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.

Unravelling the Origin of the Vibronic Spectral Signatures in an Excitonically Coupled Indocarbocyanine Cy3 Dimer.

The indocarbocyanine Cy3 dye is widely used to probe the dynamics of proteins and DNA. Excitonically coupled Cy3 dimers exhibit very unique spectral signatures that depend on the interchromophoric geometrical orientation induced by the environment, making them powerful tools to infer the dynamics of their surroundings. Understanding the origin of the dimeric spectral signatures is a necessity for an accurate interpretation of the experimental results. In this work, we simulate the vibronic spectrum of an experimentally well-studied Cy3 dimer, and we explain the origin of the experimental signatures present in its linear absorption spectrum. The Franck-Condon harmonic approximations, among other tests, are used to probe the factors contributing to the spectrum. It is found that the first peak in the absorption spectrum originates from the lower energy excitonic state, while the next two peaks are vibrational progressions of the higher energy excitonic state. The polar solvent plays a crucial role in the appearance of the spectrum, being responsible for the localized S1 minimum, which results in an increased intensity of the first peak.

Modeling the Electronic Absorption Spectra of the Indocarbocyanine Cy3.

Accurate modeling of optical spectra requires careful treatment of the molecular structures and vibronic, environmental, and thermal contributions. The accuracy of the computational methods used to simulate absorption spectra is limited by their ability to account for all the factors that affect the spectral shapes and energetics. The ensemble-based approaches are widely used to model the absorption spectra of molecules in the condensed-phase, and their performance is system dependent. The Franck-Condon approach is suitable for simulating high resolution spectra of rigid systems, and its accuracy is limited mainly by the harmonic approximation. In this work, the absorption spectrum of the widely used cyanine Cy3 is simulated using the ensemble approach via classical and quantum sampling, as well as, the Franck-Condon approach. The factors limiting the ensemble approaches, including the sampling and force field effects, are tested, while the vertical and adiabatic harmonic approximations of the Franck-Condon approach are also systematically examined. Our results show that all the vertical methods, including the ensemble approach, are not suitable to model the absorption spectrum of Cy3, and recommend the adiabatic methods as suitable approaches for the modeling of spectra with strong vibronic contributions. We find that the thermal effects, the low frequency modes, and the simultaneous vibrational excitations have prominent contributions to the Cy3 spectrum. The inclusion of the solvent stabilizes the energetics significantly, while its negligible effect on the spectral shapes aligns well with the experimental observations.

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.