No abstract

Two procedures based on the weighted least-squares (LS) and the maximum likelihood estimation (MLE) method to confidently analyze single-molecule (SM) fluorescence decays with a total number (N) of 2,500-60,000 counts have been elucidated and experimentally compared by analyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematically underestimates the fluorescence lifetimes by approximately 5%, for the range of 1,000-20,000 events, whereas the MLE method gives stable results over the whole intensity range, even at counts N less than 1,000, where the LS analysis delivers unreasonable values. This difference can be attributed to the different statistics approaches and results from improper weighting of the LS method. As expected from theory, the results of both methods become equivalent above a certain threshold of N detected photons per decay, which is here experimentally determined to be approximately 20,000. In contrast to the bulk lifetime distributions, the SM fluorescence lifetime distributions exhibit standard deviations that are sizably larger than the statistically expected values. This comparison proves the strong influence of the inhomogenuous microenvironment on the photophysical behavior of single molecules embedded in a 10-30-nm thin polymer layer.

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oris located within the repeated sequences flanking the short unique arm (Us), and one copy of OriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oris. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.The herpes simplex virus type 1 (HSV-1) genome is a 160-kilobase pair (kb) linear duplex DNA molecule consisting of two components, L and S. The L component consists of unique sequences (UL) flanked by the inverted repeated sequences ab and b'a', whereas the S component consists of unique sequences (Us) flanked by inverted repeated sequences ac and c'a' (Fig. 1A) (26,46). The "a" sequence is present as a direct repeat at both molecular termini and in inverted orientation at the L-S junction (9,22,38,56).Attempts to localize the origin(s) of viral DNA synthesis within the structurally complex HSV-1 genome have involved studies of both standard and defective genomes. Electron microscopic studies of replicating standard wildtype viral DNA extracted from infected cells have been interpreted to suggest that the genome contains two origins of viral DNA synthesis, one near the middle of UL and the other near one molecular terminus (20. 27).Indirect evidence supporting the existence of two origins comes from studies of defective molecules of HSV-1 which are generated during serial passage of the virus at high multiplicities of infection. Defective DNA molecules fall into two classes, class I and class II, each consisting of tandem duplications of small subsets of viral DNA sequences. Class I defective genomes contain sequences from the "c" repeats which bracket Us (16-18, 32, 33, 36), whereas class II defective genomes contain sequences from Ul (16,19,32,45) (Fig. IB). Both classes also contain the "a" sequence which specifies a site for the cleavage of viral DNA concatamers during encapsidation (40,55). By analogy with the defective genomes of other DNA viruses, all of which contain origins of DNA synthesis. the existence of two distinct subsets of viral DNA sequences in defective HSV-1 genomes suggests that the genome contains three origins of DNA synthesis: two in diploid "c" sequences (ois), and one in UL (oriL) (Fig. IB).Direct evidence that the repeat units of class I and II * Corresponding author.

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

Dye photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Förster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing b...

We extended the sensitivity of Raman correlation spectroscopy in solution to the single-molecule level by applying surface-and resonance-enhanced Raman scattering (SERRS) combined with time-gated, confocal signal detection. The brightness of the SERRS signal of single Rhodamine 6G molecules adsorbed on a single silver nanoparticle is comparable to fluorescence. Rare event analysis reveals the existence of few particles with simultaneous SERRS and fluorescence signal. The observation of a dynamic exchange between heterogeneous binding sites is supported by the existence of multiple SERRS brightnesses in the signal intensity distribution and by signal fluctuations in the 60 µs time range detected by autocorrelation analysis. Finally, polarization-dependent SERRS autocorrelation curves and single-particle analysis allowed us to measure individual rotational diffusion times and to directly analyze the heterogeneity of the ensemble in solution.

Abstract:The comparison of Fö rster resonance energy transfer (FRET) efficiencies between two fluorophores covalently attached to a single protein or DNA molecule is an elegant approach for deducing information about their structural and dynamical heterogeneity. For a more detailed structural interpretation of single-molecule FRET assays, information about the positions as well as the dynamics of the dye labels attached to the biomolecule is important. In this work, Rhodamine 6G (2-[3′-(ethylamino)-6′-(ethylimino)-2′,7′-dimethyl-6′H-xanthen-9′-yl]-benzoic acid) bound to the 5′-end of a 20 base pair long DNA duplex is investigated by both single-molecule multiparameter fluorescence detection (MFD) experiments and NMR spectroscopy. Rhodamine 6G is commonly employed in nucleic acid research as a FRET dye. MFD experiments directly reveal the equilibrium of the dye bound to DNA between three heterogeneous environments, which are characterized by distinct fluorescence lifetime and intensity distributions as a result of different guanine-dye excited-state electron transfer interactions. Sub-ensemble fluorescence autocorrelation analysis shows the highly dynamic character of the dye-DNA interactions ranging from nano-to milliseconds and species-specific triplet relaxation times. Two-dimensional NMR spectroscopy corroborates this information by the determination of the detailed geometric structures of the dye-nucleobase complex and their assignment to each population observed in the single-molecule fluorescence experiments. From both methods, a consistent and detailed molecular description of the structural and dynamical heterogeneity is obtained.

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.