Synthesis of Thiazoloindole α-Amino Acids: Chromophores Amenable to One- and Two-Photon Induced Fluorescence.

Thiazoloindole α-amino acids have been synthesized in four steps from tryptophan using a dual-catalytic thiolation reaction and a copper-mediated intramolecular N-arylation process. Late-stage diversification of the thiazoloindole core with electron-deficient aryl substituents produced chromophores that on one-photon excitation displayed blue-green emission, mega-Stokes shifts, and high quantum yields. The thiazoloindole amino acids could also be excited via two-photon absorption in the near-infrared, demonstrating their potential for biomedical imaging applications.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA.

Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of σ2PA(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Pulse-shaped broadband multiphoton excitation for single-molecule fluorescence detection in the far field.

Multiphoton excitation of fluorescence has many potential advantages over resonant (one-photon) excitation, but the method has not found widespread use for ultrasensitive applications. We recently described an approach to the multiphoton excitation of single molecules that uses a pulse shaper to compress and tailor pulses from an ultrafast broadband laser in order to optimise the brightness and signal-to-background ratio following non-linear excitation. Here we provide a detailed description of the setup and illustrate its use and potential by optimising two-photon fluorescence of a common fluorophore, rhodamine 110, at the single-molecule level. We also show that a DNA oligonucleotide labelled with a fluorescent nucleobase analogue, tC, can be detected using two-photon FCS, whereas one-photon excitation causes rapid photobleaching. The ability to improve the signal-to-background ratio and to reduce the incident power required to attain a given brightness can be applied to the multiphoton excitation of any fluorescent species, from small molecules with low multiphoton cross sections to the brightest nanoparticles.

Heterogeneous migration routes of DNA triplet repeat slip-outs.

It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called "mobile" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)10 repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.

Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats.

Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington's Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets.

Conformationally rigid pyrazoloquinazoline α-amino acids: one- and two-photon induced fluorescence.

The synthesis and photophysical properties of a new class of α-amino acid bearing a rigid pyrazoloquinazoline chromophore are described. Confromational constraint of the amino acid side-chains resulted in high emission quantum yields, while the demonstration of two-photon-induced fluorescence via near-IR excitation signifies their potential for sensitive bioimaging applications.

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.

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 ε 442 = 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.

Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation.

The ability to routinely detect fluorescent nucleobase analogues at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylamine-substituted extended-6-aza-uridine (DMAthaU). We show that DMAthaU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMAthaU undergoes two-photon (2P) absorption at low excitation powers, switching to three-photon (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analogue, which facilitated the first single-molecule detection of an emissive nucleoside with multiphoton excitation.

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity.

Fluorescent nucleobase analogues (FBAs) have many desirable features in comparison to extrinsic fluorescent labels, but they are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their short excitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach. Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness. We have studied the two-photon-excited fluorescence properties of pA and how they are affected by incorporation in DNA. We find that pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in an oligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context, resulting in the highest reported brightness for such a probe. The use of a two-photon microscope with ultrafast excitation and pulse shaping has allowed the detection of pA-containing oligonucleotides in solution with a limit of detection of ∼5 molecules, demonstrating that practical single-molecule detection of FBAs is now within reach.

Stacking-induced fluorescence increase reveals allosteric interactions through DNA.

From gene expression to nanotechnology, understanding and controlling DNA requires a detailed knowledge of its higher order structure and dynamics. Here we take advantage of the environment-sensitive photoisomerization of cyanine dyes to probe local and global changes in DNA structure. We report that a covalently attached Cy3 dye undergoes strong enhancement of fluorescence intensity and lifetime when stacked in a nick, gap or overhang region in duplex DNA. This is used to probe hybridization dynamics of a DNA hairpin down to the single-molecule level. We also show that varying the position of a single abasic site up to 20 base pairs away modulates the dye-DNA interaction, indicative of through-backbone allosteric interactions. The phenomenon of stacking-induced fluorescence increase (SIFI) should find widespread use in the study of the structure, dynamics and reactivity of nucleic acids.

Pentacyclic adenine: a versatile and exceptionally bright fluorescent DNA base analogue.

Emissive base analogs are powerful tools for probing nucleic acids at the molecular level. Herein we describe the development and thorough characterization of pentacyclic adenine (pA), a versatile base analog with exceptional fluorescence properties. When incorporated into DNA, pA pairs selectively with thymine without perturbing the B-form structure and is among the brightest nucleobase analogs reported so far. Together with the recently established base analog acceptor qAnitro, pA allows accurate distance and orientation determination via Förster resonance energy transfer (FRET) measurements. The high brightness at emission wavelengths above 400 nm also makes it suitable for fluorescence microscopy, as demonstrated by imaging of single liposomal constructs coated with cholesterol-anchored pA-dsDNA, using total internal reflection fluorescence microscopy. Finally, pA is also highly promising for two-photon excitation at 780 nm, with a brightness (5.3 GM) that is unprecedented for a base analog.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m-1 s-1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks.

Conformational Heterogeneity in a Fully Complementary DNA Three-Way Junction with a GC-Rich Branchpoint.

DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first 19F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.

Mitochondria-targeted spin-labelled luminescent iridium anticancer complexes.

Mitochondria generate energy but malfunction in many cancer cells, hence targeting mitochondrial metabolism is a promising approach for cancer therapy. Here we have designed cyclometallated iridium(iii) complexes, containing one TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) spin label [C43H43N6O2Ir1·PF6]˙ (Ir-TEMPO1) and two TEMPO spin labels [C52H58N8O4Ir1·PF6]˙ (Ir-TEMPO2). Electron paramagnetic resonance (EPR) spectroscopy revealed spin-spin interactions between the TEMPO units in Ir-TEMPO2. Both Ir-TEMPO1 and Ir-TEMPO2 showed bright luminescence with long lifetimes (ca. 35-160 ns); while Ir-TEMPO1 displayed monoexponential decay kinetics, the biexponential decays measured for Ir-TEMPO2 indicated the presence of more than one energetically-accessible conformation. This observation was further supported by density functional theory (DFT) calculations. The antiproliferative activity of Ir-TEMPO2 towards a range of cancer cells was much greater than that of Ir-TEMPO1, and also the antioxidant activity of Ir-TEMPO2 is much higher against A2780 ovarian cancer cells when compared with Ir-TEMPO1. Most notably Ir-TEMPO2 was particularly potent towards PC3 human prostate cancer cells (IC50 = 0.53 µM), being ca. 8× more active than the clinical drug cisplatin, and ca. 15× more selective towards cancer cells versus normal cells. Confocal microscopy showed that both Ir-TEMPO1 and Ir-TEMPO2 localise in the mitochondria of cancer cells.

Optical detection of gadolinium(iii) ions via quantum dot aggregation.

A rapid, sensitive and selective optical readout of the presence of gadolinium(iii) ions would have a wide range of applications for clinical and environmental monitoring. We demonstrate that water-soluble CdTe quantum dots (QDs) are induced to aggregate by Gd3+ ions in aqueous solution. By using a combination of photoluminescence spectroscopy, dynamic light scattering and fluorescence correlation spectroscopy (FCS) to monitor quantum dot aggregation kinetics, we correlate the efficiency of the self-quenching process with the degree of aggregation across a broad range of conditions, including different sizes of QDs. We attribute the aggregation to metal binding to the QD's surface ligands and the quenching to intra-aggregate energy transfer between QDs. When the strategy was applied to additional trivalent ions, the aggregation rate varied according to the particular trivalent metal ion used, suggesting that the selectivity can be enhanced and controlled by appropriate design of the capping ligands and solution conditions.

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.