Ultrafast Time-resolved Fluorescence Spectroscopy Research Articles

Superior Photostability of the Unnatural Base 6-Amino-5-nitropyridin-2-ol: A Case Study Using Ultrafast Broadband Fluorescence, Transient Absorption, and Theoretical Computation.

6-Amino-5-nitropyridin-2-ol (Z), a nitroaromatic compound and a base for Hachimoji nucleic acids, holds significant potential in expanding the genetic alphabet, as well as in synthetic biology and biotechnology. Despite its promising applications, the spectral characterization and photoinduced properties of Z have remained largely unexplored until now. This study presents a comprehensive investigation into its excited state dynamics in various solvents, utilizing state-of-the-art ultrafast broadband time-resolved fluorescence and transient absorption spectroscopy, complemented by computational methods. The acquired results provide direct experimental evidence that, upon photoexcitation, Z emits prompt fluorescence from a nearly planar structure in its excited state, independent of solvent properties. This state deactivates nonradiatively within sub-picoseconds through internal conversion with a unitary yield, primarily mediated by the rotation of the nitro group. This unusually rapid deactivation pathway entirely excludes the involvement of long-lived nπ* states, triplet states, and photoproducts, which are commonly observed in most nitroaromatic compounds and natural DNA and RNA bases. Our findings underscore that Z, as an unnatural base, exhibits superior photostability compared to canonical natural bases. This provides valuable insights into the photodynamics of nitroaromatic compounds, which is beneficial for strategic substitution design in environmental and biological applications.

Solvent-polarity dependence of ultrafast excited-state dynamics of trans-4-nitrostilbene.

Ultrafast excited-state dynamics of the simplest nitrostilbenes, namely trans-4-nitrostilbene (t-NSB), was studied in solvents of various polarities with ultrafast broadband time-resolved fluorescence and transient absorption spectroscopies, and by quantum-chemical computations. The results revealed that the initially excited S1(ππ*) state deactivation dynamics is strongly influenced by the solvent polarity. Specifically, the t-NSB S1-state lifetime decreases by three orders of magnitude from ∼60 ps in high-polarity solvents to ∼60 fs in nonpolar solvents. The strong solvent-polarity dependence arises from the differences in dipole moments among the S1 and relevant states, including the major intersystem crossing (ISC) receiver triplet states, and therefore, the solvent polarity can modulate their relative energies and ISC rates. In nonpolar solvents, the sub-100 fs lifetime is due to a combination of efficient ISC and internal conversion. In medium-polarity solvents, the S1-state population decays via a competing ISC relaxation mechanism in a biphasic manner, and the ISC rates are found to obey the inverse energy gap law of the strong coupling case. In high-polarity solvents, the S1 state is stabilized to a much lower energy such that ISC becomes energetically infeasible, and the S1 state decays via barrier crossing along the torsion angle of the central ethylenic bond to the nonfluorescent perpendicular configuration. Regardless of the initial S1-state deactivation pathways in various solvents, the excited-state population is ultimately trapped in the metastable T1-state perpendicular configuration, at which a slower ISC occurs to bring the system to the ground state and bifurcate into either trans or cis form of NSB.

Sequence-specific binding behavior of coralyne toward triplex DNA: An ultrafast time-resolved fluorescence spectroscopy study.

Triplex DNA structure has potential therapeutic application in inhibiting the expression of genes involved in cancer and other diseases. As a DNA-targeting antitumor and antibiotic drug, coralyne shows a remarkable binding propensity to triplex over canonical duplex and thus can modulate the stability of triplex structure, providing a prospective gene targeting strategy. Much less is known, however, about coralyne-binding interactions with triplex. By combining multiple steady-state spectroscopy with ultrafast fluorescence spectroscopy, we have investigated the binding behaviors of coralyne with typical triplexes. Upon binding with a G-containing triplex, the fluorescence of coralyne is markedly quenched owing to the photoinduced electron transfer (PET) of coralyne with the G base. Systematic studies show that the PET rates are sensitive to the binding configuration and local microenvironment, from which the coexisting binding modes of monomeric (full and partial) intercalation and aggregate stacking along the sugar-phosphate backbone are distinguished and their respective contributions are determined. It shows that coralyne has preferences for monomeric intercalation within CGG triplex and pure TAT triplex, whereas CGC+ triplex adopts mainly backbone binding of coralyne aggregates due to charge repulsion, revealing the sequence-specific binding selectivity. The triplex-DNA-induced aggregation of coralyne could be used as a probe for recognizing the water content in local DNA structures. The strong π-π stacking of intercalated coralyne monomer with base-triplets plays an important role in stabilizing the triplex structure. These results provide mechanistic insights for understanding the remarkable propensity of coralyne in selective binding to triplex DNA and shed light on the prospective applications of coralyne-triplex targeted anti-gene therapeutics.

Energy transfer dynamics in B-phycoerythrin from the red alga Porphyridium purpureum

The B-phycoerythrin hexamer (αβ)6γ of Porphyridium purpureum was isolated and purified. The absorption, circular dichroism, fluorescence and ultrafast time-resolved spectra were obtained. The results showed a double absorption peak at 545 nm and 565 nm and a shoulder peak at 498 nm, and fluorescence emission maxima at 580 nm and 620 nm were observed. The circular dichroism spectra in the near-ultraviolet region were obtained and resolved for the first time, which showed that the two peaks at 260 nm and 305 nm were considered to be correlated to phenylalanine (Phe) and tryptophan (Trp) in a conservative hydrophobic microenvironment, respectively. The circular dichroism spectra in the visible region showed that PEB139α/PEB158β and PEB82α/PEB82β existed as two exciton-coupled bilin pairs. Energy transfer within the exciton-coupled pairs was by exciton splitting, while between the exciton-coupled pairs was by Förster resonance. From the studies of the energy transfer dynamics by ultrafast time-resolved fluorescence spectroscopy, it was confirmed that the energy transfer of the B-PE hexamer had three time components of 8 ps, 60 ps, and 1200 ps. In addition, the internal energy transfer pathways of B-phycoerythrin hexamer were identified by deconvoluting the fluorescence decay curve at different detection wavelengths.

Mapping the excitation energy migration pathways in phycobilisomes from the cyanobacterium Acaryochloris marina

In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77 K. The study demonstrates that if complexes are stabilized by high phosphate (900 mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50 mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.

Ultrafast Through-Space Electronic Energy Transfer in Molecular Dyads Built around Dynamic Spacer Units.

A pair of complementary molecular dyads have been synthesized around a 1,2-diaminocyclohexyl spacer that itself undergoes ring inversion. Despite these conformational exchange processes, the donor and acceptor occupy quite restricted spatial regions, and they are not interchangeable. The donor and acceptor pair comprise disparate boron dipyrromethene dyes selected to display favorable electronic energy transfer (EET). Steady-state emission spectroscopy confirms that through-space EET from donor to acceptor is almost quantitative, aided by the relatively short separations. Ultrafast time-resolved fluorescence spectroscopy has allowed determination of the rates of EET for both dyads. Surprisingly, in view of the close proximity of donor and acceptor (center-to-center separations less than 20 Å), the EET dynamics are well-accounted for in terms of the computed molecular conformations and conventional Förster theory. One dyad appears as a single family of conformations, but EET for the second dyad corresponds to dual-exponential kinetics. In this latter case, an intramolecular hydrogen bond helps stabilize an open geometry, wherein EET is relatively slow.

The energy transfer dynamics in C-phycocyanin hexamer from <italic>Spirulina platensis</italic>

Photosynthesis is an ancient and important conversion of solar light to biological energy in photosynthetic organisms. Phycobilisome (PBS) system in cyanobacteria and red algae is one of two major light-harvesting systems of photosynthetic oxygen organisms. PBS is aggregation of water-soluble phycobiliproteins (PBPs) which are distinctively colored group of disk-shaped macromolecular proteins bearing covalently attached open-chain tetrapyrroles known as phycobilins. PBPs are capable to absorb solar light and transfer energy to the chlorophyll of photosynthetic reaction center with a high efficiency. Light energy absorbed by phycobilins is transmitted between the subunits, and then transmitted between different PBPs, finally to the photoreaction center located on the thylakoid membrane. The energy transfer between PBPs is very fast and high efficiency. Because energy can be transferred in different chromophores and there are a large number of chromophores in PBPs, the energy transfer path of PBPs is complex and diverse. In the past 20 years, energy transfer process of PBPs have been analyzed by time-resolved fluorescence spectroscopy because a large shift of the fluorescence peaks can be clearly observed. For example, Zhang et al. estimated the time constant for energy transfer from PC (rod) to APC (core) as 18 ps and 55 ps based on PBPs crystal structures and time-resolved fluorescence spectra of the rod-core complex. However, due to the technical bottleneck of theoretical simulation and spectral experimental detection, the research on the physical mechanism of photosynthetic energy transfer is relatively slow. In recent years, the ultrafast spectrum time resolution has been continuously improved, which provides the conditions for studying the energy transfer of PBPs. In this paper, the mechanism of energy transfer in C-phycocyanin hexamer from Spirulina platensis was studied by ultrafast time-resolved fluorescence spectroscopy. The energy transfer pathway and the related transfer parameters in the C-phycocyanin hexamer were identified by the fitting of the fluorescence decay curve at different detection wavelengths. It was proved that the energy transfer of C-phycocyanin hexamer had four time components from the experiment, which are 6, 22, 280 and 1470 ps, respectively, then the time components were identified and discussed. This study reflects the energy transfer kinetics between different chromophores in C-phycocyanin and provides important experimental data for solving the relationship between the structure and function of PBPs. At the same time, our study has important value for the understanding of the primary process of photosynthesis. In the future, if the resolution of a streak camera can be raised to femtoseconds, it is possible to find more energy transfer paths, and thus it is expected to make a breakthrough in the ultrafast and ultra-efficient energy transfer mechanism of PBPs.

Excitation relaxation dynamics and energy transfer in pigment-protein complexes of a dinoflagellate, revealed by ultrafast fluorescence spectroscopy.

Photosynthetic light-harvesting complexes, found in aquatic photosynthetic organisms, contain a variety of carotenoids and chlorophylls. Most of the photosynthetic dinoflagellates possess two types of light-harvesting antenna complexes: peridinin (Peri)-chlorophyll (Chl) a/c-protein, as an intrinsic thylakoid membrane complex protein (iPCP), and water-soluble Peri-Chl a-protein, as an extrinsic membrane protein (sPCP) on the inner surface of the thylakoid. Peri is a unique carotenoid that has eight C=C bonds and one C=O bond, which results in a characteristic absorption band in the green wavelength region. In the present study, excitation relaxation dynamics of Peri in solution and excitation energy transfer processes of sPCP and the thylakoid membranes, prepared from the photosynthetic dinoflagellate, Symbiodinium sp., are investigated by ultrafast time-resolved fluorescence spectroscopy. We found that Peri-to-Chl a energy transfer occurs via the Peri S1 state with a time constant of 1.5ps or 400fs in sPCP or iPCP, respectively, and that Chl c-to-Chl a energy transfer occurs in the time regions of 350-400fs and 1.8-2.6ps.

Excited-state vibrational relaxation and deactivation dynamics of trans-4-(N,N-dimethylamino)-4′-nitrostilbene in nonpolar solvents studied by ultrafast time-resolved broadband fluorescence spectroscopy

Abstract The excited-state relaxation dynamics of trans -4-( N , N -dimethylamino)-4′-nitrostilbene excited to the first absorption band in nonpolar solvents (cyclohexane and n -hexadecane) has been studied with ultrafast time-resolved broadband fluorescence spectroscopy. The temporal evolution of the time-resolved fluorescence spectra and bandwidth narrowing reveal a multiple-stage vibrational relaxation within ∼15 ps followed by slower excited-state deactivation steps (80 and 950 ps in cyclohexane). In addition, an oscillatory fluorescence with a period of about 1.1 ps was observed and attributed to the coherent vibrational motion of the nitrophenyl/aminophenyl torsion that modulates the early-time fluorescence spectra. The decay of the total fluorescence intensity with cubic frequency correction was found to be consistent with a competitive triplet–single deactivation mechanism.

Ultrafast Charge Transfer from CdSe Quantum Dots to p-Type NiO: Hole Injection vs Hole Trapping

Semiconductor quantum dot (QD) to metal oxide electron injection dynamics is well documented in the scientific literature. In contrast to that, not much is known so far about hole injection time scales in such systems. The current study fills this gap. We investigate photocathodes consisting of CdSe QDs and p-type NiO to study hole injection dynamics from the valence band of the QDs to NiO. The combination of two complementary techniques, ultrafast time-resolved absorption and fluorescence spectroscopies, enabled us to distinguish between hole trapping and injection. A kinetic component on the time scale of a few hundreds of picoseconds was identified as hole injection. By changing the size of the QDs, the driving force of the hole injection was tuned and we demonstrated that the hole injection rates are well described by the Marcus theory of charge transfer. In order to enhance the overall hole injection efficiency, we have passivated the CdSe QDs by a gradient ZnS shell. The core–shell QDs show signific...

Excited state properties of chlorophyll f in organic solvents at ambient and cryogenic temperatures

Chlorophyll f is a photosynthetic pigment that was discovered in 2010. In this study, we present investigations on spectral and dynamic characteristics of singlet-excited and triplet states of Chl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The pigment was studied at room temperature in two organic solvents: pyridine and diethyl ether that have different characters of coordination of the chlorophyll magnesium (Mg) atom (hexa- and penta-coordination, respectively). Cryogenic measurements (77K) were performed in 2-methyltetrahydrofuran (hexa-coordination). The singlet-excited state lifetime was measured to be 5.6ns at room temperature regardless of Mg coordination and 8.1ns at 77K. The fluorescence quantum yield of Chl f was also determined in pyridine to be 0.16. The triplet state lifetime was studied in detail in pyridine at room temperature, and the inherent lifetime was estimated to ~150μs. Selective measurements at 77K demonstrated that the metastability of the triplet state greatly enhances, and its lifetime increases by a factor of more than three.

Distinct Conformational Transition Patterns of Noncoding 7SK snRNA and HIV TAR RNAs upon Tat Binding

Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information.

Photophysical Properties of the Excited States of Bacteriochlorophyll f in Solvents and in Chlorosomes

Bacteriochlorophyll f (BChl f) is a photosynthetic pigment predicted nearly 40 years ago as a fourth potential member of the Chlorobium chlorophyll family (BChl c, d, and e). However, this pigment still has not been found in a naturally occurring organism. BChl c, d, and e are utilized by anoxygenic green photosynthetic bacteria for assembly of chlorosomes--large light-harvesting complexes that allow those organisms to survive in habitats with extremely low light intensities. Recently, using genetic methods on two different strains of Chlorobaculum limnaeum that naturally produce BChl e, two research groups produced mutants that synthesize BChl f and assemble it into chlorosomes. In this study, we present detailed investigations on spectral and dynamic characteristics of singlet excited and triplet states of BChl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The studies were performed on isolated BChl f in various solvents, at different temperatures, and on BChl f-containing chlorosomes in order to uncover any unusual or unfavorable properties that stand behind the lack of appearance of this pigment in natural environments.

Ultrafast Time-Resolved Broadband Fluorescence Studies of the Benzene-Tetracyanoethylene Complex: Solvation, Vibrational Relaxation, and Charge Recombination Dynamics

The charge-transfer (CT) state relaxation dynamics of the benzene-tetracyanoethylene (BZ-TCNE) complex was studied with broadband ultrafast time-resolved fluorescence spectroscopy implemented by optical Kerr gating in three solvents of different polarities. The CT state of the BZ-TCNE complex is reached via femtosecond laser excitation, and the subsequent temporal evolutions of the fluorescence spectra were measured. Analyses of various time-dependent spectral properties revealed rapid relaxations along solvent and vibrational coordinates in competition with charge recombination (CR). By comparing the results in solvents of different polarities, we partially separated solvation and vibrational relaxation dynamics and explored the solvent-dependent CR dynamics. Time-dependent dynamic fluorescence Stokes shift (TDFSS) measurements unveiled the solvation and vibrational relaxation contributions to the observed spectral relaxation. The biphasic and slow time scales of the vibrational contributions identified in TDFSS suggested nonstatistical and hindered intramolecular vibrational-energy redistribution that can be attributed to the unique structural properties of EDA complexes. The slowest spectral relaxation of 10-15 ps identified in TDFSS was ascribed to relaxation of the BZ(+)-TCNE(-) intermolecular vibrations, which is equivalent to a structural relaxation from the initial Franck-Condon configuration to the equilibrium CT-state structure. The time scales of vibrational relaxation indicate that a fraction of the CT-state population undergoes CR reactions before complete vibrational/structural equilibrium is achieved. In carbon tetrachloride, a nonexponential temporal profile was observed and attributed to vibrational nonequilibrium CR. In dichloromethane, polar solvation greatly accelerates CR reactions, and a slower reaction-field-induced structural relaxation gives rise to a pronounced biexponential decay. The equilibrium CR time constants of the BZ-TCNE CT state are 29 ps, 150 ps, and 68 ps in dichloromethane, carbon tetrachloride, and cyclohexane, respectively.

Study of Biomolecular Recognition Using Time-Resolved Optical Spectroscopy

Molecular recognition process refers to the weak non-covalent interaction, which takes place selectively and specifically between small ligand molecules with biological macromolecules. Understanding of such recognition in biological and biomimetic milieu is the central attraction for drug designing, which is crucial for the improvement of human healthcare. A thorough knowledge of the structural, dynamical and energetic parameters that dictate such molecular interactions can find immense use in the modulations of the ligand-macromolecule recognition process. In this article, we present our continuous effort to investigate the fundamental physical processes involved in the biomolecular recognition, e.g. efficiency (binding affinity and rigidity of the complex) and role of solvent molecules in the molecular recognition using steady state and predominantly, ultrafast time-resolved fluorescence spectroscopy. In this perspective, we have thoroughly investigated the molecular recognition of small ligand/drug molecules (Rifampicin; Rf, 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran; DCM, and Nile Blue; NB) by a human transporter protein, Human Serum Albumin (HSA), and also established the nonspecific type of interaction between a ligand molecule (Rf) and a biomimetic system (Sodium Dodecyl Sulfate (SDS) micelle). Simultaneous recognition of an intercalator (Ethidium Bromide, EtBr) and a DNA minor groove binder (Hoeschst 33258, H258) to a dodecamer DNA of specific sequence has also been monitored. Besides, we report an investigation on the recognition of ethidium (Et) cation, a potential mutagen, by synthetic DNA and various cell nuclei in presence of a stimulant drug, caffeine, employing the mentioned spectroscopic techniques along with NMR and fluorescence microscopy. Moreover, we have explored the differential molecular recognition of 8-anilino-1-naphthalenesulfonic acid (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS) by bovine pancreatic -chymotrypsin (CHT) upon interaction with genomic DNA. The correlation of the molecular recognition of the DNA and DNA-protein complexes with the hydration dynamics has been further exploited in our studies. In addition, we have developed functional nanoparticles/Quantum dots (QDs) that are covalently linked to biological molecules to detect the molecular interaction phenomenon between biomolecules. It should be noted that QDs have a significant contribution in the field of nano-biotechnology due to its high quantum yield, low photo-bleaching and increased biological application (cell labeling, in vivo imaging, gene delivery, sensing of fluorescence and molecular recognition). In this regard, we have exploited QDs as a potential energy donor/acceptor system and validated Forster resonance energy transfer (FRET) model over nano-surface energy transfer (NSET) technique. Therefore, the ultrafast non-radiative energy migration from tryptophan (Trp214) present in HSA to the HSA bound CdS QD, and from 4-nitrophenyl anthranilate (NPA) to Silver (Ag) nanoclusters in CHT (both NPA and Ag bound to CHT) have been investigated using FRET technique to monitor the protein folding pathway of HSA, and molecular interaction between NPA and CHT respectively. Moreover, we have also used functionalized QDs (CdSe/ZnS) for the detection of molecular recognition of ethidium bromide (EtBr) by a synthetic DNA. However, the intention of this review is to give an overview of ultrafast optical spectroscopic techniques for the exploration of biomolecular recognition, which may find potential significance for further research in the field of nano-biotechnology and medicine

Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine from Neighboring Bases in Single-Stranded DNA-Binding Protein Subsites

Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.

Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii

The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.

Dynamic Ensemble View of the Conformational Landscape of HIV-1 TAR RNA and Allosteric Recognition

RNA conformational dynamics and the resulting structural heterogeneity play an important role in RNA functions, e.g., recognition. Recognition of HIV-1 TAR RNA has been proposed to occur via a conformational capture mechanism. Here, using ultrafast time-resolved fluorescence spectroscopy, we have probed the complexity of the conformational landscape of HIV-1 TAR RNA and monitored the position-dependent changes in the landscape upon binding of a Tat protein-derived peptide and neomycin B. In the ligand-free state, the TAR RNA samples multiple families of conformations with various degrees of base stacking around the three-nucleotide bulge region. Some subpopulations partially resemble those ligand-bound states, but the coaxially stacked state is below the detection limit. When Tat or neomycin B binds, the bulge region as an ensemble undergoes a conformational transition in a position-dependent manner. Tat and neomycin B induce mutually exclusive changes in the TAR RNA underlying the mechanism of allosteric inhibition at an ensemble level with residue-specific details. Time-resolved anisotropy decay measurements revealed picosecond motions of bases in both ligand-free and ligand-bound states. Mutation of a base pair at the bulge--stem junction has differential effects on the conformational distributions of the bulge bases. A dynamic model of the ensemble view of the conformational landscape for HIV-1 TAR RNA is proposed, and the implication of the general mechanism of RNA recognition and its impact on RNA-based therapeutics are discussed.

Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine Residues Positioned Within Binding Subsites of a Single-Stranded DNA Binding Protein

DNAs bound by proteins are kinetically dynamic and exist in multiple conformations, including conformations that differ in stacking interactions. We have utilized ultrafast time resolved fluorescence spectroscopy to investigate changes in base stacking of single-stranded DNA (ssDNA) upon binding of the dimeric gene 5 protein (g5p) of fd, f1, and M13 strains of E. coli bacteriophages. DNA oligomer hairpins of 44 nucleotides were designed to have two antiparallel 16-nucleotide ssDNA tails for binding of a cluster of g5p dimers. Otherwise identical oligomers each contained a single fluorescent 2-aminopurine (2AP) base. Time domain fluorescence measurements showed that the label exists in an ensemble of conformations, including stacked, partially stacked, and unstacked species that are altered upon binding of the g5p. Two oligomers had 2AP labels at different subsite locations within one of the four-nucleotide DNA-binding sites of a given g5p monomer when the oligomers were saturated with g5p. These labels showed increases of the unstacked conformation from 22-24% for the free DNAs to 47-74% for the bound DNAs, where the extent of this increase was specific for the subsite location within the DNA-binding site of the g5p monomer. Time resolved anisotropy measurements indicated that the 2AP labels were in relatively inflexible conformations within the g5p-saturated oligomers.

UV‐Dissipation Mechanisms in the Eumelanin Building Block DHICA

The UV-dissipative mechanisms of the eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and the 4,7-dideutero derivative (DHICA-d(2)) in buffered H(2)O or D(2)O have been characterized by using ultrafast time-resolved fluorescence spectroscopy. Excitation of the carboxylate anion form, the dominating state at neutral pH, leads to dual fluorescence. The band peaking at lambda=378 nm is caused by emission from the excited initial geometry. The second band around lambda=450 nm is owed to a complex formed between the mono-anion and specific buffer components. In the absence of complex formation, the mono-anion solely decays non-radiatively or by emission with a lifetime of about 2.1 ns. Excitation of the neutral carboxylic acid state, which dominates at acidic pH, leads to a weak emission around lambda=427 nm with a short lifetime of 240 ps. This emission originates from the zwitterionic state, formed upon excitation of the neutral state by sub-ps excited-state intramolecular proton transfer (ESIPT) between the carboxylic acid group and the indole nitrogen. Future studies will unravel whether this also occurs in larger building blocks and ESIPT is a built-in photoprotective mechanism in epidermal eumelanin.