Aurimas Vyšniauskas

The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy

Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells. Herein, we report the design and development of a small fluorescent molecule, which can be used as an optical probe for G-quadruplexes. We demonstrate that the fluorescence lifetime of this new probe changes considerably upon interaction with different nucleic acid topologies. Specifically, longer fluorescence lifetimes are observed in vitro for G-quadruplexes than for double- and single-stranded nucleic acids. Cellular studies confirm that this molecule is cell permeable, has low cytotoxicity and localizes primarily in the cell nucleus. Furthermore, using fluorescence lifetime imaging microscopy, live-cell imaging suggests that the probe can be used to study the interaction of small molecules with G-quadruplexes in vivo.

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

Microviscosity is of paramount importance in materials and bio-sciences. Fluorescence imaging using molecular rotors has emerged as a versatile tool to measure microviscosity, either using a fluorescence lifetime or a ratiometric signal of the rotor; however, only a limited number of blue-to-green-emitting fluorophores with both the lifetime and the ratiometric signal sensitivity to viscosity have been reported to date. Here we report a deep red emitting dual viscosity sensor, which allows both the ratiometric and the lifetime imaging of viscosity. We study viscosity in a range of lipid-based systems and conclude that in complex dynamic systems dual detection is preferable in order to independently verify the results of the measurements as well as perform rapid detection of changing viscosity.

Molecular Rotors Provide Insights into Microscopic Structural Changes During Protein Aggregation

Changes in microscopic viscosity represent an important characteristic of structural transitions in soft matter systems. Here we demonstrate the use of molecular rotors to explore the changes in microrheology accompanying the transition of proteins from their soluble states into a gel phase composed of amyloid fibrils. The formation of beta-sheet rich protein aggregates, including amyloid fibrils, is a hallmark of a number of neurodegenerative disorders, and as such, the mechanistic details of this process are actively sought after. In our experiments, molecular rotors report an increase in rigidity of approximately three orders of magnitude during the aggregation reaction. Moreover, phasor analysis of the fluorescence decay signal from the molecular rotors suggests the presence of multiple distinct mechanistic stages during the aggregation process. Our results show that molecular rotors can reveal key microrheological features of protein systems not observable through classical fluorescent probes operating in light switch mode.

A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress

Abstract Oxidation of cellular structures is typically an undesirable process that can be a hallmark of certain diseases. On the other hand, photooxidation is a necessary step of photodynamic therapy (PDT), a cancer treatment causing cell death upon light irradiation. Here, the effect of photooxidation on the microscopic viscosity of model lipid bilayers constructed of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine has been studied. A molecular rotor has been employed that displays a viscosity‐dependent fluorescence lifetime as a quantitative probe of the bilayers viscosity. Thus, spatially‐resolved viscosity maps of lipid photooxidation in giant unilamellar vesicles (GUVs) were obtained, testing the effect of the positioning of the oxidant relative to the rotor in the bilayer. It was found that PDT has a strong impact on viscoelastic properties of lipid bilayers, which ‘travels’ through the bilayer to areas that have not been irradiated directly. A dramatic difference in viscoelastic properties of oxidized GUVs by Type I (electron transfer) and Type II (singlet oxygen‐based) photosensitisers was also detected.

Tuning the sensitivity of fluorescent porphyrin dimers to viscosity and temperature

Abstract Conjugated porphyrin dimers have emerged as versatile viscosity‐sensitive fluorophores that are suitable for quantitative measurements of microscopic viscosity by ratiometric and fluorescence lifetime‐based methods, in a concentration‐independent manner. Here, we investigate the effect of extended conjugation in a porphyrin‐dimer structure on their ability to sense viscosity and temperature. We show that the sensitivity of the fluorescence lifetime to temperature is a unique property of only a few porphyrin dimers.

Trianguleniums as Optical Probes for G-Quadruplexes: A Photophysical, Electrochemical, and Computational Study.

Abstract Nucleic acids can adopt non‐duplex topologies, such as G‐quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA‐M2 as a unique fluorescence probe for such studies. This probes emission lifetime is highly dependent on the topology of the DNA it interacts with opening up the possibility of carrying out live‐cell imaging studies. Herein, we describe the origin of its fluorescence selectivity for G‐quadruplexes. Cyclic voltammetry predicts that the appended morpholino groups can act as intra‐ molecular photo‐induced electron transfer (PET) quenchers. Photophysical studies show that a delicate balance between this effect and inter‐molecular PET with nucleobases is key to the overall fluorescence enhancement observed upon nucleic acid binding. We utilised computational modelling to demonstrate a conformational dependence of intra‐molecular PET. Finally, we performed orthogonal studies with a triangulenium compound, in which the morpholino groups were removed, and demonstrated that this change inverts triangulenium fluorescence selectivity from G‐quadruplex to duplex DNA, thus highlighting the importance of fine tuning the molecular structure not only for target affinity, but also for fluorescence response.

Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor

Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.