Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

Abstract

This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, OPEN ACCESSMolecules 2013, 18 15358 molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA

“…52 [3][4][5][6][7][8][9] Samples (500 µL each in 50 mM sodium cacodylate buffer, 50 mM NaCl, 50 mM KCl, 1 mM MgCl 2 , pH = 6.2) were prepared in quartz cells for spectrophotometry with the light path 1 cm hermetically closed by Teflon stoppers. Each sample contained 1.3 µM target duplex and increasing concentrations of polyamides from 0 up to 8-fold molar excess relatively to duplex.…”

“…For efficient use in living cells, fluorescent DNA probes must exhibit high affinity and specificity, high stability in biological medium, good cellular penetration, ability to reach chromatin in cells, minimal perturbation of DNA functions and high signal to background ratio. 3 Polyamide minor groove binders (MGB) 4,5 are promising candidates for dsDNA sequence-specific targeting in both fixed and living cells. They specifically recognize DNA sequences in the minor groove according to established rules and form stable complexes.…”

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…FISH technology was developed in 1980s and was initially used in genome imaging 27, 75 . FISH probes for RNA imaging are a group of ssDNA, ssRNA or hybrid ssRNA/DNA sequences tagged with same fluorophores, with the sequences complementary to different parts of the target RNAs (Fig.…”

“…Furthermore, we provide an outlook for the development of RNA imaging in vivo with even more sensitive imaging probes and advanced imaging technologies. There are also some other related excellent reviews, including RNA detection on a chip 20 , live-cell RNA imaging 21–27 , probes for biomolecular detection 28–31 and miRNA imaging. 32, 33 …”

New imaging reagents for lipid dense regions in live cells and the nucleus in fixed MCF-7 cells

“…1 Such processes are expected to affect the native intracellular processes while the imaging studies are performed in physiologically authentic environments and have relevance for cell/molecular biology, studying bio-chemical processes, medicine, pharmacology and diagnostics. 3,4 Stability towards photo-bleaching, cell membrane permeability, nominal cytotoxicity and luminescence in the longer wavelength following excitation with non-harmful visible light are some of the essential criteria for any such efficient imaging reagent. There are significant activities in designing appropriate imaging reagent for visualization of specific organelles with organelle-selective dyes in the membraneenclosed intracellular structures, as this helps in gaining insight for monitoring important biological processes.…”