Next-generation fluorescent nucleic acids probes for microscopic analysis of intracellular nucleic acids
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Fluorescence imaging of nucleic acids is a very important technique necessary to understand gene expression and the resulting changes in cell function. This mini-review focuses on sequence-specific fluorescence imaging of intracellular RNA and methylated DNA using fluorescent nucleic acid probes. A couple of functional fluorescent nucleic acid probes developed by our laboratory are introduced and the examples of their application to fluorescence imaging of intracellular nucleic acids are described.
KeywordsMethylated DNA RNA Fluorescence Single cell imaging Synthetic nucleic acid
The mechanism of gene expression in a various of cell events is the most important research target in molecular biology and cell biology researches. Imaging of these events within individual cells (Dirks and Tanke 2006; Silverman and Kool 2005), and even within organisms (Weigert et al. 2013), should be less invasive and more suitable for monitoring of intracellular events than the traditional biochemical methods, and thus, fluorescent signals are frequently used for monitoring of biomolecules and their functions in a cell. Visualizing the behavior of nucleic acids in cells is the best way to understand ongoing cellular function expression, and a number of fluorescent nucleic acid probes have been developed for sensing of target intracellular nucleic acids. Because hybridization of short fluorescent nucleic acids complementary to the target nucleic acid sequence is very sequence-specific, flexible design of fluorescent nucleic acid probes give us a direct method for the visualization of intracellular nucleic acids when the sequence information is available.
Design of ‘fluorescent nucleic acid probes’ which can be removed or turned off when the probe does not recognize its target nucleic acid is very important for the establishment of microscopic analysis of intracellular nucleic acids without a sensitive washing process, resulting in a highly reliable, labor-saving, and real-time fluorescence observation. The chemistry of sequence-specific fluorescence emission control is a significant key for the design of a next generation of functional fluorescent probes. Various DNA probes whose fluorescence intensity is controlled in a sequence-specific fashion have been widely used (Tyagi et al. 1998; Tyagi and Kramer 1996; Piatek et al. 1998; Fang et al. 2000; Broude 2002; Okamoto et al. 2003; Tan et al. 2004). However, many problems remain therein: (1) different types of dyes are required in a probe; (2) both strand ends are occupied by different dyes; and (3) probes require the formation of higher-order structures for efficient fluorescence quenching. Therefore, fluorescent nucleic acid probes using a new concept that solves these problems need to be developed to produce the next generation of fluorescent probes for nucleic acid sensing.
This mini-review focuses on sequence-specific fluorescence imaging of intracellular RNA and methylated DNA using fluorescent nucleic acid probes. A couple of functional fluorescent nucleic acid probes developed by our laboratory are introduced and the examples of their application to fluorescence imaging of intracellular nucleic acids are described. Their fluorescence emission was sequence-specifically controlled by the unique chemistry such as hybridization-sensitive fluorescence emission or methylation-specific metal complex formation.
Live cell imaging of intracellular RNA
Nucleic acids labeled by thiazole orange dyes (TO) are well known to have strong hybridization-dependant fluorescence. However, the fluorescence suppression observed in the single-stranded state of TO-labeled DNA derivatives is still insufficient, and a more specific design is required for effective sensing of intracellular nucleic acids without background fluorescence.
ECHO probe is very effective for sequence-specifically visualizing single-stranded nucleic acids, such as mRNA. Sequence-specific nucleic acid imaging has greatly advanced by the development of fluorescence in situ hybridization (FISH) methods (Nath and Johnson 2000; Levsky and Singer 2003; Smolina et al. 2007; Jaco et al. 2008), but conventional FISH is cumbersome and time-consuming, because it contains sensitive washing processes. On the other hand, ECHO probes have the higher sequence-specific fluorescence emission ability compared with conventional fluorescence-labeled nucleic acid probes, and thus a high-resolution FISH has become possible by adapting ECHO probes (ECHO–FISH) (Wang et al. 2012). ECHO–FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. This method does not require stringent washing steps and it is accomplished in a 25-min procedure from fixation to mounting. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons.
The character of fluorescence switching by ECHO probes is suitable for visualizing mRNA localization in a living cell. The probes were introduced into cells by using manipulator-assisted microinjection, lipofection, or electropolation to visualize intracellular mRNA localization. For example, immediately after the injection of 5′-T6D514T6–3′ (D514 is a thymine derivative doubly labeled with two TO dyes) into the nucleus of a living HeLa cell, fluorescence was observed from the nucleus (Kubota et al. 2009a). ECHO probes with a DNA backbone are digested by intracellular nucleases and its half-life time is about 3 h in living cells. In order to add nuclease resistance to ECHO probes for the long-term intracellular RNA observation, the backbone of ECHO probes was replaced with a 2′-O-methyl RNA (Kubota et al. 2009b). The high nuclease resistance of new ECHO probes with 2′-O-methyl RNA makes long-term intracellular RNA imaging possible. Intracellular mRNA behavior during cell life events, such as migration and division, was observed through long-term monitoring of the cells into which a 2′-O-methyl RNA probe, 5′-OMeU6D514OMeU6–3′, was transfected. The fluorescence of the probe in the mother cells was evenly distributed to their two daughter cells after cell division.
Desmethyl TO dye unit has been developed for a pH-sensitive ECHO probe (Okamoto et al. 2013). The DNA probe doubly modified with desmethyl TO dye units showed effective on/off switching of fluorescence at pH 5–7, which is similar to the behavior of conventional ECHO probes. On the other hand, the fluorescence from the probe at higher pH (pH 8–10) was weak even in the presence of the complementary RNA strand, because acidic pH is important for the protonation and emission of desmethyl TO dye units.
Finally, we developed a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to visualize unmodified, native RNA at intranuclear foci in living mice and chicks (Oomoto et al. 2015). We combined ECHO-FISH probe technology with in vivo electroporation to achieve microscopic imaging in native tissues. This method enabled us to observe the spatial dynamics of individual ribonuclear foci, which are regulated by cellular transcriptional activity, at the single-cell level. Confirming the physiological relevance of this technique, the in vivo RNA labeling did not interfere with the function of the target RNA or cause noticeable cytotoxicity or perturbation of cellular behavior. With ECHO-liveFISH, we also detected the spatial dynamics demonstrated by the disintegration of 28S rRNA concentration upon actinomycin D treatment. The imaging result is consistent with the previous data in other species showing that the assembly of nucleoli is triggered by activation of rDNA transcription. Many advantages are offered by ECHO-liveFISH for in vivo RNA imaging, such as easy and effective delivery, negligible toxicity, lack of perturbation of cellular functions, stability in a living cellular environment, brightness, high signal-to-noise ratio, fast hybridization and photoactivation kinetics, reversible fluorescence emission, and data acquisition with standard confocal imaging systems.
Methyl-specific fluorescent in situ hybridization
Transcription of DNA to RNA is well controlled by the epigenetic modification of DNA and histone proteins, independent of their primary sequences. One of epigenetic DNA modifications, methylation of cytosine (C), plays a crucial role in the regulation of gene expression (Jones and Wolffe 1999; Li 2002), and erroneous DNA methylation contributes to the etiopathogenesis of tumorigenesis and aging (Jones and Laird 1999; Robertson 2005). Much effort has gone into developing a simple reaction that can be used for the detection of intracellular 5-methylcytosine (5mC).
Furthermore, MeFISH was applied to detect methylation at human satellite repeat sequences. Human classical satellites 2 and 3 and alpha satellite are generally highly methylated in somatic cells from healthy individuals, but they are hypomethylated in those from patients with immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. The syndrome is classified into two types with different hypomethylation levels at satellites. When we examined MeFISH, the control cells showed fluorescent signals at both classical satellite and alpha satellite. In type 1 ICF cells, the classical satellite signals were very low although the alpha satellite MeFISH signals were detectable. On the other hand, signals were undetectable at both satellites in type 2 ICF cells under the same photographic exposure conditions. The faint MeFISH signals were detected even in the ICF specimens, which was consistent with the facts that these sequences are hypomethylated, but not completely unmethylated, in ICF cells (Jiang et al. 2005; Jeanpierre et al. 1993; Brun et al. 2011).
Furthermore, the collaboration with Abe group in RIKEN adapted MeFISH to the study of intact embryos, and established a method called “whole-mount MeFISH” (Shiura et al. 2014). The DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, was examined and a result that was consistent with previous findings was obtained. We also combined whole-mount MeFISH with immunostaining or FISH techniques. These combined whole-mount MeFISH methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution.
Fluorescent imaging of intracellular nucleic acids has made great progress over the last few decades. In this mini-review, we introduced a couple of unique fluorescent nucleic acid probes for effective microscopic imaging of intracellular RNA and methylated DNA. The ECHO probe technology, one of the TO probes, made it possible to detect the target nucleic acid and to analyze both the spatiotemporal dimensions of the diverse RNA dynamics in a living cell. The hybridization sensitivity of fluorescence emission from ECHO probes is a great advantage in live cell imaging. Further expansion of the diversity of ECHO probes should lead to further improvements in the methodology of nucleic acid imaging. The ICON probe technology and its application to MeFISH were also described in this mini-review. The sequence-specific formation of a stable osmium complex by ICON probes made the fluorescence imaging of DNA methylation at the target cytosine in a cell possible. This new concept of DNA methylation imaging technology at a single cell level will be the starting point for an epoch-making methylation-imaging assay, which will supersede conventional methods.
The author would like to thank all members of Okamoto laboratory and collaboration group staffs for their enthusiastic works and meaningful discussion.
AO was a major contributor in writing the manuscript. The author read and approved the final manuscript.
This work was supported by the Japan Society for the Promotion of Science (JSPS), the Funding Program for Next Generation World-Leading Researchers (LR036) and JSPS Grant-in-Aid for Scientific Research (A) 15H02190 and 18H03931.
The author declares that he has no competing interests.
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