| Molecular beacons (MBs) are fluorescent oligonucleotide nucleic acid probes whith a hairpin-shaped structure in which the 5'and 3'ends are self-complementary, the design of MBs is based on fluorescence resonance energy transfer (FRET).An MB contains a stem and a loop. In the MB closed state, the stem brings to proximity a fluorescent dye and a quencher located at both ends of the oligonucleotide: the fluorescence emission from the dye is consequently quenched. When the MB binding to a matching target region, the stem opens and the distance between the fluorescent dye and the quencher increases, enabling fluorescent emission to be detected. There are several advantages of MB, including excellent sensitivity, high selectivity, and real-time detection of target sequences in living cells. MB has been increasingly used in real-time monitoring of polymerase chain reactions (PCR), protein–DNA interaction studies and DNA/RNA hybridization kinetics assays.The synthesis of a typical MB is difficult and expensive. We describe an extrinsic fluorophore-based G4-MB (MBef),it consists of two parts: 30G, it with a hairpin-shaped structure and a fluorescent dye NMM (N-methyl mesoporphyrin IX). MBef and typical MBs are significantly different. A typical MB uses exogenous fluorescence labeling and the stem of it depends on Watson-Crick duplex. But the stem of MBef depends on the formation of a quadruplex motif. NMM can bind to quadruplex specifically. When quadruplex binds to NMM fluorescence intensity can change, and such a change can be used to monitor the substrate recognition of MBef.In this paper, we study the characteristics of MBef in various aspects. Firstly, we use UV spectrophotometer monitoring 30G and 30G+NMM thermal denaturizing curves .We find Tm value of 30G increasing significantly when adding NMM to 30G. This means that NMM affects 30G stability. This shows MBef differencing endogenous fluorescent labeling molecular beacon. We need to find the best working conditions of MBef. To this end, we conduct the following experiments. Next, this paper also considers the best concentration of 30G and NMM. For extrinsic fluorophore-based molecular beacons, the ratio of fluorophores to molecular beacons are the important factors affecting signal-to-noise ratio of detection. By fluorescence spectrophotometer monitors different concentrations ratio 30G and NMM. We observe the changes in fluorescence intensity. According to the experimental results, we have identified the best concentrations of 30G and NMM. When 30G and NMM in the best concentrations exist, the system can get a better signal-to-noise ratio. Finally, we study the different ions and ionic strength affecting the resolving power of MBef. This experiment selects two ions: Na+ and K+. Experimental results showed that when Na+ exists, Na+ concentration no matter how much is, the resolving power of MBef is low. But when K+ exists, the resolving power of MBef is relatively high. Meanwhile, we have found that different concentrations K+ to the resolving power of MBef have different effects. Therefore, we conduct a further analysis. Finally find the best K+ concentration. In this K+ concentration the resolving power of MBef can reach a higher level. Finally we summed up the best working condition of MBef.Through a series of studies we have found that this extrinsic fluorophore-based (MBef) molecular beacon not only maintains the high sensitivity and selectivity of the typical molecular beacon, but also has some new advantages, such as easy synthesis and low price. This experiment finds the best working condition of MBef successfully. Under this condition, the target oligonucleotide can induce the conformational change of quadruplex structure when it completely matched recognition arm of the loop of MBef, and the system transfers from high fluorescence intensity state to low fluorescence intensity state. Whereas, mismatched oligonucleotide can't accomplish preceding task. Therefore MBef is able to discriminate a single base difference in both target sequences.
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