Stem, molecular beacon

Ionic strength influences are well known with respect to the rate and energetics of nucleic acid hybridization [17]. Charge and ionic radius are both important in terms of stabilizing the structure of the duplex as well as stabilizing the stem portion of the molecular beacon [17]. The stem structure stability was increased when a divalent cation was incorporated into the hybridization buffer solution [17]. It was reported that cations were best at stabilizing the duplex formed upon hybridization in the order Ca2+ > Mg2+ K+ > Na+. The ultimate detection limit of the sensor configuration was calculated to be 1.1 nM [17]. 

Molecular beacons (MB) are stem-loop hairpin oligonucleotide structures that have a fluorescent dye at one end and a fluorescence quencher at the other. In the hairpin state, the quencher and fluorophore are in close proximity and therefore there is no fluorescence from the probe. However, when the MB binds to a complementary oligonucleotide as a duplex then the fluorophore and quencher are separated and the fluorophore can emit fluorescence. They are particularly useful in monitoring reactions with time, e.g., in PCR, " rolling circle amplification, hybridisation, telomerase activity, ligation... 

Figure 2.8a. Schematic representation of excimer-monomer switching molecular beacons (EMS MBs). EMS-MBs were dually labeled with pyrene at both 3 and S ends of single-stranded oligonucleotides with a stem-and-loop structure. In tlie absence of target DNAs. the slem-close-shaped EMS MBs predominantly emit the exclmer fluorescence (yellow-green). Upon hybridization with target DNAs, the EMS-MBs undergo the ilynamic conformational change to emit the monoincr fluorescence (pale Uue).

Thermal Denaturation Profiles. In the absence of a complimentary target, the molecular beacon expressed approximately 40% fluorescence (relative percentage) at 80°C (Figure 2), indicating that the stem structure had been denatured and the beacons had assumed a coiled conformation. As the temperature was decreased, stable hybridization of the stems was noted at 50°C, when the fluorescence had decreased to 0%. The melting temperature of the stem was determined to be 50-55°C. 

Another type of probe that utilizes a signal system of fluorescent and quenching dyes is the molecular beacon (Fig. 16.6). Molecular beacons form a stem-loop structure when free in solution. The close proximity of the fluorochrome and quenching components prevents the probe... 

A number of modifications to molecular beacons (MBs) have been reported. MBs have been synthesised using PNA (see section 1.2.1) for the detection of DNA mismatches. It is reported that DNA MBs are only able to discriminate matched from mismatched sequences within narrow temperature ranges, but using PNA MBs up to 100-fold selectivity for matched versus mismatched is described. " Sheng et al. have incorporated the unnatural base pair (93) into the stem of a MB. The (93) base pair is specific and neither base will form a base pair with the native DNA bases, therefore... 

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