Bdata_apt

Concatenated HTS sequencing data file, containing unique SELEX-enriched sequences after filtering to remove any sequences of size not matching N, where N = 30 nts (size of the random region in the naive RNA library). Bdata_apt contains the sequences enriched in the presence of NSP2

Phasor FLIM analysis in cultured HeLa cells.

(A) The phasor histogram of images shown in panel B. The grey dotted line indicates the axis used for color-coding the FLIM images in (B) and (C). (B) FLIM images 24 h after transfection of the stable control RNA, construct 1, construct 2 and the stable control RNA without TMR in cultured HeLa cells. The scale bar is 30 μm. (C) FLIM images for all measured constructs and time points. These measurements are the same as those shown in Fig 4. The scale bar is 200 μm.</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

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

Evaluation of the degradation of the dual-labeled RNAs in cell extract by different techniques.

The constructs were incubated in HeLa cell extract for 3 h and the degradation was monitored with a confocal microscope. The degradation was analyzed using FCS (A), FCCS (B) and FRET via intensity (C) and fluorescence lifetime (D). The curves were normalized to 1 for the initial data-point.</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

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

Chemical diversity in a metal-organic framework revealed by fluorescence lifetime imaging

The presence and variation of chemical functionality and defects in crystalline materials, such as metal–organic frameworks (MOFs), have tremendous impact on their properties. Finding a means of identifying and characterizing this chemical diversity is an important ongoing challenge. This task is complicated by the characteristic problem of bulk measurements only giving a statistical average over an entire sample, leaving uncharacterized any diversity that might exist between crystallites or even within individual crystals. Here we show that by using fluorescence imaging and lifetime analysis, both the spatial arrangement of functionalities and the level of defects within a multivariable MOF crystal can be determined for the bulk as well as for the individual constituent crystals. We apply these methods to UiO-67, to study the incorporation of functional groups and their consequences on the structural features.We believe that the potential of the techniques presented here in uncovering chemical diversity in what is generally assumed to be homogeneous systems can provide a new level of understanding of materials properties.</p

Chemical diversity in a metal-organic framework revealed by fluorescence lifetime imaging

The presence and variation of chemical functionality and defects in crystalline materials, such as metal–organic frameworks (MOFs), have tremendous impact on their properties. Finding a means of identifying and characterizing this chemical diversity is an important ongoing challenge. This task is complicated by the characteristic problem of bulk measurements only giving a statistical average over an entire sample, leaving uncharacterized any diversity that might exist between crystallites or even within individual crystals. Here we show that by using fluorescence imaging and lifetime analysis, both the spatial arrangement of functionalities and the level of defects within a multivariable MOF crystal can be determined for the bulk as well as for the individual constituent crystals. We apply these methods to UiO-67, to study the incorporation of functional groups and their consequences on the structural features.We believe that the potential of the techniques presented here in uncovering chemical diversity in what is generally assumed to be homogeneous systems can provide a new level of understanding of materials properties.</p

Lateral mobility of 18.5-kDa MBP in OLN-93 cells.

<p><i>z</i>-scan FCS and <i>z</i>-scan RICS measurements were performed on OLN-P, OLN-G and OLN-GS cells cultured on PLL and transiently transfected with 18.5-kDa MBP-eGFP. Experiments were performed 24 hours after transfection. <b>A</b>) A schematic of a cell showing <i>z</i>-scanning at the basal plasma membrane. <b>B</b>) The total intensity as a function of <i>z</i>-position for a typical z-scanning measurement is shown. <b>C</b>) The averaged autocorrelation curves from 10 cells were fitted with a 2D one component diffusion model. The diffusion coefficients from <i>z</i>-scan FCS are shown as a bar graph. <b>D</b>) A representative autocorrelation curve and corresponding 2D1C fit model is shown from a <i>z</i>-scan RICS measurement at the ventral plasma membrane. <b>E</b>) The diffusion coefficients for the <i>z</i>-scan RICS experiments are shown as a bar graph and represent the average of at least 10 cell measurements. Bars (<b>C,E</b>) represent the mean+SEM. The statistical analysis was performed using GraphPad Prism 5 (one-way ANOVA followed by the Newman-Keuls posttest, * p<0.05).</p