Photophysics and X-ray structure of crystalline 2-aminopurine.

To explore the effect of intermolecular interactions on the photophysics of 2-aminopurine (2AP) in a well-defined environment, we have investigated the fluorescence properties of single 2AP crystals, having determined their X-ray structure. In the crystal, 2AP is subject to base-stacking and hydrogen-bonding interactions similar to those found in DNA. The crystal shows dual fluorescence: pi-stacked molecules in the bulk of the lattice have redshifted excitation and emission spectra, while molecules at defect sites have spectra similar to those of 2AP in solution or in DNA. Heterogeneous intermolecular interactions in the crystal give rise to multiexponential fluorescence decay characteristics similar to those observed for 2AP-labelled DNA. The presence of about 13 % of the 7H tautomer in the crystal confirms that 9H-7H tautomerisation of 2AP occurs in the ground state. Long-wavelength excitation of a 2AP-labelled oligonucleotide duplex produced redshifted emission similar to that observed in the crystal, indicating that pi-stacking interaction of 2AP with nucleobases gives rise to a low energy excited state.     

Single‐Molecule Fluorescence Detection of a Synthetic Heparan Sulfate Disaccharide

The first single‐molecule fluorescence detection of a structurally‐defined synthetic carbohydrate is reported: a heparan sulfate (HS) disaccharide fragment labeled with Alexa488. Single molecules have been measured whilst freely diffusing in solution and controlled encapsulation in surface‐tethered lipid vesicles has allowed extended observations of carbohydrate molecules down to the single‐molecule level. The diverse and dynamic nature of HS–protein interactions means that new tools to investigate pure HS fragments at the molecular level would significantly enhance our understanding of HS. This work is a proof‐of‐principle demonstration of the feasibility of single‐molecule studies of synthetic carbohydrates which offers a new approach to the study of pure glycosaminoglycan (GAG) fragments.     

Dual triggering of DNA binding and fluorescence via photoactivation of a dinuclear ruthenium(II) arene complex

The dinuclear RuII arene complexes [{(eta6-arene)RuCl}2(mu-2,3-dpp)](PF6)2, arene=indan (1), benzene (2), p-cymene (3), or hexamethylbenzene (4) and 2,3-dpp=2,3-bis(2-pyridyl)pyrazine, have been synthesized and characterized. Upon irradiation with UVA light, complexes 1 and 2 readily underwent arene loss, while complexes 3 and 4 did not. The photochemistry of 1 was studied in detail. In the X-ray structure of [{(eta6-indan)RuCl}2(mu-2,3-dpp)](PF6)2 (1), 2,3-dpp bridges two RuII centers 6.8529(6) A apart. In water, aquation of 1 in the dark occurs with replacement of chloride with biexponential kinetics and decay constants of 100+/-1 min-1 and 580+/-11 min-1. This aquation was suppressed by 0.1 M NaCl. UV or visible irradiation of 1 in aqueous or methanolic solution led to arene loss. The fluorescence of the unbound arene is approximately 40 times greater than when it is complexed. Irradiation of 1 also had a significant effect on its interactions with DNA. The DNA binding of 1 is increased after irradiation. The non-irradiated form of 1 preferentially formed DNA adducts that only weakly blocked RNA polymerase, while irradiation of 1 transformed the adducts into stronger blocks for RNA polymerase. The efficiency of irradiated 1 to form DNA interstrand cross-links was slightly greater than that of cisplatin in both 10 mM NaClO4 and 0.1 M NaCl. In contrast, the interstrand cross-linking efficiency of non-irradiated 1 in 10 mM NaClO4 was relatively low. An intermediate amount of cross-linking was observed when the sample of DNA already modified by non-irradiated 1 was irradiated. DNA unwinding measurements supported the conclusion that both mono- and bifunctional adducts with DNA can form. These results show that photoactivation of dinuclear RuII arene complexes can simultaneously produce a highly reactive ruthenium species that can bind to DNA and a fluorescent marker (the free arene). Importantly, the mechanism of photoreactivity is also independent of oxygen. These complexes, therefore, have the potential to combine both photoinduced cell death and fluorescence imaging of the location and efficiency of the photoactivation process.     

Single-molecule fluorescence detection of a tricyclic nucleoside analogue

Fluorescent nucleobase surrogates capable of Watson–Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push–pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits ε₄₄₂ = 20 000 M⁻¹ cm⁻¹ and Φ_em,540 = 0.39 in water, increasing to Φ_em = 0.50–0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.

Fluorescent nucleoside analogue ABN is readily detected at the single-molecule level and retains a quantum yield >50% in duplex DNA oligonucleotides.     

Global structure of forked DNA in solution revealed by high-resolution single-molecule FRET

Branched DNA structures play critical roles in DNA replication, repair, and recombination in addition to being key building blocks for DNA nanotechnology. Here we combine single-molecule multiparameter fluorescence detection and molecular dynamics simulations to give a general approach to global structure determination of branched DNA in solution. We reveal an open, planar structure of a forked DNA molecule with three duplex arms and demonstrate an ion-induced conformational change. This structure will serve as a benchmark for DNA-protein interaction studies.     

Branchpoint expansion in a fully complementary three-way DNA junction

Branched nucleic acid molecules serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resolution single-molecule FRET, time-resolved spectroscopy, and molecular modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in solution. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the molecule. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramolecular receptors and nanosystems.     

Highly luminescent, triple- and quadruple-stranded, dinuclear Eu, Nd, and Sm(III) lanthanide complexes based on bis-diketonate ligands

The bis(beta-diketone) ligands 1,3-bis(3-phenyl-3-oxopropanoyl)benzene, H(2)L(1) and 1,3-bis(3-phenyl-3-oxopropanoyl) 5-ethoxy-benzene, H(2)L(2), have been prepared for the examination of dinuclear lanthanide complex formation and investigation of their properties as sensitizers for lanthanide luminescence. The ligands bear two conjugated diketonate binding sites linked by a 1,3-phenylene spacer. The ligands bind to lanthanide(III) or yttrium(III) ions to form neutral homodimetallic triple stranded complexes [M(2)L(1)(3)] where M = Eu, Nd, Sm, Y, Gd and [M(2)L(2)(3)], where M = Eu, Nd or anionic quadruple-stranded dinuclear lanthanide units, [Eu(2)L(1)(4)](2-). The crystal structure of the free ligand H(2)L(1) has been determined and shows a twisted arrangement of the two binding sites around the 1,3-phenylene spacer. The dinuclear complexes have been isolated and fully characterized. Detailed NMR investigations of the complexes confirm the formation of a single complex species, with high symmetry; the complexes show clear proton patterns with chemical shifts of a wide range due to the lanthanide paramagnetism. Addition of Pirkle's reagent to solutions of the complexes leads to splitting of the peaks, confirming the chiral nature of the complexes. Electrospray and MALDI mass spectrometry have been used to identify complex formulation and characteristic isotope patterns for the different lanthanide complexes have been obtained. The complexes have high molar absorption coefficients (around 13 x 10(4) M(-1)cm(-1)) and display strong visible (red or pink) or NIR luminescence upon irradiation at the ligand band around 350 nm, depending on the choice of the lanthanide. Emission quantum yield experiments have been performed and the luminescence signals of the dinuclear complexes have been found to be up to 11 times more intense than the luminescence signals of the mononuclear analogues. The emission quantum yields and the luminescence lifetimes are determined to be 5% and 220 micros for [Eu(2)L(1)(3)], 0.16% and 13 micros for [Sm(2)L(1)(3)], and 0.6% and 1.5 micros for [Nd(2)L(1)(3)]. The energy level of the ligand triplet state was determined from the 77 K spectrum of [Gd(2)L(1)(3)]. The bis-diketonate ligand is shown to be an efficient sensitizer, particularly for Sm and Nd. Photophysical studies of the europium complexes at room temperature and 77 K show the presence of a thermally activated deactivation pathway, which we attribute to ligand-to-metal charge transfer (LMCT). Quenching of the luminescence from this level seems to be operational for the Eu(III) complex but not for complexes of Sm(III) and Nd(III), which exhibit long lifetimes. The quadruple-stranded europium complex has been isolated and characterized as the piperidinium salt of [Eu(2)L(1)(4)](2-). Compared with the triple-stranded Eu(III) complex in the solid state, the quadruple-stranded complex displays a more intense emission signal with a distinct emission pattern indicating the higher symmetry of the quadruple-stranded complex.     

Two-photon luminescence from polar bis-terpyridyl-stilbene derivatives of Ir(III) and Ru(II)

Four structurally related iridium(III) and ruthenium(II) complexes bearing two polar terpyridyl-stilbene derived chromophores 4-(4-{2-[4-(methoxy)phenyl]ethenyl}phenyl)-2,2'-6',2'-terpyridine (ttpyeneanisole) and 4-(4-{2-[phenyl]ethenyl}phenyl)-2,2'-6',2'-terpyridine (tpystilbene) have been synthesised and characterised in the solid state and in solution. In the solid state, the dihedral angle subtending the pyridyl and tolyl groups of 27.1° in the Ir(III) complex [Ir(ttpyeneanisole)(2)]·3PF(6) is more acute than in the Ru(II) derivative [Ru(tpystilbene)(2)]·2PF(6) (35.5°), indicating the presence of a greater degree of π-delocalisation across the terpyridine unit in the former compound. Their luminescence properties in fluid solution have been investigated following both resonant and non-resonant excitation. We have shown that each of the complexes undergoes two-photon excitation when excited in the near infrared (740 to 820 nm), with two-photon absorption cross sections in the range 11-67 × 10(-50) cm(4) s photon(-1). The larger cross sections for the Ir(III) complexes reflect the differences observed in the solid state. This work therefore demonstrates that such complexes are promising as luminescent markers for 3D imaging and illustrates that simple functionalisation of the chromophores and the choice of metal can lead to marked enhancements in the two-photon cross sections (σ(2)) compared to those of simpler heteroleptic polypyridyl based derivatives.     

Metallation-Induced Heterogeneous Dynamics of DNA Revealed by Single-Molecule FRET

The metallation of nucleic acids is key to wide-ranging applications, from anticancer medicine to nanomaterials, yet there is a lack of understanding of the molecular-level effects of metallation. Here, we apply single-molecule fluorescence methods to study the reaction of an organo-osmium anticancer complex and DNA. Individual metallated DNA hairpins are characterised using Förster resonance energy transfer (FRET). Although ensemble measurements suggest a simple two-state system, single-molecule experiments reveal an underlying heterogeneity in the oligonucleotide dynamics, attributable to different degrees of metallation of the GC-rich hairpin stem. Metallated hairpins display fast two-state transitions with a two-fold increase in the opening rate to ≈2 s⁻¹, relative to the unmodified hairpin, and relatively static conformations with long-lived open (and closed) states of 5 to ≥50 s. These studies show that a single-molecule approach can provide new insight into metallation-induced changes in DNA structure and dynamics.