RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular 'Riboglow' platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescence turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small noncoding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye-binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

It has long been recognized that the measured magnitudes of dynamic hyperpolarizabilities (b l values) can depend sensitively on molecular structure.[1] Whether a chromophore with nonlinear optical properties (NLO chromophore) is an electronically asymmetric, dipolar, donor-linker-acceptor (D-L-A) molecule, or an electronically symmetric, yet noncentrosymmetric, D-L-D or A-L-A octopolar structure, oscillator strength and the extent to which charge is redistributed in electronic transitions depend on the degree of coupling of D and A to the conjugated L. In virtually all known NLO chromophores studied to date, the orientations of D, L, and A are not rigidly fixed; hence, the experimentally determined electronic coupling between these units is generally established by the distribution of condensed-phase conformeric populations set by the nature of D-L and L-A connectivity.[2] Further, if one considers octopolar chromophores, the requirement of noncentrosymmetry places further restrictions on important spatial relationships between D, L, and A: nearly all known NLO octopoles have either D 3h or T d symmetry.[3] It has been recognized that D 2 and D 2d symmetries could be exploited in the design of single-oscillator octopolar NLO chromophores, [4] or octopolar compounds that exhibit metal-to-ligand charge-transfer transitions; [5] however, it has been stated that no interesting chromophoric benchmarks yet have such symmetries.[6] The few established NLO octopoles with D 2 symmetry are characterized by weakly coupled oscillators in which 3D charge redistribution occurs by a through-space delocalization mechanism.[7] We report here that strongly coupled D 2 -symmetric oscillators provide an important motif for potent octopolar NLO chromophores and demonstrate the utility of hyper-Rayleigh light scattering (HRS) measurements [8] to interrogate conformeric populations of chromophores having D 2 and D 2d symmetries at ambient temperature in solution.The utility of HRS in probing structure derives from the fact that it is intrinsically sensitive to symmetry; at the molecular level, a hyperpolarizable molecule must be noncentrosymmetric. When a substantial HRS signal is observed from an isotropic solution of molecules, at least a fraction of them must have noncentrosymmetric structures. This property of even-order nonlinear optical probes of electronic structure has been utilized to characterize the electrooptic characteristics of nondipolar chromophores RuR f PZnRu, OsR f PZnOs, OsPZnOs, PZnRuPZn, and PZnOsPZn (Scheme 1).Scheme 1. Structures of investigated compounds.[*] Dr.

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and correct localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, "FR-MQV," which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (∼140 000 M −1 cm −1 ), increased radiative rate constant, and reduced nonradiative rate constant with respect to its precursor. The properties of FR-MQV derive from three mutationsM42Q, C159V, and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the para-hydroxyphenyl and the acylimine sites of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the para-hydroxyphenyl site of the chromophore were mutated. The M42Q mutation is located near the acylimine moiety of the chromophore and was discovered using site-directed mutagenesis guided by X-ray crystal structures. FR-MQV exhibits a 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells [based on fluorescence-activated cell sorting (FACS)] compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells. These properties make FR-MQV a promising template for further engineering into a new family of RFPs.

A series of mono-, bis-, tris-, and tetrakis-(porphinato)zinc(II) (PZn)-elaborated ruthenium(II) bis(terpyridine) (Ru) complexes has been synthesized in which an ethyne unit connects the macrocycle meso carbon atom to terpyridyl (tpy) 4-, 4′-, and 4″-positions. These supermolecular chromophores, based on the ruthenium(II) [5-(4′-ethynyl-(2,2′;6′,2″-terpyridinyl))-10,20-bis(2′,6′-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2′;6′,2″-terpyridine) 2+ bishexafluorophosphate (RuPZn) archetype, evince strong mixing of the PZn-based oscillator strength with ruthenium terpyridyl charge resonance bands. Potentiometric and linear absorption spectroscopic data indicate that for structures in which multiple PZn moieties are linked via ethynes to a [Ru(tpy) 2 ] 2+ core, little electronic coupling is manifest between PZn units, regardless of whether they are located on the same or opposite tpy ligand. Congruent with these experiments, pump-probe transient absorption studies suggest that the individual RuPZn fragments of these structures exhibit, at best, only modest excited-state electronic interactions that derive from factors other than the dipole-dipole interactions of these strong oscillators; this approximate independent character of the component RuPZn oscillators enables fabrication of NLO multipoles with extraordinary hyperpolarizabilities.Dynamic hyperpolarizability (β λ ) values and depolarization ratios (ρ) were determined from hyper-Rayleigh light scattering (HRS) measurements carried out at an incident irradiation wavelength (λ inc ) of 1300 nm. The depolarization ratio data provide an experimental measure of chromophore optical symmetry; appropriate coupling of multiple charge-transfer oscillators produces structures having enormous averaged hyperpolarizabilities (β HRS values), while evolving (PF 6 ) 2 possessing a β HRS value at 1300 nm more than a factor of three larger than that determined for any chromophore having octopolar symmetry examined to date. Because NLO octopoles possess omnidirectional NLO responses while circumventing the electrostatic interactions that drive bulk-phase centrosymmetry for NLO dipoles at high chromophore concentrations, the advent of octopolar NLO chromophores having vastly superior β HRS values at technologically important wavelengths will motivate new experimental approaches to achieve acentric order in both bulk-phase and thin film structures.

Temperature dependent photodegradation and recovery studies of Dipserse Orange 11 (DO11) dye dissolved in poly(methyl methacrylate) and polystyrene polymer hosts are used as a test of the recently proposed correlated chromophore domain model.[1] This model posits that dye molecules form domains or aggregates. The nature of aggregation or how it mediates self healing is not yet well understood. In this paper we present qualitative evidence that supports the hypothesis that the dye molecules undergo a change to a tautomer state with higher dipole moment and hydrogen bond with the amines and keto oxygens of the polymer. Groupings of such molecules in a polymer chain form what we call a domain, and interactions between molecules in a domain make them more robust to photodegradation and mediate self healing.

Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ∼2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.

It has long been recognized that the measured magnitudes of dynamic hyperpolarizabilities (b l values) can depend sensitively on molecular structure.[1] Whether a chromophore with nonlinear optical properties (NLO chromophore) is an electronically asymmetric, dipolar, donor-linker-acceptor (D-L-A) molecule, or an electronically symmetric, yet noncentrosymmetric, D-L-D or A-L-A octopolar structure, oscillator strength and the extent to which charge is redistributed in electronic transitions depend on the degree of coupling of D and A to the conjugated L. In virtually all known NLO chromophores studied to date, the orientations of D, L, and A are not rigidly fixed; hence, the experimentally determined electronic coupling between these units is generally established by the distribution of condensed-phase conformeric populations set by the nature of D-L and L-A connectivity.[2] Further, if one considers octopolar chromophores, the requirement of noncentrosymmetry places further restrictions on important spatial relationships between D, L, and A: nearly all known NLO octopoles have either D 3h or T d symmetry.[3] It has been recognized that D 2 and D 2d symmetries could be exploited in the design of single-oscillator octopolar NLO chromophores, [4] or octopolar compounds that exhibit metal-to-ligand charge-transfer transitions; [5] however, it has been stated that no interesting chromophoric benchmarks yet have such symmetries.[6] The few established NLO octopoles with D 2 symmetry are characterized by weakly coupled oscillators in which 3D charge redistribution occurs by a through-space delocalization mechanism.[7] We report here that strongly coupled D 2 -symmetric oscillators provide an important motif for potent octopolar NLO chromophores and demonstrate the utility of hyper-Rayleigh light scattering (HRS) measurements [8] to interrogate conformeric populations of chromophores having D 2 and D 2d symmetries at ambient temperature in solution.The utility of HRS in probing structure derives from the fact that it is intrinsically sensitive to symmetry; at the molecular level, a hyperpolarizable molecule must be noncentrosymmetric. When a substantial HRS signal is observed from an isotropic solution of molecules, at least a fraction of them must have noncentrosymmetric structures. This property of even-order nonlinear optical probes of electronic structure has been utilized to characterize the electrooptic characteristics of nondipolar chromophores RuR f PZnRu, OsR f PZnOs, OsPZnOs, PZnRuPZn, and PZnOsPZn (Scheme 1).Scheme 1. Structures of investigated compounds.[*] Dr.

The approximately linear scaling of fluorescence quantum yield (ϕ) with fluorescence lifetime (τ) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet, and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized. In this study, we focused on understanding the outcome of lifetime-based directed evolution of mCherry, which is a popular red-FP (RFP). We identified four positions (W143, I161, Q163, and I197) near the FP chromophore that can be mutated to create mCherry-XL (eXtended Lifetime: ϕ = 0.70; τ = 3.9 ns). The 3-fold higher quantum yield of mCherry-XL is on par with that of the brightest RFP to date, mScarlet. We examined selected variants within the evolution trajectory and found a near-linear scaling of lifetime with quantum yield and consistent blue-shifts of the absorption and emission spectra. We find that the improvement in brightness is primarily due to a decrease in the nonradiative decay of the excited state. In addition, our analysis revealed the decrease in nonradiative rate is not limited to the blue-shift of the energy gap and changes in the excited state reorganization energy. Our findings suggest that nonradiative mechanisms beyond the scope of energy-gap models such the Englman–Jortner model are suppressed in this lifetime evolution trajectory.