RESUMO
Near-quantitative DNA bioconjugation and detailed mechanistic investigations of reactions involving 5-(vinyl)-2'-deoxyuridine (VdU) and maleimides are reported. According to accelerated reaction rates in solvents with increasing polarity and trends in product stereochemistry, VdU-maleimide reactions proceed via a formal [4 + 2] stepwise cycloaddition. In contrast, 5-(1,3-butadienyl)-2'-deoxyuridine (BDdU) reacts with maleimides in a concerted [4 + 2] Diels-Alder cycloaddition. VdU-maleimide reactions enable high-yielding bioconjugation of duplex DNA in vitro (>90%) as well as metabolic labeling experiments in cells.
Assuntos
DNA , Desoxiuridina , Reação de Cicloadição , MaleimidasRESUMO
A new fluorescent cytosine analog "tsC" containing a trans-stilbene moiety was synthesized and incorporated into hemiprotonated base pairs that comprise i-motif structures. Unlike previously reported fluorescent base analogs, tsC mimics the acid-base properties of cytosine (pKa ≈ 4.3) while exhibiting bright (ε × Φ ≈ 1000 cm-1 M-1) and red-shifted fluorescence (λem = 440 â 490 nm) upon its protonation in the water-excluded interface of tsC+:C base pairs. Ratiometric analyses of tsC emission wavelengths facilitate real-time tracking of reversible conversions between single-stranded, double-stranded, and i-motif structures derived from the human telomeric repeat sequence. Comparisons between local changes in tsC protonation with global structure changes according to circular dichroism suggest partial formation of hemiprotonated base pairs in the absence of global i-motif structures at pH = 6.0. In addition to providing a highly fluorescent and ionizable cytosine analog, these results suggest that hemiprotonated C+:C base pairs can form in partially folded single-stranded DNA in the absence of global i-motif structures.
Assuntos
Citosina , Humanos , Concentração de Íons de Hidrogênio , Citosina/química , Sequência de Bases , Pareamento de Bases , Dicroísmo Circular , Conformação de Ácido NucleicoRESUMO
Chemical modification of nucleic acids in living cells can be sterically hindered by tight packing of bioorthogonal functional groups in chromatin. To address this limitation, we report here a dual enhancement strategy for nucleic acid-templated reactions utilizing a fluorogenic intercalating agent capable of undergoing inverse electron-demand Diels-Alder (IEDDA) reactions with DNA containing 5-vinyl-2'-deoxyuridine (VdU) or RNA containing 5-vinyl-uridine (VU). Reversible high-affinity intercalation of a novel acridine-tetrazine conjugate "PINK" (KD =5±1â µM) increases the reaction rate of tetrazine-alkene IEDDA on duplex DNA by 60 000-fold (590â M-1 s-1 ) as compared to the non-templated reaction. At the same time, loss of tetrazine-acridine fluorescence quenching renders the reaction highly fluorogenic and detectable under no-wash conditions. This strategy enables live-cell dynamic imaging of acridine-modified nucleic acids in dividing cells.
Assuntos
Compostos Heterocíclicos , Ácidos Nucleicos , Acridinas , Reação de Cicloadição , DNARESUMO
Many drugs require extensive metabolism en route to their targets. High-resolution visualization of prodrug metabolism should therefore utilize analogs containing a small modification that does not interfere with its metabolism or mode of action. In addition to serving as mechanistic probes, such analogs provide candidates for theranostics when applied in both therapeutic and diagnostic modalities. Here a traceable mimic of the widely used anticancer prodrug cytarabine (ara-C) was generated by converting a single hydroxyl group to azide, giving "AzC." This compound exhibited the same biological profile as ara-C in cell cultures and zebrafish larvae. Using azide-alkyne "click" reactions, we uncovered an apparent contradiction: drug-resistant cells incorporated relatively large quantities of AzC into their genomes and entered S-phase arrest, whereas drug-sensitive cells incorporated only small quantities of AzC. Fluorescence microscopy was used to elucidate structural features associated with drug resistance by characterizing the architectures of stalled DNA replication foci containing AzC, EdU, γH2AX, and proliferating cell nuclear antigen (PCNA). Three-color superresolution imaging revealed replication foci containing one, two, or three partially resolved replication forks. Upon removing AzC from the media, resumption of DNA synthesis and completion of the cell cycle occurred before complete removal of AzC from genomes in vitro and in vivo. These results revealed an important mechanism for the low toxicity of ara-C toward normal tissues and drug-resistant cancer cells, where its efficient incorporation into DNA gives rise to highly stable, stalled replication forks that limit further incorporation of the drug, yet allow for the resumption of DNA synthesis and cellular division following treatment.
Assuntos
Antineoplásicos/química , Azidas/química , Citarabina/química , Replicação do DNA , DNA/química , Pró-Fármacos/química , Uridina/análogos & derivados , Peixe-Zebra/crescimento & desenvolvimento , Animais , Antineoplásicos/farmacologia , Azidas/farmacologia , Ciclo Celular , Citarabina/farmacologia , Humanos , Pró-Fármacos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Uridina/química , Uridina/farmacologiaRESUMO
Fluorescent base analogs (FBAs) are powerful probes of nucleic acids' structures and dynamics. However, previously reported FBAs exhibit relatively low brightness and therefore limited sensitivity of detection. Here we report the hitherto brightest FBA that has ideal molecular rotor properties for detecting local dynamic motions associated with base pair mismatches. The new trans-stilbene annulated uracil derivative "tsT" exhibits bright fluorescence emissions in various solvents (ε × Φ = 3400-29â¯700 cm-1 M-1) and is highly sensitive to mechanical motions in duplex DNA (ε × Φ = 150-4250 cm-1 M-1). tsT is thereby a "smart" thymidine analog, exhibiting a 28-fold brighter fluorescence intensity when base paired with A as compared to T or C. Time-correlated single photon counting revealed that the fluorescence lifetime of tsT (τ = 4-11 ns) was shorter than its anisotropy decay in well-matched duplex DNA (θ = 20 ns), yet longer than the dynamic motions of base pair mismatches (0.1-10 ns). These properties enable unprecedented sensitivity in detecting local dynamics of nucleic acids.
Assuntos
DNA/análise , Corantes Fluorescentes/química , Uracila/química , Pareamento Incorreto de Bases , Teoria da Densidade Funcional , Fluorescência , Corantes Fluorescentes/síntese química , Conformação de Ácido Nucleico , Uracila/síntese químicaRESUMO
Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T-T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6-19°C), as compared to those containing C-T mismatches (ΔTm = 1-4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C-HgII-T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs - even in situations where metal binding has little or no impact on the thermal stability of the duplex.
Assuntos
Pareamento Incorreto de Bases , Citosina/química , Mercúrio/química , Timidina/química , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Prata/químicaRESUMO
Metabolic incorporation of bioorthogonal functional groups into chromatin, followed by copper-free conjugation reactions, often gives low yields due to steric hindrance. Here we report that a cationic Sondheimer diyne derivative "DiMOC" rapidly reacts with azide groups in duplex DNA that are otherwise unreactive toward aliphatic cyclooctynes such as bicyclo[6.1.0]nonyne (BCN). DiMOC reversibly intercalates into duplex DNA and undergoes tandem strain-promoted cross-linking of two different azide groups to give DNA-DiMOC-"X" cross-links, where "X" theoretically represents a fluorescent probe, affinity tag, RNA, protein, carbohydrate, and so forth. As a proof of principle, the metabolic incorporation of azide-modified nucleosides into cellular DNA or RNA, followed by treatment with DiMOC and a fluorescent azide enabled visualization of newly synthesized nucleic acids in whole cells.
Assuntos
Azidas/química , DNA/química , Di-Inos/química , Imagem Molecular/métodos , RNA/química , Reagentes de Ligações Cruzadas/química , Corantes Fluorescentes , Substâncias Intercalantes/químicaRESUMO
Previous expansions beyond nature's preferred base-pairing interactions have utilized either nonpolar shape-fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N-H with C-H at a Watson-Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3-glycosyl-5-fluoro-7-methoxy-coumarin-2'-deoxyribose (dCC ). dGTP is selectively incorporated across from a template dCC during enzymatic DNA synthesis. Likewise, dCC is selectively incorporated across from a template guanine when dCC is provided as the triphosphate dCC TP. DNA polymeraseâ I (Klenow fragment) exhibited about a 10-fold higher affinity for dCC TP than dCTP, allowing selective incorporation of dCC in direct competition experiments. These results demonstrate that a single C-H can replace N-H at a Watson-Crick-type interface with preservation of functional selectivity and enhanced activity.
Assuntos
Cumarínicos/química , DNA Polimerase I/metabolismo , DNA/química , Guanina/química , Pareamento de Bases , Catálise , DNA Polimerase I/química , Replicação do DNA , Humanos , Ligação de Hidrogênio , Modelos MolecularesRESUMO
Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl-2'-deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low-fidelity thymidine kinase, but not by wild-type HeLa cells. Here we report that membrane-permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild-type cells and animals. AmdU monophosphate derivatives bearing either 5'-bispivaloyloxymethyl (POM), 5'-bis-(4-acetoxybenzyl) (AB), or "Protide" protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative "POM-AmdU" exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than "AB-AmdU". Remarkably, the addition of POM-AmdU to the water of zebrafish larvae enabled the biosynthesis of azide-modified DNA throughout the body.
Assuntos
Azidas/química , DNA/química , Nucleotídeos/química , Zidovudina/análogos & derivados , Animais , Azidas/metabolismo , Permeabilidade da Membrana Celular , Química Click , DNA/metabolismo , Ésteres/química , Ésteres/metabolismo , Células HeLa , Humanos , Nucleotídeos/metabolismo , Peixe-Zebra , Zidovudina/química , Zidovudina/metabolismoRESUMO
DNA-DNA cross-linking agents constitute an important family of chemotherapeutics that non-specifically react with endogenous nucleophiles and therefore exhibit undesirable side effects. Here we report a cationic Sondheimer diyne derivative "DiMOC" that exhibits weak, reversible intercalation into duplex DNA (Kd =15â µm) where it undergoes tandem strain-promoted cross-linking of azide-containing DNA to give DNA-DNA interstrand crosslinks (ICLs) with an exceptionally high apparent rate constant kapp =2.1×105 m-1 s-1 . This represents a 21 000-fold rate enhancement as compared the reaction between DIMOC and 5-(azidomethyl)-2'-deoxyuridine (AmdU) nucleoside. As single agents, 5'-bispivaloyloxymethyl (POM)-AmdU and DiMOC exhibited low cytotoxicity, but highly toxic DNA-DNA ICLs were generated by metabolic incorporation of AmdU groups into cellular DNA, followed by treatment of the cells with DiMOC. These results provide the first examples of intercalation-enhanced bioorthogonal chemical reactions on DNA, and furthermore, the first strain-promoted double click (SPDC) reactions inside of living cells.
Assuntos
Azidas/química , Reagentes de Ligações Cruzadas/química , DNA/química , Desoxiuridina/análogos & derivados , Substâncias Intercalantes/química , Alcinos/química , Química Click , Reação de Cicloadição , Células HeLa , Humanos , Modelos MolecularesRESUMO
The cationic porphyrin 5,10,15,20-tetrakis (diisopropyl-guanidine)-21H,23H-porphine (DIGPor) selectively binds to DNA containing O6 -methylguanine (O6 -MeG) and inhibits the DNA repair enzyme O6 -methylguanine-DNA methyltransferase (MGMT). The O6 -MeG selectivity and MGMT inhibitory activity of DIGPor were improved by incorporating ZnII into the porphyrin. The resulting metal complex (Zn-DIGPor) potentiated the activity of the DNA-alkylating drug temozolomide in an MGMT-expressing cell line. To the best of our knowledge, this is the first example of DNA-targeted MGMT inhibition.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Metilases de Modificação do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/antagonistas & inibidores , DNA/metabolismo , Dacarbazina/análogos & derivados , Glioma/patologia , Guanina/análogos & derivados , Proteínas Supressoras de Tumor/antagonistas & inibidores , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/farmacologia , Glioma/tratamento farmacológico , Glioma/enzimologia , Glioma/genética , Guanina/metabolismo , Humanos , Temozolomida , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismoRESUMO
A new method for the post-synthetic modification of nucleic acids was developed that involves mixing a phenyl triazolinedione (PTAD) derivative with DNA containing a vinyl nucleobase. The resulting reactions proceeded through step-wise mechanisms, giving either a formal [4+2] cycloaddition product, or, depending on the context of nucleobase, PTAD addition along with solvent trapping to give a secondary alcohol in water. Catalyst-free addition between PTAD and the terminal alkene of 5-vinyl-2'-deoxyuridine (VdU) was exceptionally fast, with a second-order rate constant of 2×103 m-1 s-1 . PTAD derivatives selectively reacted with VdU-containing oligonucleotides in a conformation-selective manner, with higher yields observed for G-quadruplex versus duplex DNA. These results demonstrate a new strategy for copper-free bioconjugation of DNA that can potentially be used to probe nucleic acid conformations in cells.
Assuntos
DNA/química , Triazóis/química , Estrutura MolecularRESUMO
The thymidine analogue DMAT was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties of DMAT-A base pairs were highly sensitive to mercury binding reactions at T-T mismatches located at an adjacent site or one base pair away. This allowed for precise determination of the local kinetic and thermodynamic parameters of T-HgII-T binding reactions. The on- and off-rates of HgII were surprisingly slow, with association rate constants (kon) ≈ 104-105 M-1 s-1, and dissociation rate constants (koff) ≈ 10-4-10-3 s-1; giving equilibrium dissociation constants (Kd) = 8-50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific HgII binding affinities in the range of Kd = 0.2-2.0 µM, depending on the buffer conditions. The exceptionally high kinetic stabilities of T-HgII-T metallo-base pairs (half-lives = 0.3-1.3 h) perturbed dynamic processes including DNA strand displacement and primer extension by DNA polymerases that resulted in premature chain termination of DNA synthesis. In addition to providing the first detailed kinetic and thermodynamic parameters of site-specific T-HgII-T binding reactions in duplex DNA, these results demonstrate that T-HgII-T base pairs have a high potential to disrupt DNA metabolism in vivo.
Assuntos
DNA/química , Mercúrio/química , Timina/química , Pareamento de Bases , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
Ionized nucleobases participate in pairing interactions outside of Watson and Crick's rules. Base pairing and ionization can be coupled via global conformational changes to raise the apparent pKa of protonated nucleobases to values above physiological pH. To provide the first specific reporter of proton-coupled DNA folding, we developed a "push-pull" fluorescent nucleoside analog composed of dimethylaniline (DMA) fused to deoxycytidine. "(DMA)C" exhibits the same pKa and base pairing characteristics as native cytosine residues in the human telomeric repeat sequence, where it causes little or no perturbation of DNA structure or stability. Upon protonation of (DMA)C, enhanced charge transfer results in large red shifting (+40 nm) of its excitation/emission maxima. (DMA)C's fluorescence intensity, anisotropy, and energy transfer properties can be used to track conformational changes in real time. Strand displacement assays were conducted by mixing (DMA)C-labeled duplexes containing a 5' single-stranded overhang with an excess of unlabeled DNA to initiate thermodynamically favorable unfolding-refolding reactions that release the (DMA)C-labeled strand from its complement. Rate constants for strand displacement upon addition of i-motif DNA (k = 1.0 M(-1) s(-1), t1/2 ≈ 12 h) were 320-fold lower than those measured upon addition of unfolded DNA (k = 3.2 × 10(2) M(-1) s(-1), t1/2 ≈ 2 min). These results reveal that i-motif structures having only marginal thermodynamic stabilities (Tm < 40 °C) can still pose large kinetic barriers to duplex formation under near-physiological conditions of pH (5.75), temperature (25 °C), and salt (100 mM NaCl).
Assuntos
DNA/química , DNA/genética , Corantes Fluorescentes/química , Conformação de Ácido Nucleico , Prótons , Compostos de Anilina/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Motivos de Nucleotídeos , TermodinâmicaRESUMO
A powerful method for the synthesis of 2-oxazolines from silyl-protected ß-hydroxyamides is reported. Using diethylaminosulfur trifluoride (DAST) or its tetrafluoroborate salt (XtalFluor-E), silyl-protected ß-amidoalcohols can be in situ deprotected and dehydrated to give 2-oxazolines in good yields. The utility of this approach was demonstrated by preparing the first reported oligomer of [2,4']-coupled 2-oxazoline units. By tuning the stability of the silyl protecting groups (ex. IPDMS < TES < TBS, etc.), the deprotection rate can be optimized so that all reaction intermediates remain soluble, allowing cyclodehydration to occur at all potential sites of ring closure. N-Terminal Ser residues containing an Fmoc carbamate are converted into 2-(9'-fluorenylmethyloxy)-2-oxazoline in high yield, thereby providing a new pathway for the synthesis of peptides capped with an N-terminal 2-alkoxy-2-oxazoline or 2-oxazolidinone unit.
Assuntos
Amidas/química , Oxazóis/síntese química , Ciclização , Desidratação , Conformação Molecular , Oxazóis/químicaRESUMO
Pathogen-selective labeling was achieved by using the novel gemcitabine metabolite analogue 2'-deoxy-2',2'-difluoro-5-ethynyluridine (dF-EdU) and click chemistry. Cells infected with Herpes Simplex Virus-1 (HSV-1), but not uninfected cells, exhibit nuclear staining upon the addition of dF-EdU and a fluorescent azide. The incorporation of the dF-EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK). Crystallographic analyses revealed how dF-EdU is well accommodated in the active site of HSV-1 TK, but steric clashes prevent dF-EdU from binding human TK. These results provide the first example of pathogen-enzyme-dependent incorporation and labeling of bioorthogonal functional groups in human cells.
Assuntos
Azidas/química , Corantes Fluorescentes/química , Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Uridina/análogos & derivados , Animais , Azidas/metabolismo , Domínio Catalítico , Chlorocebus aethiops , Química Click , Corantes Fluorescentes/metabolismo , Halogenação , Células HeLa , Herpes Simples/virologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/metabolismo , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Coloração e Rotulagem , Timidina Quinase/análise , Timidina Quinase/metabolismo , Uridina/metabolismo , Células VeroRESUMO
Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) "click" reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5-azido-2'-deoxyuridine (AdU) exhibits a 4 h half-life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5-(azidomethyl)-2'-deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.
Assuntos
Azidas/química , DNA/metabolismo , Desoxiuridina/química , Alcinos/química , Azidas/metabolismo , Catálise , Cobre/química , Reação de Cicloadição , DNA/química , Replicação do DNA , Nucleotídeos de Desoxiuracil/metabolismo , Desoxiuridina/metabolismo , Corantes Fluorescentes/química , Células HeLa , HumanosRESUMO
Commonly used metabolic labels for DNA, including 5-ethynyl-2'-deoxyuridine (EdU) and BrdU, are toxic antimetabolites that cause DNA instability, necrosis, and cell-cycle arrest. In addition to perturbing biological function, these properties can prevent metabolic labeling studies where subsequent tissue survival is needed. To bypass the metabolic pathways responsible for toxicity, while maintaining the ability to be metabolically incorporated into DNA, we synthesized and evaluated a small family of arabinofuranosyl-ethynyluracil derivatives. Among these, (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) exhibited selective DNA labeling, yet had a minimal impact on genome function in diverse tissue types. Metabolic incorporation of F-ara-EdU into DNA was readily detectable using copper(I)-catalyzed azide-alkyne "click" reactions with fluorescent azides. F-ara-EdU is less toxic than both BrdU and EdU, and it can be detected with greater sensitivity in experiments where long-term cell survival and/or deep-tissue imaging are desired. In contrast to previously reported 2'-arabino modified nucleosides and EdU, F-ara-EdU causes little or no cellular arrest or DNA synthesis inhibition. F-ara-EdU is therefore ideally suited for pulse-chase experiments aimed at "birth dating" DNA in vivo. As a demonstration, Zebrafish embryos were microinjected with F-ara-EdU at the one-cell stage and chased by BrdU at 10 h after fertilization. Following 3 d of development, complex patterns of quiescent/senescent cells containing only F-ara-EdU were observed in larvae along the dorsal side of the notochord and epithelia. Arabinosyl nucleoside derivatives therefore provide unique and effective means to introduce bioorthogonal functional groups into DNA for diverse applications in basic research, biotechnology, and drug discovery.
Assuntos
DNA/química , Desoxiuridina/análogos & derivados , Nucleosídeos/química , Células 3T3 , Animais , Bromodesoxiuridina/química , Ciclo Celular , Proliferação de Células , Chlorocebus aethiops , Desoxiuridina/química , Biologia do Desenvolvimento/métodos , Relação Dose-Resposta a Droga , Fertilização , Corantes Fluorescentes/química , Células HeLa , Humanos , Camundongos , Modelos Químicos , Células Vero , Peixe-ZebraRESUMO
5-Vinyl-2'-deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels-Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5-ethynyl-2'-deoxyuridine (EdU). The VdU-tetrazine ligation reaction is rapid (k≈0.02 M(-1) s(-1)) and chemically orthogonal to the alkyne-azide "click" reaction of EdU-modified DNA. Alkene-tetrazine ligation reactions provide the first alternative to azide-alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time-resolved, multicolor labeling of DNA synthesis.
Assuntos
Alcenos/química , DNA/análise , DNA/química , Desoxiuridina/análogos & derivados , Tetrazóis/química , Química Click , DNA/síntese química , Desoxiuridina/química , Células HeLa , Humanos , Estrutura MolecularRESUMO
Alkylation at the O6 position of guanine is a common and highly mutagenic form of DNA damage. Direct repair of O6-alkylguanines by the "suicide" enzyme O6-methylguanine DNA methyltransferase (MGMT, AGT, AGAT) maintains genome stability and inhibits carcinogenesis. In this study, a fluorescent analogue of thymidine containing trans-stilbene (tsT) is quenched by O6-methylguanine residues in the opposite strand of DNA by molecular dynamics that propagate through the duplex with as much as â¼9 Å of separation. Increased fluorescence of tsT or the cytosine analogue tsC resulting from MGMT-mediated DNA repair were distinguishable from non-covalent DNA-protein binding following protease digest. To our knowledge, this is the first study utilizing molecular rotor base analogues to detect DNA damage and repair activities in duplex DNA.