixed DNA and RNA character helped reveal two main features by which the enzyme discriminates between substrates. First, the identity of the target nucleotide alone is the strongest reactivity determinant, with a preference for 5-methyldeoxycytidine, while both DNA or RNA are relatively tolerated on the rest of the target strand. Second, while a complementary strand is not required for activity, DNA is the preferred partner, and complementary RNA diminishes reactivity. Our biochemical analysis, complemented by molecular dynamics simulations, provides support for an active site optimally configured for dsDNA reactivity but permissive for various nucleic acid configurations, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.

Structural Biology of the HEAT-Like Repeat Family of DNA Glycosylases.

DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision repair (BER) pathway. The alkyl-DNA glycosylases AlkC and AlkD adopt a unique structure based on α-helical HEAT repeats. Both enzymes identify and excise their substrates without a base-flipping mechanism used by other glycosylases and nucleic acid processing proteins to access nucleobases that are otherwise stacked inside the double-helix. Consequently, these glycosylases act on a variety of cationic nucleobase modifications, including bulky adducts, not previously associated with BER. The related non-enzymatic HEAT-like repeat (HLR) proteins, AlkD2, and AlkF, have unique nucleic acid binding properties that expand the functions of this relatively new protein superfamily beyond DNA repair. Here, we review the phylogeny, biochemistry, and structures of the HLR proteins, which have helped broaden our understanding of the mechanisms by which DNA glycosylases locate and excise chemically modified DNA nucleobases.