Spectroscopic approaches for studies of site-specific DNA base and backbone 'breathing' using exciton-coupled dimer-labeled DNA.

DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites.

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of 'internally-labeled' (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.

Accurate Modeling of Excitonic Coupling in Cyanine Dye Cy3.

Accurate modeling of excitonic coupling in molecules is of great importance for inferring the structures and dynamics of coupled systems. Cy3 is a cyanine dye that is widely used in molecular spectroscopy. Its well-separated excitation bands, high sensitivity to the surroundings, and the high energy transfer efficiency make it a perfect choice for excitonic coupling experiments. Many methods have been used to model the excitonic coupling in molecules with varying degrees of accuracy. The atomic transition charge model offers a high-accuracy and cost-effective way to calculating the excitonic coupling. The main focus of this work is to generate high-quality atomic transition charges that can accurately model the Cy3 dye's transition density. The transition density of the excitation of the ground to first excited state is calculated using configuration-interaction singles and time-dependent density functional theory and is benchmarked against the algebraic diagrammatic construction method. Using the transition density we derived the atomic transition charges using two approaches: Mulliken population analysis and charges fitted to the transition electrostatic potential. The quality of the charges is examined, and their ability to accurately calculate the excitonic coupling is assessed via comparison to experimental data of an artificial biscyanine construct. Theoretical comparisons to the supermolecule ab initio couplings and the widely used point-dipole approximation are also made. Results show that using the transition electrostatic potential is a reliable approach for generating the transition atomic charges. A high-quality set of charges, that can be used to model the Cy3 dye dimer excitonic coupling with high-accuracy and a reasonable computational cost, is obtained.

Aldehyde levels in e-cigarette aerosol: Findings from a replication study and from use of a new-generation device.

PURPOSE: A recent study identified high aldehyde emissions from e-cigarettes (ECs), that when converted to reasonable daily human EC liquid consumption, 5 g/day, gave formaldehyde exposure equivalent to 604-3257 tobacco cigarettes. We replicated this study and also tested a new-generation atomizer under verified realistic (no dry puff) conditions. DESIGN: CE4v2 atomizers were tested at 3.8 V and 4.8 V, and a Nautilus Mini atomizer was tested at 9.0 W and 13.5 W. All measurements were performed in a laboratory ISO-accredited for EC aerosol collection and aldehyde measurements. RESULTS: CE4v2 generated dry puffs at both voltage settings. Formaldehyde levels were >10-fold lower, acetaldehyde 6-9-fold lower and acrolein 16-26-fold lower than reported in the previous study. Nautilus Mini did not generate dry puffs, and minimal aldehydes were emitted despite >100% higher aerosol production per puff compared to CE4v2 (formaldehyde: 16.7 and 16.5 µg/g; acetaldehyde: 9.6 and 10.3 µg/g; acrolein: 8.6 and 11.7 µg/g at 9.0 W and 13.5 W, respectively). EC liquid consumption of 5 g/day reduces aldehyde exposure by 94.4-99.8% compared to smoking 20 tobacco cigarettes. CONCLUSION: Checking for dry puffs is essential for EC emission testing. Under realistic conditions, new-generation ECs emit minimal aldehydes/g liquid at both low and high power. Validated methods should be used when analyzing EC aerosol.

Evaluation of electronic cigarette liquids and aerosol for the presence of selected inhalation toxins.

INTRODUCTION: The purpose of this study was to evaluate sweet-flavored electronic cigarette (EC) liquids for the presence of diacetyl (DA) and acetyl propionyl (AP), which are chemicals approved for food use but are associated with respiratory disease when inhaled. METHODS: In total, 159 samples were purchased from 36 manufacturers and retailers in 7 countries. Additionally, 3 liquids were prepared by dissolving a concentrated flavor sample of known DA and AP levels at 5%, 10%, and 20% concentration in a mixture of propylene glycol and glycerol. Aerosol produced by an EC was analyzed to determine the concentration of DA and AP. RESULTS: DA and AP were found in 74.2% of the samples, with more samples containing DA. Similar concentrations were found in liquid and aerosol for both chemicals. The median daily exposure levels were 56 µg/day (IQR: 26-278 µg/day) for DA and 91 µg/day (IQR: 20-432 µg/day) for AP. They were slightly lower than the strict NIOSH-defined safety limits for occupational exposure and 100 and 10 times lower compared with smoking respectively; however, 47.3% of DA and 41.5% of AP-containing samples exposed consumers to levels higher than the safety limits. CONCLUSIONS: DA and AP were found in a large proportion of sweet-flavored EC liquids, with many of them exposing users to higher than safety levels. Their presence in EC liquids represents an avoidable risk. Proper measures should be taken by EC liquid manufacturers and flavoring suppliers to eliminate these hazards from the products without necessarily limiting the availability of sweet flavors.

Contrasting photophysical properties of star-shaped vs linear perylene diimide complexes.

The absorption line shapes of a series of linear and star-shaped perylene diimide (PDI) complexes are evaluated theoretically and compared to experiment. The cyclic trimer and tetrahedral complexes are part of the symmetric series, characterized by a single interchromophoric coupling, J(0), between any two PDI chromophores. The measured spectra of all complexes show pronounced vibronic progressions based on the symmetric ring stretching mode at ~1400 cm(-1). The spectral line shapes are accurately reproduced using a Holstein Hamiltonian parametrized with electronic couplings calculated using time-dependent density functional transition charge densities. Although the "head-to-tail" linear complexes display classic J-aggregate behavior, the star-shaped complexes display a unique photophysical response, which is neither J- nor H-like. In the symmetric N-mers (N = 2-4), absorption and emission are polarized along N - 1 directions in contrast to linear complexes where absorption and emission remain polarized along the long molecular axis. In the symmetric complexes the red-shift of the 0-0 peak with increasing |J(0)|, as well as the initial linear rise of the 0-0/1-0 oscillator strength ratio with increasing |J(0)|, are independent of the number of PDI chromophores, N, and are markedly smaller than what is found in the linear series, where the shifts and ratios depend on N. Moreover, whereas the radiative decay rate, γ(r), scales with N and is therefore superradiant in linear complexes, γ(r) scales with N/(N - 1) in the symmetric complexes. Vibronic/vibrational pair states (two-particle states) are found to profoundly affect the absorption line shapes of both linear and symmetric complexes for sufficiently large coupling.

A benchmark of excitonic couplings derived from atomic transition charges.

In this report we benchmark Coulombic excitonic couplings between various pairs of chromophores calculated using transition charges localized on the atoms of each monomer chromophore, as derived from a Mulliken population analysis of the monomeric transition densities. The systems studied are dimers of 1-methylthymine, 1-methylcytosine, 2-amino-9-methylpurine, all-trans-1,3,5-hexatriene, all-trans-1,3,5,7-octatetraene, trans-stilbene, naphthalene, perylenediimide, and dithia-anthracenophane. Transition densities are taken from different single-reference electronic structure excited state methods: time-dependent density functional theory (TDDFT), configuration-interaction singles (CIS), and semiempirical methods based on intermediate neglect of differential overlap. Comparisons of these results with full ab initio calculations of the electronic couplings using a supersystem are made, as are comparisons with experimental data. Results show that the transition charges do a good job of reproducing the supersystem couplings for dimers with moderate to long-range interchromophore separation. It is also found that CIS supermolecular couplings tend to overestimate the couplings, and often the transition charges approach may be better, due to fortuitous cancellation of errors.

Complexes of 2,5-bis(α-pyridyl)pyrrolate with Pd(II) and Pt(II): a monoanionic iso-π-electron ligand analog of terpyridine.

Palladium and platinum metal complexes of 2,5-bis(α-pyridyl)pyrrolate (PDP) are reported and characterized by spectroscopic methods, single-crystal X-ray diffraction, and elemental analysis. The single-crystal X-ray structures of these complexes exhibit structural features indicative of significant π-backbonding. To illustrate the effect, bond lengths are statistically compared to unmetalated PDP and to a previously reported Zn(II) complex that exhibits no backbonding. Density functional theory calculations are used to aid understanding of the electronic structural basis of the observed phenomena.

Distinguishing between relaxation pathways by combining dissociative ionization pump probe spectroscopy and ab initio calculations: a case study of cytosine.

We present a general method for tracking molecular relaxation along different pathways from an excited state down to the ground state. We follow the excited state dynamics of cytosine pumped near the S(0)-S(1) resonance using ultrafast laser pulses in the deep ultraviolet and probed with strong field near infrared pulses which ionize and dissociate the molecules. The fragment ions are detected via time of flight mass spectroscopy as a function of pump probe delay and probe pulse intensity. Our measurements reveal that different molecular fragments show different timescales, indicating that there are multiple relaxation pathways down to the ground state. We interpret our measurements with the help of ab initio electronic structure calculations of both the neutral molecule and the molecular cation for different conformations en route to relaxation back down to the ground state. Our measurements and calculations show passage through two seams of conical intersections between ground and excited states and demonstrate the ability of dissociative ionization pump probe measurements in conjunction with ab initio electronic structure calculations to track molecular relaxation through multiple pathways.

Photophysical pathways of cytosine in aqueous solution.

The effects of aqueous solvation on the photophysical pathways involving the S(1) excited state in cytosine have been studied with a mean-field QM/MM approach. Two main pathways with small barriers were found previously in isolated cytosine, using multireference configuration interaction (MRCI) methods, that facilitate radiationless decay to the ground state. These pathways are examined in solvated cytosine using a mean-field QM/MM combined with MRCI, and it is found that barriers in each direction increase moderately. The barriers in the presence of the solvent are 0.23 eV and 0.31 eV for the two different pathways compared to 0.15 eV and 0.14 eV in the gas phase, indicating that the aqueous environment does not make one of the two directions much more preferable.

Excited-state energies and electronic couplings of DNA base dimers.

The singlet excited electronic states of two pi-stacked thymine molecules and their splittings due to electronic coupling have been investigated with a variety of computational methods. Focus has been given on the effect of intermolecular distance on these energies and couplings. Single-reference methods, CIS, CIS(2), EOM-CCSD, TDDFT, and the multireference method CASSCF, have been used, and their performance has been compared. It is found that the excited-state energies are very sensitive to the applied method but the couplings are not as sensitive. Inclusion of diffuse functions in the basis set also affects the excitation energies significantly but not the couplings. TDDFT is inadequate in describing the states and their coupling, while CIS(2) gives results very similar to EOM-CCSD. Excited states of cytosine and adenine pi-stacked dimers were also obtained and compared with those of thymine dimers to gain a more general picture of excited states in pi-stacked DNA base dimers. The coupling is very sensitive to the relative position and orientation of the bases, indicating great variation in the degree of delocalization of the excited states between stacked bases in natural DNA as it fluctuates.

Change in electronic structure upon optical excitation of 8-vinyladenosine: an experimental and theoretical study.

8-Vinyladenosine (8VA) is an adenosine analog, like 2-aminopurine (2AP), that has a red-shifted absorption and high fluorescence quantum yield. When introduced into double-stranded DNA (dsDNA), its base-pairing and base-stacking properties are similar to those of adenine. Of particular interest, the fluorescence quantum yield of 8VA is sensitive to base stacking, making it a very useful real-time probe of DNA structure. The fundamental photophysics underlying this fluorescence quenching by base stacking is not well understood, and thus exploring the excited state electronic structure of the analog is warranted. In this study, we report on changes in the electronic structure of 8VA upon optical excitation. Stark spectroscopy was performed on 8VA monomer in frozen ethanol glass at 77 K to obtain the direction and degree of charge redistribution in the form of the difference dipole moment, Deltamu(01) = 4.7 +/- 0.3 D, and difference static polarizability, tr(Delta(alpha)01) = 21 +/- 11 A(3), for the S(0)-->S(1) transition. In addition, solvatochromism experiments were performed on 8VA in various solvents and analyzed using Bakhshiev's model. High level ab initio methods were employed to calculate transition energies, oscillator strengths, and dipole moments of the ground and excited states of 8VA. The direction of Deltamu(01) was assigned in the molecular frame for the lowest optically accessible state. Our study shows that the angle between ground and excited state dipole moment plays a critical role in understanding the change in electronic structure upon optical excitation. Compared to 2AP, 8VA has a larger difference dipole moment which, with twice the extinction coefficient, suggests that 8VA is superior as a two-photon probe for microscopy studies. To this end, we have measured the ratio of the two-photon fluorescence yields of the two analogs by excitation at the respective monomer absorption maxima. We show that 8VA is indeed a significantly brighter two-photon fluorophore, based on our experimental and computational results.

Solvatochromic shifts of uracil and cytosine using a combined multireference configuration interaction/molecular dynamics approach and the fragment molecular orbital method.

A recently developed combined quantum mechanics/molecular mechanics (QM/MM) approach has been applied to the calculation of solvatochromic shifts of the excited states of the pyrimidine nucleobases uracil and cytosine in aqueous solution. In this procedure the quantum mechanical solute is described using a multireference configuration interaction method while molecular dynamics simulations are used to obtain the structure of the solvent around the solute. The fragment molecular orbital multiconfiguration self-consistent field (FMO-MCSCF) method of Fedorov and Kitaura is also used and compared with the QM/MM results. The two methods give similar results. The solvatochromic shifts in uracil are found to be +0.41 (+0.44) eV for the S(1) excited state and -0.05 (-0.19) eV for the S(2) state at the QM/MM (FMO-MCSCF) level. Solvatochromic shifts in cytosine are calculated to be +0.25 (+0.19), +0.56 (+0.62), and +0.83 (+0.83) eV for the S(1), S(2), and S(3) states, respectively, at the QM/MM (FMO-MCSCF) level.

Three-state conical intersections in cytosine and pyrimidinone bases.

Three-state conical intersections have been located and characterized for cytosine and its analog 5-methyl-2-pyrimidinone using multireference configuration-interaction ab initio methods. The potential energy surfaces for each base contain three different three-state intersections: two different S(0)-S(1)-S(2) intersections (gs/pi pi(*)/n(N)pi(*) and gs/pi pi(*)/n(O)pi(*)) and an S(1)-S(2)-S(3) intersection (pi pi(*)/n(N)pi(*)/n(O)pi(*)). Two-state seam paths from these intersections are shown to be connected to previously reported two-state conical intersections. Nonadiabatic coupling terms have been calculated, and the effects of the proximal third state on these quantities are detailed. In particular, it is shown that when one of these loops incorporates more than one seam point, there is a profound and predictable effect on the phase of the nonadiabatic coupling terms, and as such provides a diagnostic for the presence and location of additional seams. In addition, it is shown that each of the three three-state conical intersections located on cytosine and 5-methyl-2-pyrimidinone is qualitatively similar between the two bases in terms of energies and character, implying that, like with the stationary points and two-state conical intersections previously reported for these two bases, there is an underlying pattern of energy surfaces for 2-pyrimidinone bases, in general, and this pattern also includes three-state conical intersections.

2-Aminopurine excited state electronic structure measured by stark spectroscopy.

2-Aminopurine (2AP) is an adenine analogue that has a high fluorescence quantum yield. Its fluorescence yield decreases significantly when the base is incorporated into DNA, making it a very useful real-time probe of DNA structure. However, the basic mechanism underlying 2AP fluorescence quenching by base stacking is not well understood. A critical element in approaching this problem is obtaining an understanding of the electronic structure of the excited state. We have explored the excited state properties of 2AP and 2-amino,9-methylpurine (2A9MP) in frozen solutions using Stark spectroscopy. The experimental data were correlated with high level ab initio (MRCI) calculations of the dipole moments, mu0 and mu1, of the ground and excited states. The magnitude and direction of the dipole moment change, Deltamu01 = mu1 - mu0, of the lowest energy optically allowed transition was determined. While other studies have reported on the magnitude of the dipole moment change, we believe that this is the first report of the direction of Deltamu, a quantity that will be of great value in interpreting absorption spectral changes of the 2AP chromophore. Polarizability changes due to the transition were also obtained.

Cytosine in context: a theoretical study of substituent effects on the excitation energies of 2-pyrimidinone derivatives.

The ultrafast radiationless decay mechanism for cytosine has been shown to be in part dependent upon high vertical excitation, while slower fluorescence displayed in some cytosine analogs is generally linked to lower vertical excitation energies. To probe how excitation energies relate to pyrimidine structure, substituent effects on the vertical excitation energies for a number of derivatives of 2-pyrimidin-(1H)-one (2P) have been calculated using multireference configuration-interaction ab initio methods. Substitutions using groups with pi electron donating, withdrawing and conjugation-extending properties at the C(4) and C(5) positions on the 2P system give predictive trends for the first three singlet excited-state energies. The S(1) pipi* energies of 2P derivatives involving C4 substitution vary linearly with the Hammett substituent parameter sigma(P)+. Cytosine is shown to have the highest bright pipi* energy of the 2P derivatives presented, with that energy being strongly dependent on the position, orientation, and geometry of the C4-amino. A simple description of the predictive energetic trends for the bright pipi* energies using frontier molecular orbital theory is presented, based on the character of the HOMO and LUMO orbitals for each derivative. The results of this study expand the current understanding of the photophysical behavior of the DNA pyrimidine bases and could be useful in the design of analogs where particular spectral properties are desired.

Radiationless decay mechanism of cytosine: an ab initio study with comparisons to the fluorescent analogue 5-methyl-2-pyrimidinone.

The ultrafast radiationless decay mechanism of photoexcited cytosine has been theoretically supported by exploring the important potential energy surfaces using multireference configuration-interaction ab initio methods for the gas-phase keto-tautomer free base. At vertical excitation, the bright state is S1 (pipi*) at 5.14 eV, with S2 (nNpi*) and S3 (nOpi*) being dark states at 5.29 and 5.93 eV, respectively. Minimum energy paths connect the Franck-Condon region to a shallow minimum on the pipi* surface at 4.31 eV. Two different energetically accessible conical intersections with the ground state surface are shown to be connected to this minimum. One pathway involves N3 distorting out of plane in a sofa conformation, and the other pathway involves a dihedral twist about the C5-C6 bond. Each of these pathways from the minimum contains a low barrier of 0.14 eV, easily accessed by low vibronic levels. The path involving the N3 sofa distortion leads to a conical intersection with the ground state at 4.27 eV. The other pathway leads to an intersection with the ground state at 3.98 eV, lower than the minimum by about 0.3 eV. Comparisons with our previously reported study of the fluorescent cytosine analogue 5-methyl-2-pyrimidinone (5M2P) reveal remarkably similar conformational distortions throughout the decay pathways of both bases. The different photophysical behavior between the two molecules is attributed to energetic differences. Vertical excitation in cytosine occurs at a much higher energy initially, creating more vibrational energy than 5M2P in the Franck-Condon region, and the minimum S1 energy for 5M2P is too low to access an intersection with the ground state, causing population trapping and fluorescence. Calculations of vertical excitation energies of 5-amino-2-pyrimidinone and 2-pyrimidinone reveal that the higher excitation energy of cytosine is likely due to the presence of the amino group at the 4-position.

The fluorescence mechanism of 5-methyl-2-pyrimidinone: an ab initio study of a fluorescent pyrimidine analog.

The photophysically important potential energy surfaces of the fluorescent pyrimidine analog 5-methyl-2-pyrimidinone have been explored using multireference configuration-interaction ab initio methods at three levels of dynamical correlation, all of which support a fluorescence mechanism. At vertical excitation S1 (dark, n(N)pi*) and S2 (bright, pipi*) are almost degenerate at 4.4 eV, with S3 (dark, n(O)pi*) at 5.1 eV. The excited system can follow the S1-S2 seam of conical intersections, accessible from the Franck-Condon region, to its minimum and then evolve from this conical intersection on the S1 (pipi*) surface to a global minimum. At lower levels of correlation, the S1 surface shows two minima separated by a barrier of up to 0.18 eV. The secondary minimum found at the lower levels of correlation becomes the global minimum with higher correlation. The S1 population at this minimum can be trapped from accessing the lowest energy S0-S1 (pipi*/gs) conical intersection by an energy gap at least 0.3-0.4 eV higher than the S1 minimum. The calculated emission energy from this minimum is 2.80 eV. Gradient pathways connecting important S1 geometries are presented, as well as other excited state conical intersections.