Fluorescent molecular rotors detect O6-methylguanine dynamics and repair in duplex DNA.

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 ∼9 Šof 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.

PARP-1 improves leukemia outcomes by inducing parthanatos during chemotherapy.

Previous chemotherapy research has focused almost exclusively on apoptosis. Here, a standard frontline drug combination of cytarabine and idarubicin induces distinct features of caspase-independent, poly(ADP-ribose) polymerase 1 (PARP-1)-mediated programmed cell death "parthanatos" in acute myeloid leukemia (AML) cell lines (n = 3/10 tested), peripheral blood mononuclear cells from healthy human donors (n = 10/10 tested), and primary cell samples from patients with AML (n = 18/39 tested, French-American-British subtypes M4 and M5). A 3-fold improvement in survival rates is observed in the parthanatos-positive versus -negative patient groups (hazard ratio [HR] = 0.28-0.37, p = 0.002-0.046). Manipulation of PARP-1 activity in parthanatos-competent cells reveals higher drug sensitivity in cells that have basal PARP-1 levels as compared with those subjected to PARP-1 overexpression or suppression. The same trends are observed in RNA expression databases and support the conclusion that PARP-1 can have optimal levels for favorable chemotherapeutic responses.

DNA templated Click Chemistry via 5-vinyl-2'-deoxyuridine and an acridine-tetrazine conjugate induces DNA damage and apoptosis in cancer cells.

AIMS: Click Chemistry is providing valuable tools to biomedical research, but its direct use in therapies remains nearly unexplored. For cancer treatment, nucleoside analogues (NA) such as 5-vinyl-2'-deoxyuridine (VdU) can be metabolically incorporated into cancer cell DNA and subsequently "clicked" to form a toxic product. The inverse electron-demand Diels-Alder (IEDDA) reaction between VdU and an acridine-tetrazine conjugate (PINK) has previously been used to label cell nuclei of cultured cells. Here, we report tandem usage of VdU and PINK to induce cytotoxicity. MAIN METHODS: Cell lines were subsequently treated with VdU and PINK, and cell viability was measured via well confluency and 3D tumor spheroid assays. DNA damage and apoptosis were evaluated using Western Blotting and cell cycle analysis by flow cytometry. Double stranded DNA break (DSB) formation was measured using the comet assay. Apoptosis was assessed by fluorescent detection of externalized phosphatidylserine residues. KEY FINDINGS: We report that the combination of VdU and PINK synergistically induces cytotoxicity in cultured human cells. The combination of VdU and PINK strongly reduced cell viability in 2D and 3D cultured cancer cells. Mechanistically, the compounds induced DNA damage through DSB formation, which leads to S-phase accumulation and apoptosis. SIGNIFICANCE: The combination of VdU and PINK represents a novel and promising DNA-templated "click" approach for cancer treatment via selective induction of DNA damage.

Formal [4 + 2] Cycloadditions of Maleimides on Duplex DNA.

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.

A Bright and Ionizable Cytosine Mimic for i-Motif Structures.

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 → 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.

[4 + 2] and [2 + 4] cycloaddition reactions on single- and double-stranded DNA: a dual-reactive nucleoside.

Here we report dual reactivity of diene-modified duplex DNA containing 5-(1,3-butadienyl)-2'-deoxyuridine "BDdU". Normal-electron demand [4 + 2] cycloaddition proceeded upon addition of a maleimide, whereas inverse-electron demand [2 + 4] cycloaddition occurred upon addition of a tetrazine to give a novel, photoswitchable product.

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo.

Modified nucleoside triphosphates (NTPs) are powerful probes and medicines, but their anionic character impedes membrane permeability. As such, invasive delivery techniques, transport carriers, or prodrug strategies are required for their in vivo use. Here, we present a fluorescent 2'-deoxyribonucleoside triphosphate "TAMRA-dATP" that exhibits surprisingly high bioavailability in vivo. TAMRA-dATP spontaneously forms nanoparticles in Mg+2-containing buffers that are taken into the vesicles of living cells and animals by energy-dependent processes. In cell cultures, photochemical activation with yellow laser light (561 nm) facilitated endosomal escape of TAMRA-dATP, resulting in its metabolic incorporation into DNA in vitro. In contrast, in vivo studies revealed that TAMRA-dATP is extensively trafficked by active pathways into cellular DNA of zebrafish (Danio rerio) and Caenorhabditis elegans where DNA labeling was observed in live animals, even without photochemical release. Metabolic labeling of DNA in whole, living animals can therefore be achieved by simply soaking animals in a buffer containing TAMRA-dATP or a structurally related compound, Cy3-dATP.

A Kinetic and Fluorogenic Enhancement Strategy for Labeling of Nucleic Acids.

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±1 µM) increases the reaction rate of tetrazine-alkene IEDDA on duplex DNA by 60 000-fold (590 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.

DNA template strand segregation in developing zebrafish.

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light sheet microscopy to track embryonic DNA in whole zebrafish. Larval development results in rapid depletion of older DNA template strands from stem cell niches in the retina, brain, and intestine. Prolonged label retention occurs in quiescent progenitors that resume replication in later development. High-resolution microscopy reveals no evidence of asymmetric template strand segregation in >100 daughter cell pairs, making it improbable that asymmetric DNA segregation prevents mutational burden according to the immortal strand hypothesis in developing zebrafish.

RNA Targeting in Acute Myeloid Leukemia.

Nucleosides and their analogues constitute an essential family of anticancer drugs. DNA has been the presumptive target of the front-line prodrug for acute myeloid leukemia (AML), cytarabine (ara-C), since the 1980s. Here, the biomolecular targeting of ara-C was evaluated in primary white blood cells using the ara-C mimic "AzC" and azide-alkyne "click" reactions. Fluorescent staining and microscopy revealed that metabolic incorporation of AzC into primary white blood cells was unexpectedly enhanced by the DNA polymerase inhibitor aphidicholine. According to RNaseH digestion and pull-down-and-release experiments, AzC was incorporated into short RNA fragments bound to DNA in peripheral blood monocytes (PBMCs) collected from all six healthy human donors tested. Samples from 22 AML patients (French-American-British classes M4 and M5) exhibited much more heterogeneity, with 27% incorporating AzC into RNA and 55% into DNA. The overall survival of AML patients whose samples incorporated AzC into RNA was approximately 3-fold higher as compared to that of the DNA cohort (p ≤ 0.056, χ2 = 3.65). These results suggest that the RNA primers of DNA synthesis are clinically favorable targets of ara-C, and that variable incorporation of nucleoside drugs into DNA versus RNA may enable future patient stratification into treatment-specific subgroups.

Fluorescent dATP for DNA Synthesis In Vivo.

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (Caenorhabditis elegans). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo.

A Highly Fluorescent Nucleobase Molecular Rotor.

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 = 3400-29 700 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.

Cross-linking cellular nucleic acids via a target-directing double click reagent.

Bioorthogonal ligation reactions are powerful tools for characterizing DNA metabolism, DNA-protein binding interactions, and they even provide new leads for therapeutic strategies. Nucleoside analogs can deliver bioorthogonal functional groups into chromatin via cellular metabolic pathways, however, insufficient phosphorylation by endogenous kinases often limits the efficiency of their incorporation. Even when successfully metabolized into biopolymers, steric hindrance of the modified nucleotide by chromatin can inhibit subsequent click reactions. In this chapter, we describe methods that overcome these limitations. Nucleotide monophosphate triesterers can bypass the need for cellular nucleoside kinase activity and thereby enable efficient incorporation of azide groups into cellular DNA. Steric access to and modification of the azide groups within natively folded chromatin can then be accomplished using a bioorthogonal "intercalating reagent" comprised of a cationic Sondheimer diyne that reversibly intercalates into duplexes where it undergoes tandem, strain-promoted cross-linking of two azides to give DNA-DNA interstrand crosslinks or DNA-fluorophore conjugation, depending on the relative number and spatial orientation of the azide groups in the DNA.

Three-Component Bioorthogonal Reactions on Cellular DNA and RNA.

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.

Concerted dynamics of metallo-base pairs in an A/B-form helical transition.

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-HgII-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-HgII-(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-HgII-(N4)C base pairs and greater A-form characteristics. Ten-fold different 1J coupling constants (15N,199Hg) are observed for (N3)C-HgII (114 Hz) versus (N4)C-HgII (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-HgII-T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding HgII, demonstrating that C-HgII-T has unique switching potential in DNA-based materials and devices.

Enzymatic Incorporation of a Coumarin-Guanine Base Pair.

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 polymerase 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.

Intercalation-enhanced "Click" Crosslinking of DNA.

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 =15 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.

In Vivo Incorporation of Azide Groups into DNA by Using Membrane-Permeable Nucleotide Triesters.

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.

HgII binds to C-T mismatches with high affinity.

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.

Superresolution imaging of individual replication forks reveals unexpected prodrug resistance mechanism.

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.