Binary probes

The noncovalent specific labeling of NA probes based on the aptamer strategy has also been reported. The malachite green (37) aptamer, which was designed as a binary probe, assembles only in the presence of the target sequences and induces an increase in the intensity of the signal emitted by the F bound aptamer. 

The design of hybridization formats that enable important changes (fluorescence intensity increase or wavelength shifts) in the presence of the targets is the focus of intense research because their use can simplify the analyses in vitro and provide the possibility of applications in living organisms. The most frequently used involve binary probes (35), competitive hybridization probes (18), linear probes with only one F (2, 5, 16, 29, 31), and molecular beacons (MBs) (39-41), (Fig. 3). 

Increasing the specificity of detection with binary probes... 

Figure 3 Schematic representation of the main hybridization formats, (a) Binary probes, (b) Competitive hybridization probes, (c) Linear probe with one F. (d) Moiecuiar Beacon, (e) Aptamer.

Detection of RNAs In living cells Autoligating Binary probes Uniprobe Two F and one Q (at selected positions) MB or MB pair F at internal position FRET, flow cytometry and confocal microscopy 19, 29, 40-42,... 

Visualization of mRNA in cellular extracts Binary probes Two Pyrene. One at the 5 -end of one ON probe and the second at the 3 -end of the second ON probe Excimer detection Time-resolved microscopy 35... 

An important factor for in vivo studies is the auto fluorescent cellular background. To overcome this problem, pyrene-labeled binary probes have been used to take advantage of the long fluorescence lifetime (>40ns) of the pyrene excimer, compared with that of the cellular extracts (7 ns), that allows selective detection of the excimer using time-resolved spectroscopy (35). 

Marti AA, Li X, Jockusch S, Li Z, Raveendra B, Kalachikov S, Russo JJ., Morozova I, Puthanveettil SV, Ju J, Turro NJ. Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy. Nucleic Acids Res. 2006 34 3161-3168. 

Kolpashchikov, D. Binary probes for fluorescent analysis of nucleic acids. PCT Int. Appl. WO 2007115242,2007 Chem. Abstr. 2007,147,441322. 

Marti, A. A. Puckett, C. A. Dyer, J. Stevens, N. Jockusch, S. Ju, J. Barton, J. K. Turro, N. J. Inorganic-organic hybrid luminescent binary probe for DNA detection based on spin-forbidden resonance energy transfer. J. Am. Chem. Soc. 2007,129, 8680-8681. 

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