Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells

Abstract

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

Dynamic association–dissociation and harboring of endogenous mRNAs in stress granules

“…1A). We chose 29-Omethyl RNA as the backbone of the probe, because this artificial nucleic acid is resistant to nucleases in living cells and it has a high affinity for nucleic acids (Molenaar et al, 2001;Okabe et al, 2011). The poly(U) 22 29-O-methyl RNA probe has previously been used to detect nuclear poly(A) + RNA (Molenaar et al, 2004;Ishihama et al, 2008).…”

“…To visualize endogenous mRNAs, we used a linear antisense 29-O-methyl RNA probe labeled with a fluorescent dye that can bind to native mRNA in a sequence-specific way (Molenaar et al, 2001;Molenaar et al, 2004;Ishihama et al, 2008;Okabe et al, 2011). A linear antisense probe was advantageous because of its ability to target native mRNA and its excellent hybridization kinetics in living cells (Okabe et al, 2011;Molenaar et al, 2001).…”

P53-dependent activation of a molecular beacon in tumor cells following exposure to doxorubicin chemotherapy

“…Previous research described the tendency of molecular beacons to shuttle into the nucleus soon after they entered the cell. [15][16][17] In light of this, we identified the intron/exon boundaries in the mRNA and designed our target sequence to be contained only in the third exon. In anticipation of the beacon encountering mostly unprocessed RNA in the nucleus, a target sequence involving two adjacent exon sequences was avoided.…”

Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons

“…However, the long-term, real-time monitoring of viral RNA in living cells has not been possible because of the relatively short half-life (Ϸ30 min) of MBs due to endogenous nuclease degradation, resulting in false-positive signals (6,7). It has been shown that modifications of MBs with 2Ј-Omethyl RNA bases and phosphorothioate internucleotide linkages can be used to significantly improve duplex stability and nuclease resistance (8)(9)(10)(11)(12)(13). It is easy to envision that such an MB design can be adapted for the real-time monitoring of viral infection.…”