Hybrid materials bring several advantages in improving the sensitivity of MBs, and many different probes have been developed. Ī recent advancement in MB technology involves the use of hybrid materials for DNA and RNA detection. Making better MBs involves improving the quenching efficiency, which is related to developing new PLS or quenchers, as well as innovative PLS and PLS–quencher systems. MB probes have high specificity toward single-base mismatch however, their sensitivity (defined as the ratio of the photoluminescence in the open versus the closed form signal-to-background ratio) is limited in many cases owing to incomplete quenching in the closed form. The first reported MB consisted of a five-base-pair stem and an 18-base loop section with 5-(2’-aminoethyl)aminonaphthalene-1-sulfonic acid as the PLS at the 5’ end and 4-(4’-dimethylaminophenylazo)benzoic acid (DABCYL) at the 3’ end acting as the quencher. This selective conformational change permits the observation of photoluminescence from the PLS in principle, only after the probe has selectively hybridized to the target. However, after hybridization with the target, the MB changes its conformation, forcing the PLS and quencher far apart and resulting in restoration of the MB photoluminescence. Owing to the close proximity enforced structurally by the stem, the quencher deactivates the PLS excited state generally by energy transfer or collisional quenching, resulting in a strong quenching of the photoluminescence. In the absence of the target, the complementary parts of the stem hybridize together, prompting the formation of a hairpin structure (loop–stem), bringing the PLS and the quencher into close proximity. Footnote 1 The MB has two distinctive parts: the loop portion, which is the probe part and is designed complementary to a desired target nucleic acid sequence, and the stem, which is formed of two self-complementary regions composed of five to six nucleotides at opposite ends of the strand. Briefly, a MB is an oligonucleotide that contains a photoluminescent species (PLS) and a quencher at different ends of the strand. The hairpin structures of MBs resemble the hairpin secondary structures commonly found in RNA, which lead to the unique properties of the probe. 1) which have been widely used in many areas, such as the detection of PCR products, mutational analysis, clinical diagnosis, genotyping, and allele discrimination. Molecular beacons (MBs), first proposed by Tyagi and Kramer in 1996, are hairpin-structured probes (Fig. On the basis of their high specificity, selectivity, and sensitivity, MBs are developed as a general platform for sensing, producing, and carrying molecules other than oligonucleotides. Nanoparticles, nanowires, graphene, metal films, and many other media have also been introduced to quench photoluminescence. Heavy-metal complexes, nanocrystals, pyrene compounds, and other materials with excellent photophysical properties have been applied as PLS of MBs. Making better MBs involves reducing the background photoluminescence and increasing the brightness of the PLS, which therefore involves the development of new PLS and quenchers, as well as innovative PLS–quencher systems. In a recognition and detection process, the hybridization of MBs with target DNA sequences restores the strong photoluminescence, which is quenched before hybridization. A molecular beacon (MB) is a hairpin-structured oligonucleotide probe containing a photoluminescent species (PLS) and a quencher at different ends of the strand.
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