Sequence dependent UV damage of complete pools of oligonucleotides.

Understanding the sequence-dependent DNA damage formation requires probing a complete pool of sequences over a wide dose range of the damage-causing exposure. We used high throughput sequencing to simultaneously obtain the dose dependence and quantum yields for oligonucleotide damages for all possible 4096 DNA sequences with hexamer length. We exposed the DNA to ultraviolet radiation at 266 nm and doses of up to 500 absorbed photons per base. At the dimer level, our results confirm existing literature values of photodamage, whereas we now quantified the susceptibility of sequence motifs to UV irradiation up to previously inaccessible polymer lengths. This revealed the protective effect of the sequence context in preventing the formation of UV-lesions. For example, the rate to form dipyrimidine lesions is strongly reduced by nearby guanine bases. Our results provide a complete picture of the sensitivity of oligonucleotides to UV irradiation and allow us to predict their abundance in high-UV environments.

Electronic and Geometric Characterization of TICT Formation in Hemithioindigo Photoswitches by Picosecond Infrared Spectroscopy.

Deciphering the exact electronic and geometric changes of photoexcited molecules is an important task not only to understand the fundamental atomistic mechanisms but also to rationally design molecular properties and functions. Here, we present a combined experimental and theoretical study of the twisted intramolecular charge transfer (TICT) process in hemithioindigo photoswitches. Using ultrafast transient IR spectroscopy as the main analytical method, a detailed understanding of the extent and direction of charge transfer within the excited molecule is obtained. At the same time, the geometrical distortion is monitored directly via changes of indicative vibrational modes over the time course of the photoreaction. These high-resolution data deliver a detailed molecular movie of the TICT process in this important class of chromophores with picosecond time resolution.

Folding and Unfolding of the Short Light-Triggered ß-Hairpin Peptide AzoChignolin Occurs within 100 ns.

To map the underlying molecular mechanisms of folding dynamics in proteins, light-operated peptides have emerged as promising tools. In this study, we reveal the complete sequence of light-induced structural changes of AzoChignolin, a short ß-hairpin peptide containing an azobenzene photoswitch in its loop region. Light-triggered structural changes were monitored by time-resolved IR spectroscopy. Formation and destruction of the hairpin structure is very fast and occurs within 100 ns for AzoChignolin in methanol. Atomistic molecular dynamics simulations using two explicit solvents, methanol and water, revealed the underlying molecular processes and allowed us to gain further insight into the reaction mechanism. Despite its rapid reaction time, hairpin formation in these solvents is not force-driven by the molecular switch but proceeded via formation of interstrand hydrogen bonds and contacts between aromatic residues. Moreover, the combined experimental and theoretical study demonstrates that the solvent (methanol vs water) does not dictate the velocity of ß-hairpin formation in the AzoChignolin peptide comprising only a few hydrophobic residues in the strands.

UV-Induced Charge-Transfer States in Short Guanosine-Containing DNA Oligonucleotides.

Charge transfer has proven to be an important mechanism in DNA photochemistry. In particular, guanine (dG) plays a major role as an electron donor, but the photophysical dynamics of dG-containing charge-transfer states have not been extensively investigated so far. Here, we use UV pump (266 nm) and picosecond IR probe (∼5-7 µm) spectroscopy to study ultrafast dynamics in dG-containing short oligonucleotides as a function of sequence and length. For the pure purine oligomers, we observed lifetimes for the charge-transfer states of the order of several hundreds of picoseconds, regardless of the oligonucleotide length. In contrast, pyrimidine-containing dinucleotides d(GT) and d(GC) show much faster relaxation dynamics in the 10 to 30 ps range. In all studied nucleotides, the charge-transfer states are formed with an efficiency of the order of ∼50 %. These photophysical characteristics will lead to an improved understanding of DNA damage and repair processes.

Triplet-Induced Lesion Formation at CpT and TpC Sites in DNA.

UV irradiation induces DNA lesions particularly at dipyrimidine sites. Using time-resolved UV pump (250 nm) and mid-IR probe spectroscopy the triplet pathway of cyclobutane pyrimidine dimer (CPD) formation within TpC and CpT sequences was studied. The triplet state is initially localized at the thymine base but decays with 30 ns under formation of a biradical state extending over both bases of the dipyrimidine. Subsequently this state either decays back to the electronic ground state on the 100 ns time scale or forms a cyclobutane pyrimidine dimer lesion (CPD). Stationary IR spectroscopy and triplet sensitization via 2'-methoxyacetophenone (2-M) in the UVA range shows that the lesions are formed with an efficiency of approximately 1.5 %. Deamination converts the cytosine moiety of the CPD lesions on the time scale of 10 hours into uracil which gives CPD(UpT) and CPD(TpU) lesions in which the coding potential of the initial cytosine base is vanished.

The Photoaddition of a Psoralen to DNA Proceeds via the Triplet State.

Psoralens are natural compounds that serve in the light dependent treatment of certain skin diseases (PUVA therapy). They are DNA intercalators that upon photoexcitation form adducts with thymine bases. For one psoralen derivative, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), the photoreactions are characterized here by nanosecond UV-vis and IR absorption spectroscopy. The triplet state of AMT is identified as the reactive one. On the 1-10 µs time scale this local triplet state transforms into a triplet biradical bearing one single bond between the addends. Within ∼50 µs this biradical forms the final adduct featuring a cyclobutane ring. This kinetic behavior is in stark contrast to the closely related photoaddition of two thymine moieties within the DNA. Origins of the differences are discussed.

Decay Pathways of Thymine Revisited.

The decay of electronically excited states of thymine (Thy) and thymidine 5'-monophosphate (TMP) was studied by time-resolved UV/vis and IR spectroscopy. In addition to the well-established ultrafast internal conversion to the ground state, a so far unidentified UV-induced species is observed. In D2O, this species decays with a time constant of 300 ps for thymine and of 1 ns for TMP. The species coexists with the lowest triplet state and is formed with a comparably high quantum yield of about 10% independent of the solvent. The experimentally determined spectral signatures are discussed in the light of quantum chemical calculations of the singlet and triplet excited states of thymine.

Photophysics of diphenyl-pyrazole compounds in solutions and α-synuclein aggregates.

BACKGROUND: Recently diphenyl-pyrazole (DPP) compounds and especially anle138b were found to reduce the aggregation of α-synuclein or Tau protein in vitro as well as in a mouse model of neurodegenerative diseases [1,2]. Direct interaction of the DPPs with the fibrillar structure was identified by fluorescence spectroscopy. Thereby a strong dependence of the fluorescence on the surroundings could be identified [3]. METHODS: Stationary and time-resolved emission experiments were performed on DPP compounds substituted by different halogens. RESULTS: The compounds reveal a pronounced dependence of the fluorescence on the surrounding solvent. In non-polar solvents they show strong emission in the blue part of the spectrum while in polar and proton donating solvents, such as water or acetic acid a dual fluorescence can be observed where a red-shifted emission points to a charge transfer in the excited state with large dipole moment. Non-radiative processes including photochemical reactions are observed for DPP substituted with heavy halogens. Upon binding of anle138b and its derivatives to protein fibrils in aqueous buffer, strong enhancement of the fluorescence at short wavelengths is found. CONCLUSION: The investigations of the DPPs in different surroundings lead to a detailed model of the fluorescence characteristics. We propose a model for the binding in fibrils of different proteins, where the DPP is located in a hydrophobic groove independent of the specific sequence of the amino acids. GENERAL SIGNIFICANCE: These investigations characterize the binding site of the DPP anle138b in protein aggregates and contribute to the understanding of the therapeutic mode of action of this compound.

Ingredients to TICT Formation in Donor Substituted Hemithioindigo.

Twisted intramolecular charge transfer (TICT) formation in hemithioindigo photoswitches has recently been reported and constitutes a second deexcitation pathway complementary to photoisomerization. Typically, this behavior is not found for this type of photoswitches, and it takes special geometric and electronic conditions to realize it. Here we present a systematic study that identifies the molecular preconditions leading to TICT formation in donor substituted hemithioindigo, which can thus serve as a frame of reference for other photoswitching systems. By varying the substitution pattern and providing an in-depth physical characterization including time-resolved and quantum yield measurements, we found that neither ground-state pretwisting along the rotatable single bond nor the introduction of strong push-pull character across the photoisomerizable double bond alone leads to formation of TICT states. Only the combination of both ingredients produces light-induced TICT behavior in polar solvents.

Twisted Hemithioindigo Photoswitches: Solvent Polarity Determines the Type of Light-Induced Rotations.

Controlling the internal motions of molecules by outside stimuli is a decisive task for the generation of responsive and complex molecular behavior and functionality. Light-induced structural changes of photoswitches are of special high interest due to the ease of signal application and high repeatability. Typically photoswitches use one reaction coordinate in their switching process and change between two more or less-defined states. Here we report on new twisted hemithioindigo photoswitches enabling two different reaction coordinates to be used for the switching process. Depending on the polarity of the solvent, either complete single bond (in DMSO) or double bond (in cyclohexane) rotation can be induced by visible light. This mutually independent switching establishes an unprecedented two-dimensional control of intramolecular rotations in this class of photoswitches. The mechanistic explanation involves formation of highly polar twisted intramolecular charge-transfer species in the excited state and is based on a large body of experimental quantifications, most notably ultrafast spectroscopy and quantum yield measurements in solvents of different polarity. The concept of pre-twisting in the ground state to open new, independent reaction coordinates in the excited state should be transferable to other photoswitching systems.

UV-Induced Charge Transfer States in DNA Promote Sequence Selective Self-Repair.

Absorption of UV-radiation in nucleotides initiates a number of photophysical and photochemical processes, which may finally cause DNA damage. One major decay channel of photoexcited DNA leads to reactive charge transfer states. This study shows that these states trigger self-repair of DNA photolesions. The experiments were performed by UV spectroscopy and HPLC on different single and double stranded oligonucleotides containing a cyclobutane pyrimidine dimer (CPD) lesion. In a first experiment we show that photoexcitation of adenine adjacent to a CPD has no influence on this lesion. However, excitation of a guanine (G) adenine (A) sequence leads to reformation of the intact thymine (T) bases. The involvement of two bases for the repair points to a long-living charge transfer state between G and A to be responsible for the repair. The negatively charged A radical anion donates an electron to the CPD, inducing ring splitting and repair. In contrast, a TA sequence, having an inverted charge distribution (T radical anion, A radical cation), is not able to repair the CPD lesion. The investigations show that the presence of an adjacent radical ion is not sufficient for repair. More likely it is the driving power represented by the oxidation potential of the radical ion, which controls the repair. Thus, repair capacities are strongly sequence-dependent, creating DNA regions with different tendencies of self-repair. This self-healing activity represents the simplest sequence-dependent DNA repair system.

Quantum Yield of Cyclobutane Pyrimidine Dimer Formation Via the Triplet Channel Determined by Photosensitization.

UV-induced formation of the cyclobutane pyrimidine dimer (CPD) lesion is investigated by stationary and time-resolved photosensitization experiments. The photosensitizer 2'-methoxyacetophenone with high intersystem crossing efficiency and large absorption cross-section in the UV-A range was used. A diffusion controlled reaction model is presented. Time-resolved experiments confirmed the validity of the reaction model and provided information on the dynamics of the triplet sensitization process. With a series of concentration dependent stationary illumination experiments, we determined the quantum efficiency for CPD formation from the triplet state of the thymine dinucleotide TpT to be 4 ± 0.2%.

Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies.

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.

2'-Methoxyacetophenone: An Efficient Photosensitizer for Cyclobutane Pyrimidine Dimer Formation.

Stationary and time-resolved experiments show that 2'-methoxyacetophenone (2-M) is an interesting compound for the investigation of triplet states in thymine samples. Time-resolved emission experiments show that the fluorescence lifetime of 2-M is 660 ps. A similar time constant of 680 ps is found in transient IR experiments. The data indicate efficient intersystem crossing (≈97%) from the fluorescent singlet state to the triplet state. The lifetime of the triplet state of 2-M dissolved in D2O at room temperature and ambient oxygen concentration is 400 ns. 2-M has a strong absorption in the UV-A range and can photosensitize the triplet state of a thymidine dinucleotide with light at a wavelength of 320 nm. The experiments show that 2-M is well-suited for time-resolved experiments on the triplet-sensitizing process.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates.

BACKGROUND: Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. METHODS: Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. RESULTS: The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd=190±120nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. CONCLUSION: Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. GENERAL SIGNIFICANCE: The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes.

Dewar Lesion Formation in Single- and Double-Stranded DNA is Quenched by Neighboring Bases.

UV-induced Dewar lesion formation is investigated in single- and double-stranded oligonucleotides with ultrafast vibrational spectroscopy. The quantum yield for the conversion of the (6-4) lesion to the Dewar isomer in DNA strands is reduced by a factor of 4 in comparison to model dinucleotides. Time resolved spectroscopy reveals a fast process in the excited state with spectral characteristics of bases which are adjacent to the excited (6-4) lesion. These kinetic components have large amplitudes and indicate that an additional quenching channel acts in the stranded DNA systems and reduces the Dewar formation yield. Presumably relaxation evolves via a charge transfer to the neighboring guanine and the paired cytosine participates in a double-stranded oligomer. Changes in the decay of the relaxed excited electronic state of the (6-4) chromophore point to modifications in the excited state energy landscape which may lead to an additional reduction of the Dewar formation yield.

A magnetic stirring setup for applications in ultrafast spectroscopy of photo-sensitive solutions.

An exchange system is presented, which allows ultrafast experiments with high excitation rates (1 kHz) on samples with reaction cycles in the range of a few seconds and small sample volumes of about 0.3 ml. The exchange is accomplished using a commercially available cuvette by the combination of a special type of magnetic stirring with transverse translational motion of the sample cuvette.

Early events of DNA photodamage.

Ultraviolet (UV) radiation is a leading external hazard to the integrity of DNA. Exposure to UV radiation triggers a cascade of chemical reactions, and many molecular products (photolesions) have been isolated that are potentially dangerous for the cellular system. The early steps that take place after UV absorption by DNA have been studied by ultrafast spectroscopy. The review focuses on the evolution of excited electronic states, the formation of photolesions, and processes suppressing their formation. Emphasis is placed on lesions involving two thymine bases, such as the cyclobutane pyrimidine dimer, the (6-4) lesion, and its Dewar valence isomer.

Identification of charge separated states in thymine single strands.

UV excitation of the DNA single strand (dT)18 leads to electronically excited states that are potential gateways to DNA photolesions. Using time-resolved infrared spectroscopy we characterized a species with a lifetime of ∼100 ps and identified it as a charge separated excited state between two thymine bases.

Making fast photoswitches faster--using Hammett analysis to understand the limit of donor-acceptor approaches for faster hemithioindigo photoswitches.

Hemithioindigo (HTI) photoswitches have a tremendous potential for biological and supramolecular applications due to their absorptions in the visible-light region in conjunction with ultrafast photoisomerization and high thermal bistability. Rational tailoring of the photophysical properties for a specific application is the key to exploit the full potential of HTIs as photoswitching tools. Herein we use time-resolved absorption spectroscopy and Hammett analysis to discover an unexpected principal limit to the photoisomerization rate for donor-substituted HTIs. By using stationary absorption and fluorescence measurements in combination with theoretical investigations, we offer a detailed mechanistic explanation for the observed rate limit. An alternative way of approaching and possibly even exceeding the maximum rate by multiple donor substitution is demonstrated, which give access to the fastest HTI photoswitch reported to date.