Monitoring oligonucleotide degradation using FCS, FCCS and FRET.

<p>The stability of various RNAs was measured as a function of incubation time in cell extracts. The main changes and parameters corresponding to RNA degradation are shown exemplary for construct 2, representing: (A) the diffusion time from the autocorrelation function (FCS), (B) the amplitude of the cross-correlation function (FCCS), (C) an apparent FRET efficiency determined from the fluorescence intensity and (D) the donor fluorescence lifetime based FRET using a phasor analysis. The colored crosses represent the center of mass in the phasor plot of measurements after 1 min (blue), 60 min (green), 120 min (orange) and 180 min (magenta). The grey arrows indicate the direction of the main changes.</p

Fluorescence intensities of HeLa cells in culture after transfection with oligomer <i>278</i>.

<p>(A) A U-shaped, sequence defined cationizable lipo-oligomer <b><i>278</i></b> for complexation of the dual-labeled RNAs (C: cysteine, K: lysine, Stp: succinoyl-tetraethylene pentamine, linA: linoleic acid). (B) Fluorescence intensity images of the HeLa cells, 15 min, 1 h, 6 h and 24 h after transfection of the four different modifications patterns. The contrast level is equal for all images. The scale bar represents 200 μm. (C) Average fluorescence count rate of the cells at the different conditions shown in (B). The error bars represent the standard deviation of three independent measurements.</p

Quantification of the fluorescence lifetime measurements.

<p>(A-D) Distribution of the pixels along the line connecting the mono-exponential decays at 4.1 ns and at 1.25 ns in the phasor plot for the four modification patterns. (E) Summary of the average fluorescence lifetimes of the cell populations shown in panels A-D using a Gaussian fit to the distribution. The error bars represent the standard deviations of three independent measurements.</p

Design of the dual-labeled RNA oligonucleotide.

<p>(A) 23 nucleotide RNA oligonucleotide conjugated to tetramethylrhodamine (TMR) at its 5’ end <i>via</i> a thioether bond and to Atto488 at its 3’ end <i>via</i> an amide bond. Upon exposure to the cellular environment, the oligonucleotide can be degraded by various RNases. (B) Modification patterns selected to monitor intracellular localization and integrity of the oligonucleotide. RNA backbone modifications to modulate stability towards nucleolytic degradation: 2’-F, 2’-O-Me and phosphorothioate.</p

Three-Dimensional Tracking of Carbon Nanotubes within Living Cells

Three-dimensional tracking of single-walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map corral volumes (0.27–1.32 μm³), determine an active transport velocity (455 nm/s), and calculate local viscosities (54–179 cP) within the cell. With respect to the future use of SWNTs as sensors in living cells, we conclude that the sensor must change the fluorescence signal by at least 4–13% to allow separation of the sensor signal from fluctuations due to rotation of the SWNT when measuring with a time resolution of 32 ms

CD spectra of labeled S-peptide with and without Atto655 stacking partner Trp15.

<p>The peak at 220 nm indicates residual <i>α</i>-helix formation and less <i>β</i>-sheet contribution in the labeled S-peptide without Trp15.</p

Atto655/Trp15 fluorescence quenching autocorrelation data fitted with a two-state exponential model function.

<p>Data and fits are shown for MD simulations (A, B) and experimental PET-FCS measurement (C). (A) Data from MD calculated over the whole simulation time (30 μs). (B) Data from MD where the initial 12 μs of the simulation was omitted. (C) Dynamic part of the correlation curve from experimental PET-FCS measurement (red) overlayed with the fitted MD data collected after 12 μs (black).</p

Experimentally obtained FCS curve and model fit function for labeled S-peptide in the presence (black) and absence (gray) of the quenching tryptophan.

<p>Indicated are the four main time regimes of the relevant processes. (I) Photon antibunching: The lifetime of the excited state of the fluorophore determines the shortest temporal separation between two photon emission events. This leads to a decrease in the correlation function for correlation times faster than the lifetime of the excited state. (II) Timescale of the internal conformational dynamics that lead to quenching/unquenching of the fluorophore and dominate the autocorrelation function. These timescales are to be compared with the MD simulations. (III) Photophysical artifacts: Intersystem crossing from the excited singlet state into a dark triplet state with lifetimes in the range of several μs (IV) This correlation regime is dominated by the diffusion of labeled peptides through the confined detection volume of the FCS setup.</p

The RMSD of non-hydrogen (heavy) atoms of residues 1-14 with respect to the unfolded starting structure for simulations with (lower panel) and without (upper panel) labels.

<p>The mean structures of the respectively four largest clusters are shown and their cluster index is indicated (#). Additionally, the simulation time when the clusters mean structures were observed during the simulation is indicated at the bottom right of each cluster structure.</p

The population size of the ten largest clusters in percentage of lifetime compared to the whole trajectory from simulations without (left panel) and with (right panel) fluorescence labels.

<p>Clustering was based on the RMSD and the single linkage algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177139#pone.0177139.ref035" target="_blank">35</a>] with a 0.25 nm cutoff was used.</p