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Nucleic Acids Research Oct 2023Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their...
Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their biophysical properties. Here, we compare the stability and mechanism of acid-mediated degradation of α-l-threose nucleic acid (TNA) to that of natural DNA and RNA. Under acidic conditions and elevated temperature (pH 3.3 at 90°C), TNA was found to be significantly more resistant to acid-mediated degradation than DNA and RNA. Mechanistic insights gained by reverse-phase HPLC and mass spectrometry indicate that the resilience of TNA toward low pH environments is due to a slower rate of depurination caused by induction of the 2'-phosphodiester linkage. Similar results observed for 2',5'-linked DNA and 2'-O-methoxy-RNA implicate the position of the phosphodiester group as a key factor in destabilizing the formation of the oxocarbenium intermediate responsible for depurination and strand cleavage of TNA. Biochemical analysis indicates that strand cleavage occurs by β-elimination of the 2'-phosphodiester linkage to produce an upstream cleavage product with a 2'-threose sugar and a downstream cleavage product with a 3' terminal phosphate. This work highlights the unique physicochemical properties available to evolvable non-natural genetic polymers currently in development for biomedical applications.
PubMed: 37650628
DOI: 10.1093/nar/gkad716 -
Nucleosides, Nucleotides & Nucleic Acids May 2024The -methyladenosine (mA) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are...
The -methyladenosine (mA) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are required for projects aimed at studying biological processes, mechanisms, and pathogenesis related to mA. Existing methods for the synthesis of mA containing RNAs use tetrabutylammonium fluoride (TBAF) for the deprotection of the 2'-silyl protecting groups. Since TBAF is nonvolatile, and is relatively non-polar, its use in the desilylation of RNA requires repeated desalting, which is tedious and gives low yields. Here we report the use of the volatile and neat triethylamine hydrogen fluoride (TEA-HF) for desilylation of mA RNA synthesis. We found that the method is much simpler, and-in our hands-give significantly higher yield of RNA. Two major concerns for mA RNA synthesis are depurination and Dimroth rearrangement. HPLC and MALDI MS of the RNA indicated that depurination is not a problem for the new method. The absence of Dimroth rearrangement is proven by RNA digestion followed by HPLC analysis of the nucleosides.
PubMed: 38735066
DOI: 10.1080/15257770.2024.2353181 -
Chemistry, An Asian Journal Dec 2022Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase...
Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase (depurination) at the sarcin/ricin loop of the ribosomal RNA. In this work, we describe the preparation and optimization process of a fluorescent probe that can be used to visualize depurination. Starting from a fluorescent thiophene nucleobase analog, various RNA probes that fluoresce exclusively in the presence of a depurinated sarcin/ricin-loop RNA were designed and characterized. The main challenge in this process was to obtain a high fluorescence signal in the hybridized state with an abasic RNA strand, while keeping the background fluorescence low. With our new RNA probes, the fluorescence intensity and lifetime can be used for efficient monitoring of depurinated RNA.
Topics: Ricin; RNA Probes; RNA; Fluorescence; Purines
PubMed: 36321802
DOI: 10.1002/asia.202201077 -
Chemical Research in Toxicology Oct 2019N3-Methyl-2'-deoxyadenosine (MdA) is the major dA methylation product in duplex DNA. MdA blocks DNA replication and undergoes depurination at significantly higher rates...
N3-Methyl-2'-deoxyadenosine (MdA) is the major dA methylation product in duplex DNA. MdA blocks DNA replication and undergoes depurination at significantly higher rates than the native nucleotide from which it is derived. Recent reports on the effects of the nucleosome core particle (NCP) environment on the reactivity of N7-methyl-2'-deoxyguanosine (MdG) inspired this investigation concerning the reactivity of MdA in NCPs. NCPs containing MdA at selected positions were produced using a strategy in which the minor groove binding Me-Lex molecule serves as a sequence specific methylating agent. Hydrolysis of the glycosidic bond in MdA to form abasic sites (AP) is suppressed in a NCP. Experiments using histone variants indicate that the proximal, highly basic N-terminal tails are partially responsible for the decreased depurination rate constant. MdA also forms cross-links with histone proteins. The levels of MdA-histone DNA-protein cross-links (DPC) decrease significantly over time and are replaced by those involving AP. The time dependent decrease in DPC is attributed to the reversibility of their formation and the relatively rapid rate of AP formation from MdA. Overall, MdA reactivity in NCPs qualitatively resembles that of MdG.
Topics: DNA; DNA-Binding Proteins; Deoxyadenosines; Nucleic Acid Conformation; Nucleosomes
PubMed: 31565933
DOI: 10.1021/acs.chemrestox.9b00299 -
Journal of the American Chemical Society Sep 2020Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis but suffers from...
Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis but suffers from scalability limitations and high reagent consumption. Liquid-phase oligonucleotide synthesis (LPOS) uses soluble polymer supports and has the potential of being scalable. However, at present, LPOS requires 3 separate reaction steps and 4-5 precipitation steps per nucleotide addition. Moreover, long acid exposure times during the deprotection step degrade sequences with high A content (adenine) due to depurination and chain cleavage. In this work, we present the first one-pot liquid-phase DNA synthesis technique which allows the addition of one nucleotide in a one-pot reaction of sequential coupling, oxidation, and deprotection followed by a single precipitation step. Furthermore, we demonstrate how to suppress depurination during the addition of adenine nucleotides. We showcase the potential of this technique to prepare high-purity 4-arm PEG-T (T = thymine) and 4-arm PEG-A building blocks in multigram scale. Such complementary 4-arm PEG-DNA building blocks reversibly self-assemble into supramolecular model network hydrogels and facilitate the elucidation of bond lifetimes. These model network hydrogels exhibit new levels of mechanical properties (storage modulus, bond lifetimes) in DNA bonds at room temperature (melting at 44 °C) and thus open up pathways to next-generation DNA materials programmable through sequence recognition and available for macroscale applications.
Topics: DNA; Hydrogels; Models, Molecular; Molecular Structure; Polyethylene Glycols
PubMed: 32902960
DOI: 10.1021/jacs.0c05488 -
Chemistry (Weinheim An Der Bergstrasse,... Jun 2022Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved...
Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with G and G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.
Topics: Nucleosides; Plant Proteins; RNA; RNA, Ribosomal; Ribosome Inactivating Proteins; Ribosomes
PubMed: 35390188
DOI: 10.1002/chem.202200994 -
The Analyst Oct 2019Depurination occurs via hydrolysis of the purine-deoxyribose glycosyl bond and causes nucleic acid damage. In particular, the DNA sequences that can undergo a...
Depurination occurs via hydrolysis of the purine-deoxyribose glycosyl bond and causes nucleic acid damage. In particular, the DNA sequences that can undergo a self-catalyzed depurination (SCD) will cause a great uncertainty in duplicating, separating, purifying, and storing the DNA samples. Therefore, there is a great demand to develop a rapid detection method for SCD events. Herein, the use of a convenient fluorescence method to follow the site-specific SCD was demonstrated. We found that the resultant apurine site (AP site) from depurination can be selectively recognized by a fluorescent probe of palmatine (PAL) with a turn-on fluorescence response. The dependence of SCD on the bases of the depurination site, pH, metal ions, and time shows that our method can be used to rapidly evaluate the depurination process. Furthermore, the depurination process can be photo-switched using a photoacid as an external initiator. Our work will find wide applications in preliminarily identifying the DNA depurination.
Topics: Berberine Alkaloids; Catalysis; DNA Glycosylases; DNA, Catalytic; Fluorescent Dyes; Molecular Structure
PubMed: 31482933
DOI: 10.1039/c9an01412h -
Toxins Aug 2022Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new...
Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of L. Sodins are able to release Endo's fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC = 4.83-79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC = 0.41-1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against .
Topics: Amino Acid Sequence; Animals; HeLa Cells; Humans; N-Glycosyl Hydrolases; Plant Proteins; Rabbits; Ribosome Inactivating Proteins; Ribosome Inactivating Proteins, Type 1; Ribosomes; Salsola
PubMed: 36006228
DOI: 10.3390/toxins14080566 -
International Journal of Molecular... Jul 2021This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the... (Review)
Review
This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the form of catechol estrogen-3,4-quinones, play a central role in this pathway of cancer initiation. The catechol estrogen-3,4-quinones react with specific purine bases in DNA to form depurinating estrogen-DNA adducts that generate apurinic sites. The apurinic sites can then lead to cancer-causing mutations. The process of cancer initiation has been demonstrated using results from test tube reactions, cultured mammalian cells, and human subjects. Increased amounts of estrogen-DNA adducts are found not only in people with several different types of cancer but also in women at high risk for breast cancer, indicating that the formation of adducts is on the pathway to cancer initiation. Two compounds, resveratrol, and -acetylcysteine, are particularly good at preventing the formation of estrogen-DNA adducts in humans and are, thus, potential cancer-prevention compounds.
Topics: Acetylcysteine; Animals; Antioxidants; Carcinogenesis; DNA Adducts; Estradiol; Estrogens; Estrone; Humans; Quinones; Resveratrol
PubMed: 34361004
DOI: 10.3390/ijms22158238 -
Chemistry (Weinheim An Der Bergstrasse,... May 2020MicroRNAs (miRNAs) modulate the expression of over 30 % of mammalian genes during development and apoptosis, and abnormal expression of miRNAs may lead to a range of...
MicroRNAs (miRNAs) modulate the expression of over 30 % of mammalian genes during development and apoptosis, and abnormal expression of miRNAs may lead to a range of human pathologies. Therefore, analysis of miRNAs is valuable for disease diagnostics. In this work, a novel one-pot fluorescence derivatization strategy was developed for miRNA analysis. The mechanism of the derivatization reaction was explored by using instrumental methods, including liquid chromatography, fluorescence spectroscopy, and mass spectrometry. Highly fluorescent N -ethenoadenine (ϵ-adenine) was formed and detached from the miRNA sequence through the reaction of adenine in nucleic acids with 2-chloroacetaldehyde (CAA) at 100 °C. This is the first experimental evidence that the cooperation of formed ϵ-adenine and water-mediated hydrogen-bond interaction between the proton at the 2'- and the oxyanion at 3'-positions stabilized the oxocarbenium significantly, which makes the depurination and derivatization of miRNA highly effective. Based on this derivatization strategy, a facile and sensitive high-performance liquid chromatography method was developed for quantitative assay of miRNAs. In combination with magnetic solid-phase extraction (MSPE), the HPLC method was shown to be useful for the determination of microRNAs at sub-picomolar level in serum samples.
Topics: Acetaldehyde; Adenine; Chromatography, High Pressure Liquid; Fluorescence; Humans; Mass Spectrometry; MicroRNAs; Spectrometry, Fluorescence; Water
PubMed: 31953882
DOI: 10.1002/chem.201905639