Fig 1.
Design of the dual-labeled RNA oligonucleotide.
(A) 23 nucleotide RNA oligonucleotide conjugated to tetramethylrhodamine (TMR) at its 5’ end via a thioether bond and to Atto488 at its 3’ end via 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.
Fig 2.
Monitoring oligonucleotide degradation using FCS, FCCS and FRET.
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.
Fig 3.
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.
Fig 4.
Fluorescence intensities of HeLa cells in culture after transfection with oligomer 278.
(A) A U-shaped, sequence defined cationizable lipo-oligomer 278 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.
Fig 5.
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.
Fig 6.
Quantification of the fluorescence lifetime measurements.
(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.