-
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 -
Current Topics in Microbiology and... 2012Ricin and Shiga toxins designated as ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine (A₄₃₂₄ in rat 28S rRNA) in... (Review)
Review
Ricin and Shiga toxins designated as ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine (A₄₃₂₄ in rat 28S rRNA) in the conserved α-sarcin/ricin loop of the large rRNA, inhibiting protein synthesis. Evidence obtained from a number of studies suggests that interaction with ribosomal proteins plays an important role in the catalytic activity and ribosome specificity of RIPs. This review summarizes the recent developments in identification of the ribosomal proteins that interact with ricin and Shiga toxins and the principles governing these interactions.
Topics: Enzyme Activation; Protein Binding; Ribosomal Proteins; Ribosome Inactivating Proteins; Ribosomes; Ricin; Shiga Toxins
PubMed: 21910078
DOI: 10.1007/82_2011_174 -
Clinical and Translational Medicine Mar 2016Estrogens can initiate cancer by reacting with DNA. Specific metabolites of endogenous estrogens, the catechol estrogen-3,4-quinones, react with DNA to form depurinating... (Review)
Review
Estrogens can initiate cancer by reacting with DNA. Specific metabolites of endogenous estrogens, the catechol estrogen-3,4-quinones, react with DNA to form depurinating estrogen-DNA adducts. Loss of these adducts leaves apurinic sites in the DNA, generating mutations that can lead to the initiation of cancer. A variety of endogenous and exogenous factors can disrupt estrogen homeostasis, which is the normal balance between estrogen activating and protective enzymes. In fact, if estrogen metabolism becomes unbalanced and generates excessive catechol estrogen 3,4-quinones, formation of depurinating estrogen-DNA adducts increases and the risk of initiating cancer is greater. The levels of depurinating estrogen-DNA adducts are high in women diagnosed with breast cancer and those at high risk for the disease. High levels of depurinating estrogen-DNA adducts before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Women with thyroid or ovarian cancer also have high levels of estrogen-DNA adducts, as do men with prostate cancer or non-Hodgkin lymphoma. Depurinating estrogen-DNA adducts are initiators of many prevalent types of human cancer. These findings and other discoveries led to the recognition that reducing the levels of estrogen-DNA adducts could prevent the initiation of human cancer. The dietary supplements N-acetylcysteine and resveratrol inhibit formation of estrogen-DNA adducts in cultured human breast cells and in women. These results suggest that the two supplements offer an approach to reducing the risk of developing various prevalent types of human cancer. Graphical abstract Major metabolic pathway in cancer initiation by estrogens.
PubMed: 26979321
DOI: 10.1186/s40169-016-0088-3 -
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 -
Biochemistry Oct 2022Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those...
Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, 7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.
Topics: Chromatin; DNA; Deoxyguanosine; Fluorescent Dyes; Histones; Nucleosomes; Osmolar Concentration
PubMed: 36136907
DOI: 10.1021/acs.biochem.2c00342 -
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 -
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 -
OncoTargets and Therapy 2019Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with... (Review)
Review
Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with development of breast cancer. Both estrogen receptor-dependent and independent mechanisms can contribute to the carcinogenic effects of estrogens. Among them, the oxidative metabolism of estrogens plays a key role in the initiation of estradiol-induced breast cancer by generation of reactive estrogen quinones as well as the associated formation of oxygen free radicals. These genotoxic metabolites can react with DNA to form unstable DNA adducts which generate mutations leading to the initiation of breast cancer. A variety of endogenous and exogenous factors can alter estrogen homeostasis and generate genotoxic metabolites. The use of specific phytochemicals and dietary supplements can inhibit the risk of breast cancer not only by the modulation of several estrogen-activating enzymes (CYP19, CYP1B1) but also through the induction of various cytoprotective enzymes (eg, SOD3, NQO1, glutathione S-transferases, OGG-1, catechol--methyltransferases, CYP1B1A, etc.) that reestablish the homeostatic balance of estrogen metabolism via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent and independent mechanisms.
PubMed: 31388303
DOI: 10.2147/OTT.S183192