Oxazine dye-conjugated dna oligonucleotides: Förster resonance energy transfer in view of molecular dye-DNA interactions

In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive F?rster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large F?rster distance of ~7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.     

The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer

A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5-300 nM and a high sensitivity with the limit of detection of 4.9 nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer-target and antibody-antigen recognitions.     

Steady‐state and time‐resolved fluorescence of tetramethylrhodamine attached to DNA: correlation with DNA sequences

Time‐resolved fluorescence as well as steady‐state absorption and fluorescence were detected in order to study the interactions between tetramethylrhodamine (TAMRA) and DNA when TAMRA was covalently labeled on single‐ and double‐stranded oligonucleotides. Fluorescence intensity quenching and lifetime changes were characterized and correlated with different DNA sequences. The results demonstrated that the photoinduced electron transfer interaction between guanosine residues and TAMRA introduced a short lifetime fluorescence component when guanosine residues were at the TAMRA‐attached terminal of the DNA sequences. The discrepancy of two‐state and three‐state models in previous studies was due to the DNA sequence selection and sensitivity of techniques used to detect the short lifetime component. The results will help the design of fluorescence‐based experiments related to a dye labeled probe. Copyright © 2012 John Wiley & Sons, Ltd.     

Dynamics of mismatched base pairs in DNA

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.     

Fluorescence determination of tryptophan side-chain accessibility and dynamics in triple-helical collagen-like peptides

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.     

Fluorescence quenching dynamics of tryptophan in proteins. Effect of internal rotation under potential barrier.

In many proteins fluorescence from single tryptophan exhibits a nonexponential decay function. To elucidate the origin of this nonexponential decay, we have examined the fluorescence decay function and time-resolved fluorescence anisotropy of a fluorophore covalently bound to a macromolecule by solving a rotational analogue of the Smoluchowski equation. An angular-dependent quenching constant and potential energy for the fluorophore undergoing internal rotation were introduced into the equation of motion for fluorophore. Results of numerical calculations using the equations thus obtained predict that both the fluorescence decay function and time-resolved anisotropy are dependent on rotational diffusion coefficients of fluorophore and potential energy for the internal rotation. The method was applied to the observed fluorescence decay curve of the single tryptophan in apocytochrome c from horse heart. The calculated decay curves fit the observed ones well.     

Environmental modulation of M13 coat protein tryptophan fluorescence dynamics

The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic tyrosine fluorescence as a tool to study the interaction of the shaker B "ball" peptide with anionic membranes

Steady-state and time-resolved fluorescence from the single tyrosine in the inactivating peptide of the Shaker B potassium channel (ShB peptide) and in a noninactivating peptide mutant, ShB-L7E, has been used to characterize their interaction with anionic phospholipid membranes, a model target mimicking features of the inactivation site on the channel protein. Partition coefficients derived from steady-state anisotropy indicate that both peptides show a high affinity for anionic vesicles, being higher in ShB than in ShB-L7E. Moreover, differential quenching by lipophilic spin-labeled probes and fluorescence energy transfer using trans-parinaric acid as the acceptor confirm that the ShB peptide inserts deep into the membrane, while the ShB-L7E peptide remains near the membrane surface. The rotational mobility of tyrosine in membrane-embedded ShB, examined from the decay of fluorescence anisotropy, can be described by two different rotational correlation times and a residual constant value. The short correlation time corresponds to fast rotation reporting on local tyrosine mobility. The long rotational correlation time and the high residual anisotropy suggest that the ShB peptide diffuses in a viscous and anisotropic medium compatible with the aliphatic region of a lipid bilayer and support the hypothesis that the peptide inserts into it as a monomer, to configure an intramolecular beta-hairpin structure. Assuming that this hairpin structure behaves like a rigid body, we have estimated its dimensions and rotational dynamics, and a model for the peptide inserted into the bilayer has been proposed.     

Fluorescence anisotropy decay studies upon hemoglobin A and its subunits

Fluorescent conjugates of hemoglobin A, its isolated β-chain, and the apo-derivative of the β-chain have been prepared in which the β-93 sulfhydryl was conjugated with 1,5-AEDANS. Radiationless enery transfer to the heme group results in a major decrease in fluorescence intensity and decay time. Measurements of the time decay of fluorescence anisotropy, employing single-photon counting, indicate that the apparent rotational correlation time is, in each case, substantially reduced from the value expected for a rigid molecule of the same molecular weight. This observation raises the possibility that internal degrees of rotational freedom exist.     

Interactions of sodium and potassium ions with oligonucleotides carrying human telomeric sequence and pyrene moieties at both termini

The organization of human telomeric DNA is of intense interest because of its role in aging, cancer research and bioanalytical applications. The Htelom sequence 5'-G(3)(T(2)AG(3))(3)-3' has been use to prepare two pyrene-modified fluorescence probes with three- and six-carbon linkers: Py-Htelom-Py(C3) and Py-Htelom-Py(C6), respectively. Results of the circular dichroism (CD), native PAGE, steady-state fluorescence, and anisotropy measurements of sodium and potassium quadruplex formation by these pyrene-modified conjugates are presented and discussed in order to clarify which conformation facilitates or renders the pyrene/pyrene or G-tetrad/pyrene stacking interaction. The CD spectra and native PAGE images suggested that conjugation of pyrene moieties has negligible effect on the folding properties of Htelom oligonucleotide. CD melting profiles and thermodynamic parameters revealed that both sodium and potassium quadruplexes are stabilized by the anchoring of pyrene tags with potassium ion being more effective than its sodium counterpart. Monomer emission of pyrene dominated in all investigated systems with fluorescence intensity being sensitive to the nature and concentration of cation and this phenomenon was attributed to the quenching processes and to the particular topologies of sodium and potassium quadruplexes. Strong quenching observed in the presence of KCl was attributed to the peculiarity of the potassium hybrid-type quadruplex, which enables effective stacking of pyrene moieties on the exposed guanine tetrads, thus facilitating static or electron transfer quenching. Plausibility of stacking interactions between pyrene and G-tetrad in a hybrid-type potassium quadruplex was further supported by the anisotropy measurements and molecular modeling results.     

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

Tetramethyl-6-carboxyrhodamine quenching-based aptasensing platform for aflatoxin B1: Analytical performance comparison of two aptamers

In this study, a simple TAMRA (tetramethyl-6-carboxyrhodamine) quenching-based aptasensing platform was designed for the detection of aflatoxin B1 (AFB1). Here, we compared the analytical performance of two aptamer sequences: seqA and seqB. The AFB1 detection was based on the interactions of FAM (carboxyfluorescein)-labeled aptamer with TAMRA-labeled DNA complementary strand in the presence and absence of target analyte. Under optimized experimental conditions, TAMRA-labeled strand quenched the fluorescence response of FAM-labeled aptamer due to the noncovalent interaction between the two DNA strands. The binding of AFB1 induced the complex formation and weakened the interaction between FAM-labeled aptamer and TAMRA-labeled complementary strand, resulting in the fluorescence recovery. By using this principle concept, an assay was constructed for the detection of AFB1. The method exhibited good sensitivity, good selectivity with a limit of detection of 0.2 ng ml⁻¹, and a wide linear range from 0.25 to 32 ng ml⁻¹. For real sample application, the aptasensors were tested in beer and wine samples, with good recovery rates obtained for AFB1 detection.     

Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.     

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.     

Luminescent metal-ligand complexes as probes of macromolecular interactions and biopolymer dynamics

The knowledge of microsecond dynamics is important for an understanding of the mechanism and function of biological systems. Fluorescent techniques are well established in biophysical studies, but their applicability to probe microsecond timescale processes is limited. Luminescent metal-ligand complexes (MLCs) have created interest mainly due to their unique luminescent properties, such as the exceptionally long decay times and large fundamental anisotropy values, allowing examination of microsecond dynamics by fluorescence methods. MLC properties also greatly simplify instrumentation requirements and enable the use of light emitting diode excitation for time-resolved measurements. Recent literature illustrates how MLC labels take full advantage of well developed fluorescence techniques and how those methods can be extended to timescales not easily accessible with nanosecond probes. MLCs are now commercially available as reactive labels which give researchers access to methods that previously required more complex approaches. The present paper gives an overview of the applications of MLC probes to studies of molecular dynamics and interactions of proteins, membranes and nucleic acids.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Biophysical characterization of DNA and RNA aptamer interactions with hen egg lysozyme

This work characterized the binding of an RNA aptamer recognizing hen egg white lysozyme, as well as a literature-reported single-stranded DNA analog of sequence identical to the original RNA aptamer, using fluorescence anisotropy, isothermal titration calorimetry (ITC) and analytical ultracentrifugation. The polyanionic DNA aptamer analog is selective for lysozyme even over cationic cytochrome c and has been reported to be successfully used in biosensing applications. The association however, is predominantly of electrostatic character, strongly salt-sensitive and entropically-driven, in contrast to previously described enthalpically-driven antibody-lysozyme and DNA aptamer-VEGF interactions. With a moderate selectivity for their target, high salt-sensitivity along with fast association and dissociation behavior, these molecules might serve as pseudo-affinity ligands for biomolecular separations.     

Solvent-exposed tryptophans probe the dynamics at protein surfaces.

The dynamics of single tryptophan (W) side chain of protease subtilisin Carlsberg (SC) and myelin basic protein (MBP) were used for probing the surface of these proteins. The W side chains are exposed to the solvent, as shown by the extent of quenching of their fluorescence by KI. Time-resolved fluorescence anisotropy measurements showed that the rotational motion of W is completely unhindered in the case of SC and partially hindered in the case of MBP. The rotational correlation time (phi) associated with the fast local motion of W did not scale linearly with the bulk solvent viscosity (eta) in glycerol-water mixtures. In contrast, phi values of either W side chains in the denatured proteins or the free W scaled almost linearly with eta, as expected by the Stokes-Einstein relationship. These results were interpreted as indicating specific partitioning of water at the surface of the proteins in glycerol-water mixtures.     

Fluorescence polarization study of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli

The lipoic acids of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli have been modified with two fluorescent probes, N-(1-pyrenyl)-maleimide and 5-[[[(iodoacetyl)amino]ethyl]amino]-naphthylene-1-sulfonic acid. Time-resolved fluorescence polarization of partially labeled complexes (18-77% inhibition of enzyme activity) reveals a complex depolarization process: one component of the anisotropy is characterized by a rotational correlation time much longer than the time scale of the measurements (less than or equal to 400 ns), reflecting the overall rotation of the complex, while a second component of the anisotropy decays with a rotational correlation time of 320 (+/- 50) ns. This decay is essentially independent of viscosity and is consistent with a model in which the depolarization is due to the dissociation from and rotation of lipoic acids between binding sites on the multienzyme complex. The sum of the rate constants characterizing the association and dissociation with the binding sites is approximately 3 x 10(6) s-1. In addition, approximately 5% of the anisotropy of the N-(1-pyrenyl)maleimide-labeled complex decays with a rotational correlation time of 25 ns; this can be attributed to local motion of the probe. At high extents of N-(1-pyrenyl)maleimide labeling (90-95% inhibition of enzyme activity), the anisotropy decay can be described by a constant term plus a rotational correlation time of about 1 microseconds. The increase in the correlation time probably reflects interactions between pyrene moieties. The N-(1-pyrenyl)maleimide-labeled dihydrolipoyl transsuccinylase core of the multienzyme complex has been isolated, and the anisotropy is constant over the observed time range of 300 ns. This suggests that the native structure is necessary for observation of lipoic acid movement within the complex. Fluorescent-labeled limited trypsin digestion fragments of the alpha-ketoglutarate dehydrogenase complex also have been isolated, and anisotropy measurements reveal substantial mobility of the label within the fragments. The time-resolved anisotropy of FAD in the native complex and in the isolated dihydrolipoyl dehydrogenase indicates some rapid local mobility of the FAD (rotational correlation time of 12 ns) that is viscosity independent, as well as a component of the anisotropy that is constant over the 35-ns time scale of the experiments.     

141 DNA origami as a platform for assembly of nanophotonic elements

Achieving DNA-functionalized semiconductor quantum dots (QDs) that are robust enough to be compatible with the DNA nanotechnology that withstand precipitation at high temperature and ionic strength is a challenge. Here we report a method that facilitates the synthesis of stable core/shell (1–20 monolayers) QD-DNA conjugates in which the end part (5–10 nucleotides) of the phosphorothiolated oligonucleotides is embedded within the shell of the QD. These reliable QD-DNA conjugates exhibit excellent chemical, colloidal and photonic stability over a wide pH range (4–12) and at high salt concentrations (>100?mM Na⁺ or Mg²⁺), bright fluorescence emission with quantum yields of upto 70%, and broad spectral tunability with emission ranging from UV to NIR (360–800?nm). The assembly of these different QDs into DNA origami in a well-controlled pattern was demonstrated (Deng, Samanta, Nangreave, Yan, & Liu, 2012). We also used DNA origami as a platform to co-assemble a gold nanoparticle with 20?nm diameter (AuNP) and an organic fluorophore (TAMRA) and studied the distance dependent plasmonic interactions between the particle and the dye using steady state fluorescence and lifetime measurements. Greater fluorescence quenching was found at smaller inter-particle distances, which was accompanied by an enhancement of the decay rate. We further fabricated 20?nm and 30?nm AuNP homodimers with different inter-particle distances using DNA origami scaffolds and positioned a Cy3 fluorophore between the AuNPs in both the assemblies. Up to 50% enhancement of the Cy3 fluorescence quantum efficiency was observed for the dye between the 30?nm AuNPs. These results are in good agreement with the theoretical simulations (Pal et al., 2013).