Deciphering the pH-dependence of ground- and excited-state equilibria of thienoguanine

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

HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other\u27s transcription through HnRNPK-mediated post-transcriptional regulation. HnRNPK controls the mRNA stability and colocalization of IER3 and IER3-AS1. HnRNPK interaction with IER3 and IER3-AS1 determines their oncogenic functions by maintaining them in a single-stranded form. hnRNPK depletion neutralizes their oncogenic functions through promoting dsRNA formation and cytoplasmic accumulation. Intriguingly, hnRNPK loss-of-function and gain-of-function experiments reveal its role in maintaining global single- and double-stranded RNA. Thus, our data unveil the critical role of HnRNPK in maintaining single-stranded RNAs and their physiological functions by blocking RNA-RNA interactions

A new phosphoramidite enables orthogonal double labelling to form combination oligonucleotide probes

Oligonucleotides labelled with thiazole orange intercalator and a reporter dye on the same thymine base have been synthesized. The key phosphoramidite (AP-C3 dT) contains an alkyne and amine, enabling dual orthogonal labelling of the nucleobase. Multiple monomers can be added to produce heavily functionalised oligonucleotides. In their DNA and 2′-OMe RNA formats these combination probes display high duplex stability and fluorescence when bound to complementary DNA and RNA

Vesicles Formation by Zwitterionic Micelle and Poly‑l‑lysine: Solvation and Rotational Relaxation Study

The stable unilamellar vesicles formation, having large potential applications in biological as well as biomedical fields, has been investigated in aqueous solution composed of a zwitterionic surfactant, <i>N</i>-hexadecyl-<i>N</i>,<i>N</i>-dimethylammonio-1-propanesulfonate (SB-16), and water-soluble cationic poly­(amino acid), poly-l-lysine (PLL). Dynamic light scattering (DLS), transmission electron microscopy (TEM), and other optical spectroscopic techniques revealed the transformation of SB-16 micelles in aqueous solutions into stable unilamellar vesicles above a certain concentration (0.008 to 0.1% w/v) of PLL. Solvation and rotational dynamics of coumarin 480 (C-480) give the information on hydration behavior around the headgroup regions of SB-16 micelle and SB-16/PLL vesicle. It was observed that the hydration nature around the headgroup regions of SB-16/PLL vesicular system is higher than the head group regions of micellar system. Thus, PLL permits more water molecules in the headgroup regions of vesicular system

Graphene Oxide and Pluronic Copolymer Aggregates–Possible Route to Modulate the Adsorption of Fluorophores and Imaging of Live Cells

In recent years, amphiphilic triblock copolymers have attracted increasing attention due to their tunable properties and biocompatible nature, and the degree of hydrophobicity of these block copolymers can be modulated by varying the hydrophobic poly­(propylene oxide) (PPO) blocks and hydrophilic poly­(ethylene oxide) (PEO) moieties. Beside these, compared to the conventional micelles, block copolymer aggregates are more heterogeneous. For this reason, we have chosen two different fluorophores with different hydrophobicity so that we can monitor the different regions into the aggregates. We have shown the effect of theses Pluronic block copolymer aggregates on the adsorption of two fluorophores on the graphene oxide (GO) surface. The PPO segment of the block copolymer strongly interact with the hydrophobic basal plane of GO. Thus, in the presence of these aggregates the interaction between the GO and fluorophores is restricted depending on their location into the aggregates. The adsorption of the fluorophores is also dependent on the hydrophobicity of the aggregates. In most of the cases, the adsorption phenomena follow the traditional Langmuir isotherm. Further, fluorescence correlation spectroscopy (FCS) study successfully provides insight into the molecular diffusion of these fluorophores adsorbed on GO surface. In water, almost equal amount of fluorophores are adsorbed irrespective of their nature. However, in pluronic aggregates, the amount of adsorbed fluorophores decreases significantly depending on their position and hydrophobicity. In addition, our FCS result indicates that the molecular diffusion of these fluorophores in the presence of GO and triblock copolymer deviate from the normal Fickian diffusion and show anomalous superdiffusion. Finally, we have also demonstrated that fluorophore loaded block copolymer and GO can be used as an effective tool for the live cell imaging. In the presence of pluronic aggregates, fluorophores can be distributed in most of the cell surface, and cellular uptake of GO is also increased. Furthermore, due to the biocompatible nature of these pluronics, GO-P123 can serve as a drug delivery vehicle

Cholesterol Based Surface Active Ionic Liquid That Can Form Microemulsions and Spontaneous Vesicles

In this article, we have reported the synthesis and physicochemical characterization of a novel l-glycine amino acid derived cholesterol based surface active ionic liquid (SAIL). This SAIL has been explored for the preparation of ionic liquid (IL)-in-oil microemulsions and vesicles. The formation of IL-in-oil microemulsion is characterized by construction of a ternary phase diagram, dynamic light scattering (DLS) measurement, proton nuclear magnetic resonance (¹H NMR) study, fluorescence measurement using coumarin 480 (C-480) as a molecular probe, and also by recording the diffusion behavior of the molecular probe rhodamine 6G (R6G) in microemulsion droplets through the fluorescence correlation spectroscopy (FCS) technique. Similarly, the spontaneous vesicle formation from the SAIL in water has been established using DLS, transmission electron microscopy (TEM), cryogenic-transmission electron microscopy (cryo-TEM), field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), FCS, and fluorescence lifetime imaging microscopy (FLIM) measurements. These aggregates may potentially serve as good biomimicking models and possible drug carriers

Spectroscopy and Fluorescence Lifetime Imaging Microscopy To Probe the Interaction of Bovine Serum Albumin with Graphene Oxide

The interaction of graphene oxide (GO) with bovine serum albumin (BSA) in aqueous buffer solution has been investigated with various spectroscopic and imaging techniques. At single molecular resolution this interaction has been performed using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) techniques. The conformational dynamics of BSA on GO’s influence have been explored by FCS and circular dichroism (CD) spectroscopy. For the FCS studies BSA was labeled covalently by a fluorophore, Alexa Fluor 488. On the addition of GO in phosphate buffer of 10 mM at pH 7.4 the diffusion time (τ_D) and the hydrodynamic radius (<i>R</i>_h) of BSA increase due to adsorption of BSA. Conformational relaxation time components of native BSA drastically vary with the addition of GO, signifying the change of conformational dynamics of BSA after addition of GO. The adsorption isotherm also indicates significant adsorption of BSA on the GO surface. Adsorption of BSA on the GO surface has shown in direct images of atomic force microscopy (AFM) and FLIM. Fluorescence quenching study of BSA with addition of GO also indicates that there is strong interaction between BSA and GO

Unique Photophysical Behavior of 2,2′-Bipyridine-3,3′-diol in DMSO–Water Binary Mixtures: Potential Application for Fluorescence Sensing of Zn²⁺ Based on the Inhibition of Excited-State Intramolecular Double Proton Transfer

In this work we have investigated the anomalous behavior of DMSO–water binary mixtures using 2,2′-bipyridine-3,3′-diol (BP­(OH)₂) as a microenvironment-sensitive excited-state-intramolecular-double-proton-transfer (ESIDPT) probe. Here we present results on the UV–vis absorption and fluorescence properties of BP­(OH)₂ in the binary solutions. DMSO–water binary mixtures at various compositions are an intriguing hydrogen bonded system, where DMSO acts to diminish the hydrogen bonding ability of water with the dissolved solutes. As a result, we observe unusual changes in the photophysical properties of BP­(OH)₂ with increasing DMSO content in complete correlation with the prior simulation and experimental results on the solvent structures and dynamics. The fluorescence quantum yield and fluorescence lifetime of BP­(OH)₂ depend strongly on the DMSO content and become maximum at very low mole fraction (∼0.12) of DMSO. The anomalous behavior at this particular region likely arises from the enhanced pair hydrophobicity of the medium as demonstrated by Bagchi and co-workers (Banerjee, S.; Roy, S.; Bagchi, B. <i>J. Phys. Chem. B</i> <b>2010</b>, <i>114</i>, 12875–12882). In addition we have also shown the utilization of BP­(OH)₂ as a potential Zn²⁺-selective fluorescent sensor in a 1:1 DMSO–water binary mixture useful for biological applications. We observed highly enhanced fluorescence emission of BP­(OH)₂ selectively for binding with the Zn²⁺ metal ion. Moreover, the fluorescence emission maximum of BP­(OH)₂-Zn²⁺ is significantly blue-shifted with a reduced Stokes shift due to the inhibition of the ESIDPT process of BP­(OH)₂ through strong coordination

Excited-State Proton Transfer Dynamics of Firefly’s Chromophore D‑Luciferin in DMSO–Water Binary Mixture

In this article we have investigated intermolecular excited-state proton transfer (ESPT) of firefly’s chromophore D-luciferin in DMSO–water binary mixtures using steady-state and time-resolved fluorescence spectroscopy. The unusual behavior of DMSO–water binary mixture as reported by Bagchi et al. (<i>J. Phys. Chem. B</i> <b>2010</b>, <i>114</i>, 12875–12882) was also found using D-luciferin as intermolecular ESPT probe. The binary mixture has given evidence of its anomalous nature at low mole fractions of DMSO (below <i>X</i>_D = 0.4) in our systematic investigation. Upon excitation of neutral D-luciferin molecule, dual fluorescence emissions (protonated and deprotonated form) are observed in DMSO–water binary mixture. A clear isoemissive point in the time-resolved area normalized emission spectra further indicates two emissive species in the excited state of D-luciferin in DMSO–water binary mixture. DMSO–water binary mixtures of different compositions are fascinating hydrogen bonding systems. Therefore, we have observed unusual changes in the fluorescence emission intensity, fluorescence quantum yield, and fluorescence lifetime of more hydrogen bonding sensitive anionic form of D-luciferin in low DMSO content of DMSO–water binary mixture