Fluorescence energy transfer as a probe for nucleic acid structures and sequences

Abstract

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

“…This is in contrast with other combinations of fluorophores where the acceptor dyes fluoresce due to direct excitation. 11,30 Another advantage of the BP-2d system is that the donor-acceptor emission profiles are widely spectrally separated, resulting in a large "Stokes' shift" and avoiding fluorescence overlap, which increases the sensitivity of the detection of the hybridization event. Addition of target mRNA causes hybridization of the probes to mRNA bringing together Alexa488 and Cy5.…”

“…However, this achievement also poses a challenge for the development of new and improved methods for the detection and monitoring of DNA or RNA either in vitro or in vivo. Different approaches have been taken in the design of oligonucleotide probes for DNA and RNA detection including molecular beacons (MBs), [5][6][7][8] binary probes (BPs), [9][10][11][12][13] and quenched autoligation probe pairs (QUAL) [14][15][16] . Other probes like scorpion primers, 17 5′-nuclease probes, 18 and cyclicons 19 have been used in vitro to detect amplification products in polymerase chain reactions (PCR).…”

“…9 BPs rely mostly on FRET for target detection. [9][10][11][12] In general, one of the sequences in the BP contains a donor fluorophore, while the second sequence should have an acceptor fluorophore, as shown schematically in Figure 1d. In the absence of the target, the probes are dispersed in solution and no FRET is observed.…”

“…An ideal BP will show strong donor fluorescence and little or no acceptor fluorescence in the absence of target; the converse is expected when the target is present. 11 These conditions are generally fulfilled when there is no light absorption by the acceptor at the donor excitation wavelength, when the distance between the fluorophores is optimal, and when there is a good overlap between the donor fluorescence spectrum and the acceptor absorption spectrum. 27 One of the advantages of BPs is their high specificity since two distinct probes must bind the target to produce the characteristic detection signal.…”

“…28 Some potential applications of BPs include in vitro monitoring of RNA transcription, 12 cellular imaging, 27 and identification of gene translocation. 11 Nevertheless, there is still much room for improvement in the design of BPs. For example, BPs present relatively slow hybridization kinetics, since two probes must hybridize to the target for the signal to be detected.…”