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.

Studies of DNA 'Breathing' by Polarization-Sweep Single-Molecule Fluorescence Microscopy of Exciton-Coupled (iCy3)2 Dimer-Labeled DNA Fork Constructs.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA 'breathing') play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here, we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we tested here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one antisymmetric (-) exciton, with the respective transition dipole moments oriented perpendicular to one another. We excite single-molecule samples using a continuous-wave linearly polarized laser, with the polarization direction continuously rotated at the frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and antisymmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and the conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined, the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome-regulatory proteins.

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.

Studies of Local DNA Backbone Conformation and Conformational Disorder Using Site-Specific Exciton-Coupled Dimer Probe Spectroscopy.

The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

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.

Temperature-dependent local conformations and conformational distributions of cyanine dimer labeled single-stranded-double-stranded DNA junctions by 2D fluorescence spectroscopy.

DNA replication and the related processes of genome expression require binding, assembly, and function of protein complexes at and near single-stranded (ss)-double-stranded (ds) DNA junctions. These central protein-DNA interactions are likely influenced by thermally induced conformational fluctuations of the DNA scaffold across an unknown distribution of functionally relevant states to provide regulatory proteins access to properly conformed DNA binding sites. Thus, characterizing the nature of conformational fluctuations and the associated structural disorder at ss-dsDNA junctions is critical for understanding the molecular mechanisms of these central biological processes. Here, we describe spectroscopic studies of model ss-dsDNA fork constructs that contain dimers of "internally labeled" cyanine (iCy3) chromophore probes that have been rigidly inserted within the sugar-phosphate backbones of the DNA strands. Our combined analyses of absorbance, circular dichroism, and two-dimensional fluorescence spectroscopy permit us to characterize the local conformational parameters and conformational distributions. We find that the DNA sugar-phosphate backbones undergo abrupt successive changes in their local conformations-initially from a right-handed and ordered DNA state to a disordered splayed-open structure and then to a disordered left-handed conformation-as the dimer probes are moved across the ss-dsDNA junction. Our results suggest that the sugar-phosphate backbones at and near ss-dsDNA junctions adopt specific position-dependent local conformations and exhibit varying extents of conformational disorder that deviate widely from the Watson-Crick structure. We suggest that some of these conformations can function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites.

Entangled two-photon absorption by atoms and molecules: A quantum optics tutorial.

Two-photon absorption (TPA) and other nonlinear interactions of molecules with time-frequency-entangled photon pairs have been predicted to display a variety of fascinating effects. Therefore, their potential use in practical quantum-enhanced molecular spectroscopy requires close examination. This Tutorial presents a detailed theoretical study of one- and two-photon absorption by molecules, focusing on how to treat the quantum nature of light. We review some basic quantum optics theory and then we review the density-matrix (Liouville) derivation of molecular optical response, emphasizing how to incorporate quantum states of light into the treatment. For illustration, we treat in detail the TPA of photon pairs created by spontaneous parametric down conversion, with an emphasis on how quantum light TPA differs from that with classical light. In particular, we treat the question of how much enhancement of the TPA rate can be achieved using entangled states. This Tutorial includes a review of known theoretical methods and results as well as some extensions, especially the comparison of TPA processes that occur via far-off-resonant intermediate states only and those that involve off-resonant intermediate states by virtue of dephasing processes. A brief discussion of the main challenges facing experimental studies of entangled two-photon absorption is also given.

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.

Submillisecond Conformational Transitions of Short Single-Stranded DNA Lattices by Photon Correlation Single-Molecule Förster Resonance Energy Transfer.

Thermally driven conformational fluctuations (or "breathing") of DNA play important roles in the function and regulation of the "macromolecular machinery of genome expression." Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here, we use microsecond-resolved single-molecule Förster resonance energy transfer (smFRET) experiments to investigate the backbone fluctuations of short [oligo(dT)n] templates within DNA constructs that also serve as models for ss-dsDNA junctions. Such junctions, together with the attached ssDNA sequences, are involved in interactions with the ssDNA binding (ssb) proteins that control and integrate the functions of DNA replication complexes. We analyze these data using a chemical network model based on multiorder time-correlation functions and probability distribution functions that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs interconvert, on submillisecond time scales, between three macrostates with distinctly different end-to-end distances. These are (i) a "compact" macrostate that represents the dominant species at equilibrium; (ii) a "partially extended" macrostate that exists as minority species; and (iii) a "highly extended" macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA-associating proteins.

Quantifying the enhancement of two-photon absorption due to spectral-temporal entanglement.

When a low flux of time-frequency-entangled photon pairs (EPP) illuminates a two-photon transition, the rate of two-photon absorption (TPA) can be enhanced considerably by the quantum nature of photon number correlations and frequency correlations. We use a quantum-theoretic derivation of entangled TPA (ETPA) and calculate an upper bound on the amount of quantum enhancement that is possible in such systems. The derived bounds indicate that in order to observe ETPA the experiments would need to operate at a combination of significantly higher rates of EPP illumination, molecular concentrations, and conventional TPA cross sections than are achieved in typical experiments.

Dinucleotides as simple models of the base stacking-unstacking component of DNA 'breathing' mechanisms.

Regulatory protein access to the DNA duplex 'interior' depends on local DNA 'breathing' fluctuations, and the most fundamental of these are thermally-driven base stacking-unstacking interactions. The smallest DNA unit that can undergo such transitions is the dinucleotide, whose structural and dynamic properties are dominated by stacking, while the ion condensation, cooperative stacking and inter-base hydrogen-bonding present in duplex DNA are not involved. We use dApdA to study stacking-unstacking at the dinucleotide level because the fluctuations observed are likely to resemble those of larger DNA molecules, but in the absence of constraints introduced by cooperativity are likely to be more pronounced, and thus more accessible to measurement. We study these fluctuations with a combination of Molecular Dynamics simulations on the microsecond timescale and Markov State Model analyses, and validate our results by calculations of circular dichroism (CD) spectra, with results that agree well with the experimental spectra. Our analyses show that the CD spectrum of dApdA is defined by two distinct chiral conformations that correspond, respectively, to a Watson-Crick form and a hybrid form with one base in a Hoogsteen configuration. We find also that ionic structure and water orientation around dApdA play important roles in controlling its breathing fluctuations.

Fluorescence-detected Fourier transform electronic spectroscopy by phase-tagged photon counting.

Fluorescence-detected Fourier transform (FT) spectroscopy is a technique in which the relative paths of an optical interferometer are controlled to excite a material sample, and the ensuing fluorescence is detected as a function of the interferometer path delay and relative phase. A common approach to enhance the signal-to-noise ratio in these experiments is to apply a continuous phase sweep to the relative optical path, and to detect the resulting modulated fluorescence using a phase-sensitive lock-in amplifier. In many important situations, the fluorescence signal is too weak to be measured using a lock-in amplifier, so that photon counting techniques are preferred. Here we introduce an approach to low-signal fluorescence-detected FT spectroscopy, in which individual photon counts are assigned to a modulated interferometer phase ('phase-tagged photon counting,' or PTPC), and the resulting data are processed to construct optical spectra. We studied the fluorescence signals of a molecular sample excited resonantly by a pulsed coherent laser over a range of photon flux and visibility levels. We compare the performance of PTPC to standard lock-in detection methods and establish the range of signal parameters over which meaningful measurements can be carried out. We find that PTPC generally outperforms the lock-in detection method, with the dominant source of measurement uncertainty being associated with the statistics of the finite number of samples of the photon detection rate.

Local DNA Base Conformations and Ligand Intercalation in DNA Constructs Containing Optical Probes.

Understanding local conformations of DNA at the level of individual nucleic acid bases and base pairs is important for elucidating molecular processes that depend on DNA sequence. Here, we apply linear absorption and circular dichroism measurements to the study of local DNA conformations, using the guanine base analog 6-methyl isoxanthopterin (6-MI) as a structural probe. We show that the spectroscopic properties of this probe can provide detailed information about the average local base and basepair conformations as a function of the surrounding DNA sequence. Based on these results we apply a simple theoretical model to calculate the circular dichroism spectra of 6-MI-substituted DNA constructs and show that our model can be used to extract information about how the local conformations of the 6-MI probe are influenced by the local base or basepair environment. We also use this probe to examine the pathway for the insertion (intercalation) of a tethered acridine ligand (9-amino-6-chloro methoxyacridine) into duplex DNA. We show that this model intercalator interacts with duplex DNA by a "displacement insertion intercalation" mechanism, whereby the acridine moiety is inserted into the DNA structure and displaces the base located opposite its attachment site. These findings suggest that site-specifically positioned base analog probes can be used to characterize the molecular and structural details of binding ligand effects on local base stacking and unstacking reactions in single- and double-stranded DNA and thus may help to define the molecular mechanisms of DNA-protein interactions that involve the site-specific intercalation of aromatic amino acid side chains into genomic DNA.

Measuring local conformations and conformational disorder of (Cy3)2 dimer labeled DNA fork junctions using absorbance, circular dichroism and two-dimensional fluorescence spectroscopy.

The sugar-phosphate backbone of DNA near single-stranded (ss)-double-stranded (ds) junctions likely fluctuates within a broad distribution of conformations to permit the proper binding of genome regulatory proteins that function at these sites. In this work we use absorbance, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS) to study the local conformations and conformational disorder within chromophore-labeled DNA constructs. These constructs employ dimers of the fluorescent chromophore Cy3 that are site-specifically incorporated into the sugar-phosphate backbones of DNA strands at ss-ds DNA fork junctions. We show that these data can be analyzed to determine the local conformations of the (Cy3)2 dimer, and the degree of conformational disorder. Our analysis employs an essential-state Holstein-Frenkel Hamiltonian model, which takes into account the internal electronic-vibrational motions within each Cy3 chromophore, and the resonant electronic interaction that couples the two chromophores together. Our results suggest that this approach may be applied generally to understand local backbone conformation and conformational disorder at ss-ds DNA fork junctions.

Temperature-dependent conformations of exciton-coupled Cy3 dimers in double-stranded DNA.

Understanding the properties of electronically interacting molecular chromophores, which involve internally coupled electronic-vibrational motions, is important to the spectroscopy of many biologically relevant systems. Here we apply linear absorption, circular dichroism, and two-dimensional fluorescence spectroscopy to study the polarized collective excitations of excitonically coupled cyanine dimers (Cy3)2 that are rigidly positioned within the opposing sugar-phosphate backbones of the double-stranded region of a double-stranded (ds)-single-stranded (ss) DNA fork construct. We show that the exciton-coupling strength of the (Cy3)2-DNA construct can be systematically varied with temperature below the ds-ss DNA denaturation transition. We interpret spectroscopic measurements in terms of the Holstein vibronic dimer model, from which we obtain information about the local conformation of the (Cy3)2 dimer, as well as the degree of static disorder experienced by the Cy3 monomer and the (Cy3)2 dimer probe locally within their respective DNA duplex environments. The properties of the (Cy3)2-DNA construct we determine suggest that it may be employed as a useful model system to test fundamental concepts of protein-DNA interactions and the role of electronic-vibrational coherence in electronic energy migration within exciton-coupled bio-molecular arrays.

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks.

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.