Target molecular beacon

Fujimoto K, Shimizu H, Inouye M (2004) Unambiguous detection of target DNAs by excimer—monomer switching molecular beacons. J Org Chem 69 3271-3275... 

Molecular beacons are single-stranded hairpin-shaped nucleotide probes. In the presence of the target nucleotide sequence the molecular beacon unfolds, binds and fluoresces (Figure 6.13). 

Loop this is the nucleotide region of the molecular beacon, which is complementary to the target nucleotide sequence. 

Figure 6.13 Mode of action of a molecular beacon. Fluorescence is only observed when the beacon and the target nucleotide bind together...

A simple diagnostic test has been devised for prostate cancer, using a specific molecular beacon mixed with the target DNA on a microscope slide. The DNA is treated to separate the strands and, provided there is the correct correspondence between the bases, in situ combination occurs between the bases on the molecular beacon and those on the strands. Thus, if fluorescence is observed, the DNA sample must contain the base sequence indicative of prostate cancer. 

Thermal denaturation profiles were done using one of the molecular beacon structures in solution, without the alkyl chain linker attached to the 3 -thymidine residue. The fully complementary and single base pair mismatched sequence targets were investigated. The differences of Tm from the melt profiles showed that single base pair mismatches could not be discriminated from the complementary target material. Melt curves were also... 

Tsourkas et al. (2003) reported dual FRET molecular beacon assays, where the donor probe was labeled with either Eu3+ or Tb3+ complex of DTPA-csl24-ethylenediamine (and no quenchers attached). For the Eu3+ complex, the acceptor probe was Cy5-labeled (and no quenchers attached) and for the Tb complex, the acceptor probe was labeled with Cy3 or ROX as a fluorophore and with dabcyl as a quencher. They demonstrated that these pairs of probes detected DNA targets ( 50-mer) with high S/N. 

Fig. B.18.1. Molecular beacons are virtually non-fluorescent in the unhybridized form 1. Target binding opens the molecular beacon to form 2 and fluorescence occurs.

Usnally a nncleic acid probe that emits a signal only when bound to a specific target. An example is a molecular beacon. See Stohr, K., Hafner, B., Nolle., O. et al.. Species-specific identification of mycobacterial 16S rRNA PCR amplicons nsing smart probes. Anal. Chem. 77, 7195-7203, 2005. There are other examples of smart probes inclnding proteins (Wunder, A., Tung, C.-H., Mtiller-Ladner, U., Weissleder, R., and Mahmood, U., In vivo imaging of protease activity in arthritis, a novel approach for monitoring treatment response, Arthritis Rheumatism 50, 2459-2465, 2004) and chiral compounds (Tsukube, H. and Shinoda, S., Lanthanide complexes as smart... 

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). 

Kuhn H, Demidov W, Coull IM, Eiandaca MI, Gildea BD, Frank-Kamenetskii MD. Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. 42. J. Am. Chem. Soc. 2002 124 1097-1103. 

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. The MBs have been immobihzed by Tan and co-workers [27] onto ultrasmall optical fibre probes through avidin-biotin binding. The MB-DNA biosensor detected its target DNA molecules, in real time, with selectivity for a single base-pair mismatch. This MB-DNA-biosensor was used by Perlette and Tan [28] for real-time monitoring of mRNA-DNA hybridization inside a living cell. 

For studies of hematological malignancies, the sequence-specific fluorescence probe-based systems provide the advantage of an important further test for identification of the sequence of interest by virtue of hybridization of fluo-rescently labeled internal probes to the amplified target sequence. The most commonly used sequence-specific chemistries include the exonuclease (TaqMan), the linear hybridization probe, and the hairpin-based (Molecular Beacon) systems. (See Chapter 37 for further information on nucleic acid techniques.)... 

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