Technical Reference #220

220.

Visualizing RNA Molecules Inside the Nucleus of Living Cells Roeland W. Dirks; Chris Molenaar; Hans J. Tanke, Leiden University Medical Center, Methods, 29(220), (2003)
Link To Paper

Abstract:
Fluorescence in situ hybridization is a widely used technique in cell biology providing insight into the spatial organization ofspecific RNA transcripts in the cell nucleus. However to further investigate the dynamics of the transcription process and t

Keywords:
In vivo hybridization; living cell; 2'-O Methyl RNA; molecular beacon; oligodeoxyneucleotides; peptide nucleic acid; fluorescence resonance energy tranfer; microinjection; live cell imaging; RNA fluorescence in situ hybridization

Materials & Methods:
Major drawbacks associated with the use of linear DNA probes for detecting RNA molecules in living cells are the inability to wash out nonhybridized probes and the instability of DNA probes inside cells. Consequently the amount of probe that should be introduced into a cell must be experimentally determined such that nonhybridized free diffusing probes do not obscure specific hybridization signals. Furthermore sequence composition and length of oligonucleotides need to be considered as nonspecific hybridization to nontarget RNAs cannot be eliminated by stringent washes. Fluorescently labeled oligodeoxynucleotide probes are extremely prone to degradation by nucleases. This instability of oligodeoxyribonucleotide probes requires that on probe delivery cell hybridization can be monitored only for short periods. Once they accumulate in the cell nucleus they are degraded with a half-time of 15–20 min [21] leading to a nearly complete intracellular degradation within a few hours. The fact that oligonucleotides accumulate rapidly in the nucleus implies the existence of nuclear binding sites. Indeed it has been shown that oligodeoxynucleotides bind to nuclear proteins by electrostatic interactions [22]. Taking into account that oligodeoxynucleotides show a rather low affinity for complementary target RNA sequences binding to these proteins may prevail. This notion is consistent with our own experience with oligodeoxynucleotides complementary to the poly(A) tail of mRNAs to rRNA and to various snRNAs. On injection none of these probes revealed specific hybridization patterns in cell nuclei but rather revealed a diffuse nuclear staining [23]. Taken together the properties of oligodeoxynucleotides are generally not compatible with specific and reproducible detection of RNAs in the nucleus of living cells. 3.2. Linear 20-O-methyl RNA probes Compared with 20-deoxyoligoribonucleotide probes 20-OMe RNA probes (Fig. 1) exhibit faster hybridization kinetics increased melting temperatures enhanced binding specificity and the ability to bind structured molecules. Furthermore 20-OMe RNA probes are completely resistant to degradation by RNA-specific nucleases [24] making them an ideal probe type for living cell studies. Considering the fact that 20-OMe RNA binds much more stably to target RNA sequences compared with oligodeoxynucleotide probes in living cells binding to nuclear proteins is less favored leading to better signal-to-noise ratios. Comparison between the melting properties of 20-OMe RNA and deoxyoligonucleotides hybridized to RNA targets shows that the Tm value of a 15-bp 20-OMe RNA probe is about 22 C higher than that of an oligodeoxynucleotide with the same sequence and length and the Tm value of a 19-bp 20-OMe RNA probe is about 19 C higher [25]. Also 20- OMe RNA probes discriminate much better between matched and mismatched RNA targets further improving their specificity compared with oligodeoxynucleotide probes [25]. 20-OMe RNA probes were first applied in living cell studies to detect snRNAs [26] and later by Molenaar et al. [23] to detect a variety of RNA types includingsnRNAs rRNA poly(A) RNA (Fig. 2A) and a specific human cytomegalovirus (CMV) immediate early mRNA transcript (Fig. 2B).

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
COS-7