Electronic Excitation Energy Transfer is an important intermolecular photophysical process that can affect the excited state lifetime of a chromophore. A molecule in an electronically excited state can return to the ground state by radiative as well as non-radiative processes. During the excited state lifetime, if the chromophore (energy donor) finds a suitable species (energy acceptor) nearby with resonant energy levels, it can transfer the excitation energy to that species and return to the ground state. This process is called Electronic Excitation Energy Transfer. When the energy donor is fluorescent, the process is called Fluorescence Resonance Energy Transfer (FRET) [1]. FRET is a non-radiative process that affects the fluorescence intensity as well as the excited state lifetime of the donor. It occurs due to the electrostatic coulombic interaction between the transition charge densities of the donor and the acceptor. The rate of energy transfer can be evaluated using the Fermi golden rule of quantum mechanics [2]. When the donor and the acceptor are separated by distances that are much larger in comparison with the sizes of the donor and the acceptor, the interaction between them can be thought of as that between their transition dipoles. In such a case, the interaction between the donor and the acceptor is dipolar and the rate of energy transfer has an R−6 dependence, where R is the distance between the donor and the acceptor [3]. This dependence has first been suggested theoretically by Forster in 1947 [4] followed by the experimental verification by Stryer and Haugland [5]. Since then the process has been used as a spectroscopic ruler to study the conformational dynamics of biopolymers like DNA, RNA, proteins etc [6]. A variety of dye molecules have been explored for donors and acceptors in FRET and the range of distances that can be measured using FRET involving dyes is in the range 1 − 10 nm. When the distances between the donor and the acceptor are not much larger in comparison with their sizes, the dipolar approximation to the interaction is not a very good approximation, thereby leading to deviations from the traditional R-6 dependence. Such non-R-6dependencies are found for polymers, quantum wells, quantum wires etc [7–9]. The interest in such dependencies is due to the need for developing nanoscopic rulers that can measure distances well beyond 10 nm. The objective of our work has been to study energy transfer from fluorophores to various kinds of acceptors that have extended charge densities and understand the distance dependence of the rate of energy transfer [10]. We use the Fermi golden rule as the starting point and develop analytical models for evaluating the rate as a function of the distance between the donor and the acceptor. We study the process of energy transfer from fluorescent dye molecules that serve as energy donors to a variety of energy acceptors namely, graphene, doped graphene, single-walled carbon nanotubes and metal nanoparticles. We also study transfer from fluorophores to a semiconducting sheet and a semiconducting tube of electronic charge density. There have been experimental studies in the literature of the fluorescence quenching of dyes near single-walled carbon nanotubes [11–13]. But, there are no studies of the distance dependence of rate. Single-walled carbon nanotubes can be thought of as rolled up sheets of graphene. However, interestingly, there were no reports of fluorescence quenching by graphene at the time when we thought of this possibility. Therefore, we first study the process of energy transfer from a fluorophore, which is kept at a distance z above a layer of graphene to the electronic energy levels of graphene. We find that the long range behavior of the rate has an z -4 dependence on the distance [14, 15]. From our study of transfer from pyrene to graphene, we find that fluorescence quenching can be experimentally observed up to a distance of ~ 30 nm, which is quite large in comparison with the traditional FRET limit (10 nm). Recent experiments that have been performed after our theory was reported have in fact observed the fluorescence quenching of dyes near graphene. Further, the process has been found to be very useful in fabricating devices based on graphene [16], in eliminating fluorescence signals in resonance Raman spectroscopy [17] and in visualizing graphene based sheets using fluorescence quenching microscopy [18]. The process has also been found to be useful in quantitative DNA analysis [19, 20]. We study the transfer of an amount of energy hΩ from a dye molecule to doped graphene [21]. We consider the shift of the Fermi level from the K-point into the conduction band of graphene as a result of doping and evaluate the rate of transfer. We find a crossover of the distance dependence of the rate from z -4 to exponential as the Fermi level is increasingly shifted into the conduction band, with the crossover occurring at a shift of the Fermi level by an amount hΩ/2. We study the process of transfer of excitation energy from a fluorophore kept at a distance d away from the surface of a carbon nanotube to the electronic energy levels of the nanotube. We find both exponential and d−5 behavior of the rate [22]. For the case of metallic nanotubes, when the emission energy of the fluorophore is less than a threshold, the dependence is exponential. Otherwise, it is d−5 . For the case of semiconducting nanotubes, we find that the rate follows an exponential dependence if the amount of energy that is transferred can cause only the excitonic transition of the tube. However, if any other band gap transition is allowed, the rate follows a d−5 dependence. For the case of transfer from pyrene to a (6, 4) nanotube, we find that energy transfer is appreciable up to a distance of ~ 17 nm. We then study the process of energy transfer from a fluorophore to a semiconducting sheet of electronic charge density [10]. We find that the rate has an z-4 dependence. For the case of transfer to a semiconducting tube, we find that the rate has a d -5dependence. The dependencies are in agreement with those obtained for graphene and carbon nanotubes respectively. This shows that the asymptotic distance dependencies are a consequence of the dimensionality of the transition charge densities and are robust. Strouse et al. [23, 24] have studied the process of energy transfer from the dye fluorescein to a 1.4 nm diameter gold nanoparticle. Double-stranded DNA molecules of various lengths were used to fix the distances between the donor and the acceptor. The rate was found to have a d-4distance dependence. They refer to this process as Nanoparticle Surface Energy Transfer (NSET) and the range of distances that can be measured using NSET is more than double that of the traditional FRET experiments. However, theoretical studies that consider the transfer to the plasmonic modes of the nanoparticle find a predominant R-6 dependence [25]. We study the process of energy transfer from the dye fluorescein to a 1.4 nm diameter gold nanoparticle considering the excitation of plasmons as well as electron-hole pairs of the nanoparticle [26]. We find that the rate follows the usual Forster type R−6 distance dependence at large distances. But, at short distances, there are contributions of the form R−-n with n > 6. This is due to the quadrupolar and octupolar modes of excitation of the nanoparticle, the rates corresponding to which have R-8 and R−-10 dependencies respectively. Recent calculations using DFT also find similar deviations at short distances [27].

Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules. In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase. I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.

I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.

A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.

Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin were consistent with the actoS1 docking model. However, the neck region was much closer to the actin filament than predicted by static atomic models. The efficiency of energy transfer between Cys 374 and the regulatory light chain was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached crossbridges which appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin. The resonance energy transfer data exclude a number of versions of the swinging lever arm model, and indicate that actin participation is indispensable for conformational changes leading to force generation. The conformational selection during weak binding at the actomyosin interface may precock the myosin head for the ensuing powerstroke.

<p>A RET network consists of a network of photo-active molecules called chromophores that can participate in inter-molecular energy transfer called resonance energy transfer (RET). RET networks are used in a variety of applications including cryptographic devices, storage systems, light harvesting complexes, biological sensors, and molecular rulers. In this dissertation, we focus on creating a RET device called closed-diffusive exciton valve (C-DEV) in which the input to output transfer function is controlled by an external energy source, similar to a semiconductor transistor like the MOSFET. Due to their biocompatibility, molecular devices like the C-DEVs can be used to introduce computing power in biological, organic, and aqueous environments such as living cells. Furthermore, the underlying physics in RET devices are stochastic in nature, making them suitable for stochastic computing in which true random distribution generation is critical.</p><p>In order to determine a valid configuration of chromophores for the C-DEV, we developed a systematic process based on user-guided design space pruning techniques and built-in simulation tools. We show that our C-DEV is 15x better than C-DEVs designed using ad hoc methods that rely on limited data from prior experiments. We also show ways in which the C-DEV can be improved further and how different varieties of C-DEVs can be combined to form more complex logic circuits. Moreover, the systematic design process can be used to search for valid chromophore network configurations for a variety of RET applications.</p><p>We also describe a feasibility study for a technique used to control the orientation of chromophores attached to DNA. Being able to control the orientation can expand the design space for RET networks because it provides another parameter to tune their collective behavior. While results showed limited control over orientation, the analysis required the development of a mathematical model that can be used to determine the distribution of dipoles in a given sample of chromophore constructs. The model can be used to evaluate the feasibility of other potential orientation control techniques.</p> / Dissertation

To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances.

Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.

More than one-third of all Magnetic Resonance Imaging (MRI) scans employ image-enhancing contrast agents to increase the differential signal intensity between diseased and normal tissue. Because current clinical contrast agents exhibit low relaxivity (mM-1 s-1), low dose efficiency, and rapid secretion, we have designed a group of protein-based MRI contrast agents with multiple gadolinium binding sites. In this study, the developed purification method for Class ProCA-3 agents allows for a quick and cost-effective way to abstract up to 109 mg of pure, soluble protein from a 1L E. Coli cell pellet devoid of DNA or RNA “contamination” for extensive animal studies. Circular dichroism far-UV spectra ensure the metal stability of the agents, revealing maintenance of their native α-helical structure in the presence and absence of metal ions. Furthermore, substantial evidence supports the high dose efficiency of these agents, exhibiting up to five folds higher relaxivity than their analogous commercial competitors.

Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.