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The Biochemical Journal May 2023Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened...
Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2'-fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2'-F-N7-alkylG DNA into 2'-F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supF-based colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2'-F-N7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5'-GGT(methyl-FapyG)G-3' context were single nucleotide deletions at the 5'-G of the lesion. Overall, our results demonstrate that 2'-fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions.
Topics: DNA Damage; Alkylation; DNA; Guanine
PubMed: 37078496
DOI: 10.1042/BCJ20220460 -
Nucleosides, Nucleotides & Nucleic Acids 2024This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite...
This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.
Topics: Solid-Phase Synthesis Techniques; Organophosphorus Compounds; Deoxyribonucleotides; Base Sequence; Oligonucleotides; Chromatography, High Pressure Liquid
PubMed: 38116988
DOI: 10.1080/15257770.2023.2295478 -
Frontiers in Chemistry 2022DNA-encoded libraries are a prime technology for target-based small molecule screening. Native DNA used as genetic compound barcode is chemically vulnerable under many...
DNA-encoded libraries are a prime technology for target-based small molecule screening. Native DNA used as genetic compound barcode is chemically vulnerable under many reaction conditions. DNA barcodes that are composed of pyrimidine nucleobases, 7-deazaadenine, and 7-deaza-8-azaguanine have been investigated for their suitability for encoded chemistry both experimentally and computationally. These four-letter barcodes were readily ligated by T4 ligation, amplifiable by Taq polymerase, and the resultant amplicons were correctly sequenced. Chemical stability profiling showed a superior chemical stability compared to native DNA, though higher susceptibility to depurination than a three-letter code based on pyrimidine DNA and 7-deazaadenine.
PubMed: 35755251
DOI: 10.3389/fchem.2022.894563 -
Methods in Molecular Biology (Clifton,... 20215-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC),...
5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.
Topics: 5-Methylcytosine; Animals; Antibodies; Cell Nucleus; Cytosine; DNA; DNA Methylation; Dioxygenases; Embryo, Nonmammalian; Immunohistochemistry; Zebrafish
PubMed: 32822033
DOI: 10.1007/978-1-0716-0876-0_16 -
ACS Chemical Biology Sep 2022Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The...
Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.
Topics: Adenosine; Aniline Compounds; Antiviral Agents; DNA; High-Throughput Nucleotide Sequencing; Immunotoxins; Oligonucleotides; Plant Proteins; RNA; RNA, Ribosomal, 28S; Ribosome Inactivating Proteins; Ribosome Inactivating Proteins, Type 1; Ricin; Saporins
PubMed: 35969718
DOI: 10.1021/acschembio.2c00531 -
RNA Biology Jan 2024RNA modifications, including -7-methylguanosine (mG), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal mG...
RNA modifications, including -7-methylguanosine (mG), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal mG modifications is of paramount significance, given recent associations between altered mG deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust mG detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of mG modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of mG modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH, neutral pH and the addition of 7-methylguanosine monophosphate (mGMP) during the reducing reaction. Notably, compared to NaBH-based methods, Bo-Seq enhances the mG detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of mG relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA mG methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of mG in gene expression and its implications in cancer biology.
Topics: Humans; RNA; Nucleotides; Methylation; Methyltransferases; RNA Processing, Post-Transcriptional; Guanosine
PubMed: 38566310
DOI: 10.1080/15476286.2024.2337493 -
Analytical Chemistry Dec 2022DNA nanoframeworks, with great biological information and controlled framework structures, exhibit great potentials in biological applications. Their applications are...
DNA nanoframeworks, with great biological information and controlled framework structures, exhibit great potentials in biological applications. Their applications are normally limited by unstable structures susceptible to hydrolysis, depurination, depyrimidination, oxidation, alkylation, or nuclease degradations. Herein, to ensure the mechanical and chemical stabilities of DNA nanoframeworks for intracellular applications, biomineralization of multifunctional DNA nanoframeworks with a tetrahedral skeleton is employed. Via silicification, the S-S bond is simultaneously introduced to obtain the silica-armored DNA nanoframeworks (Si-DNA nanoframeworks), mechanically and chemically stabilized for efficient intracellular deliveries. This successfully prevents degradations and leakages of reagents loaded on Si-DNA nanoframeworks, including biomolecular siRNA and small DOX drugs. Furthermore, the nucleic acid strands of the nanoframeworks are labeled with FAM and the quencher, facilitating miRNA detection upon "turn-on" signals from hybridizations. Therefore, the nanoframeworks collapse via double responses of the silica coating (silica acidic dissolution and S-S reduction by GSH) in cancer cells, realizing on-demand reagent release for miRNA detection and synergistic treatments (by siRNA and DOX). Demonstrated by both in vivo and in vitro experiments, the biomineralization has stabilized DNA nanomaterials for biological applications.
Topics: Doxorubicin; RNA, Small Interfering; Nanoparticles; Biomineralization; Silicon Dioxide; DNA; MicroRNAs; Neoplasms
PubMed: 36342409
DOI: 10.1021/acs.analchem.2c03726 -
International Journal of Molecular... Mar 2022The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA...
The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA polymerases are specifically responsible for the translesion synthesis (TLS) of specific DNA damage, such as AP site damage, generally with relatively low fidelity. The Y-family DNA polymerases are the main error-prone DNA polymerases, and they employ three mechanisms to perform TLS, including template-skipping, dNTP-stabilized misalignment, and misincorporation-misalignment. The bypass mechanism of the dinB homolog (Dbh), an archaeal Y-family DNA polymerase from , is unclear and needs to be confirmed. In this study, we show that the Dbh primarily uses template skipping accompanied by dNTP-stabilized misalignment to bypass AP site analogs, and the incorporation of the first nucleotide across the AP site is the most difficult. Furthermore, based on the reported crystal structures, we confirmed that three conserved residues (Y249, R333, and I295) in the little finger (LF) domain and residue K78 in the palm subdomain of the catalytic core domain are very important for TLS. These results deepen our understanding of how archaeal Y-family DNA polymerases deal with intracellular AP site damage and provide a biochemical basis for elucidating the intracellular function of these polymerases.
Topics: DNA Damage; DNA Polymerase beta; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Sulfolobus acidocaldarius
PubMed: 35269871
DOI: 10.3390/ijms23052729 -
Chembiochem : a European Journal of... Oct 2022DNA long-term stability and integrity is of importance for applications in DNA based bio-dosimetry, data-storage, pharmaceutical quality-control, donor insemination and...
DNA long-term stability and integrity is of importance for applications in DNA based bio-dosimetry, data-storage, pharmaceutical quality-control, donor insemination and DNA based functional nanomaterials. Standard protocols for these applications involve repeated freeze-thaw cycles of the DNA, which can cause detrimental damage to the nucleobases, as well as the sugar-phosphate backbone and therefore the whole molecule. Throughout the literature three hypotheses can be found about the underlying mechanisms occurring during freeze-thaw cycles. It is hypothesized that DNA single-strand breaks during freezing can be induced by mechanical stress leading to shearing of the DNA molecule, by acidic pH causing damage through depurination and beta elimination or by the presence of metal ions catalyzing oxidative damage via reactive oxygen species (ROS). Here we test these hypotheses under well defined conditions with plasmid DNA pUC19 in high-purity buffer (1xPBS) at physiological salt and pH 7.4 conditions, under pH 6 and in the presence of metal ions in combination with the radical scavengers DMSO and Ectoine. The results show for the 2686 bp long plasmid DNA, that neither mechanical stress, nor pH 6 lead to degradation during repeated freeze-thaw cycles. In contrast, the presence of metal ions (Fe ) leads to degradation of DNA via the production of radical species.
Topics: Reactive Oxygen Species; Dimethyl Sulfoxide; DNA; Ions; Pharmacy; Phosphates; Pharmaceutical Preparations; Sugars
PubMed: 35972228
DOI: 10.1002/cbic.202200391 -
Toxins Jan 2021Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1...
Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.
Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Survival; Erythrocyte Aggregation; Humans; Molecular Weight; Neuroblastoma; Passifloraceae; Protein Biosynthesis; Protein Synthesis Inhibitors; Ribosome Inactivating Proteins, Type 2; Ribosomes
PubMed: 33499082
DOI: 10.3390/toxins13020081