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Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.

Thienoguanosine (thG) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, 20 DNA duplexes, where the bases facing and neighboring thG were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations, and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, thG's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of thG independently of its flanking nucleobases. thG can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of thG's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of thG. Due to this exquisite sensitivity, thG appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, thG's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlight thG as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes, and exquisite sensitivity to local structural changes.

The thienoguanine nucleobase (thGb) is an isomorphic fluorescent analogue of guanine. In aqueous buffer at neutral pH, thGb exists as a mixture of two ground-state H1 and H3 keto-amino tautomers with distinct absorption and emission spectra and high quantum yield. In this work, we performed the first systematic photophysical characterization of thGb as a function of pH (2 to 12). Steady-state and time-resolved fluorescence spectroscopies, supplemented with theoretical calculations, enabled us to identify three additional thGb forms, resulting from pH-dependent ground-state and excited-state reactions. Moreover, a thorough analysis allowed us to retrieve their individual absorption and emission spectra as well as the equilibrium constants which govern their interconversion. From these data, the complete photoluminescence pathway of thGb in aqueous solution and its dependence as a function of pH was deduced. As the identified forms differ by their spectra and fluorescence lifetime, thGb could be used as a probe for sensing local pH changes under acidic conditions.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine ( th G), a fluorescent analogue of guanosine, overcomes all these limitations. We recorded the TRFA decays of th G -labelled dsDNA of different lengths. th G behaved as a rigid, non-perturbing reporter, since no fast correlation time was recorded for any tested dsDNA. Due to its perfect stacking, only two correlation times, instead of the typical three, describe th G -labelled dsDNA rotational dynamics. Thanks to these features, we provided a complete description of the tumbling of the different dsDNA and their complexes with the Set and Ring Associated (SRA) domain of UHRF1, a key epigenetic regulator, obtaining values in excellent agreement with theoretical predictions. Moreover, th G was also found sensitive to SRA-induced base flipping of neighboring nucleobases. In the DNA label toolbox, th G thus stands out as a unique reporter for investigating the rotational dynamics of dsDNA and protein/dsDNA complexes. Thienoguanosine, a fluorescent analogue of guanosine, stands out in the DNA probe toolkit, as a rigid, non-perturbing reporter, with only two correlation times to describe dsDNA rotational dynamics. It allows a comprehensive description of the tumbling of dsDNA and their complexes with proteins, fully matching theoretical predictions. [Display omitted] • Thienoguanosine ( th G) behaves as a rigid, non-perturbing reporter in dsDNA. • Time-resolved fluorescence anisotropy of th G allows full dsDNA tumbling description. • Rotational correlation times of thG-labelled dsDNA ± protein fully match with theory • th G is sensitive to protein-induced base flipping of neighboring nucleobases • th G is unique in the toolkit of fluorescent DNA labels for monitoring DNA dynamics. [ABSTRACT FROM AUTHOR]

In this work, we used firefly oxyluciferin (OxyLH2) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, OxyLH2, and its two analogues, 4-MeOxyLH and 4,6'-DMeOxyL, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study in vitro biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (5,5-Cpr-OxyLH) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that OxyLH2 and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions. [ABSTRACT FROM AUTHOR]

The Gag precursor of HIV-1, formed of the four proteic regions matrix (MA), capsid (CA), nucleocapsid (NC) and p6, orchestrates virus morphogenesis. This complex process relies on three major interactions, NC-RNA acting as a scaffold, CA-CA and MA-membrane that targets assembly to the plasma membrane (PM). The characterization of the molecular mechanism of retroviral assembly has extensively benefited from biochemical studies and more recently an important step forward was achieved with the use of fluorescence-based techniques and fluorescently labeled viral proteins. In this review, we summarize the findings obtained with such techniques, notably quantitative-based approaches, which highlight the role of the NC region in Gag assembly. [ABSTRACT FROM AUTHOR]

Abstract: Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52–96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52–96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52–96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52–96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates. [Copyright &y& Elsevier]