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, 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.
Highlights
The mechanisms of cell development, reproduction, differentiation, functioning and evolution, as well as those of gene expression in living organisms are important subjects of modern molecular and cellular biology researches
They managed to express in living cells five different DNA-binding proteins participating in the chromosome segregation (CENP-A, centromere-specific histone, histone H3, importin-α and α-tubulin) fused with fluorescent proteins of various colors
We address the reader to several recent reviews in which all the achievements and the problems of the methods are thoroughly described [1,162,163,164]
Summary
The mechanisms of cell development, reproduction, differentiation, functioning and evolution, as well as those of gene expression in living organisms are important subjects of modern molecular and cellular biology researches. Direct visual observation of nucleic acids in vivo requires new specific and sensitive probes for studies of DNA and RNA structure and their interactions with other cell components, their stability, their mobility and dynamics, and their functions in living cells. Labeling of specific DNA regions, such as centromeres, telomeres or other repeated sequences, by stable tightly bound probes that do not disturb drastically their biological properties will allow to observe them in dynamics in real time and to get ideas about their functional roles. This review deals with the fluorescent probes used in fixed and living cells that are adapted or could potentially be adapted for direct dynamic observation of native DNA and RNA during cell cycle using fluorescence microscopy. It covers the principles of probe design for this purpose as well
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