The latest development of molecular beacons.

first molecular beacon probes were established in 1996 by Tyagi and Kramer, with the initial aim of quantifying the amount of the target in the liquid phase. Molecular beacon technology, with its simple operation, high sensitivity, strong specificity, real-time quantitative determination of nucleic acids, and even for live analysis, is not only widely used in biological research, but also in disease
gene testing
and diagnosis, such as
biomical
foundation and clinical research will also play an important role.
recently, many new types of molecular beacons, such as ss, have been designed by changing the structure of
. DNA
as a ring, RNA-DNA double strands as stems of RNA-DNA chimed molecular beacons, PNA chains instead of ss DNA formed PNA molecular beacons. The emergence of new molecular beacons has broadened the field for the further application of molecular beacons.
(1) Molecular Beacon
Molecular Beacon
is a cleverly designed fluorescent probe. At both ends of the 15-30mer oligonutochonic acid probe, add a stem area with a complementary 5-8mer sequence. In free state, the probe molecule is also called a hairpin probe because of the combination of complementary sequences in the stem region.
5' and 3' ends of the probe are jointly used with luciferin molecules and shredding agent molecules respectively. For the classic molecular beacon structure, wherein 1-amino-Chua-8-rebel acid (EDANS) is luciferin, methamphetamine-animate phenol (DABSYI.) for annihilation agent. In a free state, the two ends of the hairpin structure are close, so that the fluorescent molecule is close to the crushing molecule (approximately 7-1Onm). At this time, fluorescence resonance energy transfer occurs, so that the fluorescence emitted by the fluorescent molecules is absorbed by the crushed molecules and emitted in the form of heat, fluorescence is almost completely annihilation, fluorescence background is very low.
is how molecular beacons work, and when molecular beacons and target molecules with fully complementary sequences combine to form a double-stranded hybrid, the complementary areas of beacon stems are pulled apart and the distance between fluorescent and fragmented molecules increases. According to Foerster's theory, the central fluorescent energy transfer efficiency is inversely inversely related to the six times the distance between the two. After hybridization, the fluorescence of beacon molecules is almost 100% restored. And the detected fluorescence intensity is directly related to the amount of the target in the solution.
most commonly used annihilation agent in molecular beacons is DABSYL, which has a strong fluorescent annihilation efficiency for a variety of luciferins. Recently, Daubertret and others used gold nanoparticle clusters instead of DABSYL as an annihilation agent, people can also adjust the shape, size and composition of metal nanoclusters to obtain different annihilation agents. Because gold nanoclusters have a higher annihilation efficiency to fluorescent
reagements
, the sensitivity and specificity of molecular beacons are greatly improved by replacing DABSYL with gold nanoparticles.
the fluorescent signal conduction mechanism of the molecular beacon is based on the transfer of fluorescent energy. There may be two forms of energy transfer: direct energy transfer and fluorescent resonance energy transfer. When fluorescent and annihilation groups are very close together, direct energy transfer can be generated due to the collision of two group molecules.
Fluorescence resonance energy transfer occurs when the distance between the two substrings is at a greater extent and the emission
spectra
of the energy-giving body (fluopho corpus) overlaps with the absorption spectrum of the energy subject (annihilation group). Since it has been found that amethyst benzoic acid can be used as a common annihilation group for molecular beacons, it has a good annihilation effect on a variety of fluorescent groups with different emission spectra. Therefore, direct energy transfer may be a major fluorescent energy transfer mechanism.
molecular beacon was originally used as a fluorescent probe for polymerase chain reaction (
PCR
), molecular beacon works, it can not only quantitatively detect the amplification of the production, but also can monitor the amplification process in real time recently, such as the design of tagMan molecular beacon, is also a good performance of PCR fluorescent probe.
(2) fluorescent wavelength metastatic molecular beacon
. After inventing the traditional molecular beacon, Tyagi and others designed a fluorescent wavelength metastatic molecular beacon that connects two different fluorophor groups at one end of the molecular beacon: the fluorescent collection base and the fluorescent emission base, and the other end of the molecular beacon connects the annihilation group.
When the target molecule is not combined, it is the same as the traditional molecular beacon, the energy absorbed by the fluorescent collection group is transmitted to the annihilation group, which is released in the form of heat and does not produce fluorescence;
this way, by selecting different wavelengths of fluorescent emission groups, so that molecular beacons are designed to emit different colors of fluorescence. At the same time, this new type of molecular beacon has a large Stoke displacement, which solves the problem that the difference between excitation wavelength and emission wavelength in traditional molecular beacon is small, so that some excitation light reaches the detector through reflection and scattering, which affects sensitivity.
(3) TagMan molecular beacon
. The TagMan molecular beacon still retains the stem-ring structure of the classic molecular beacon, except that the TagMan molecular beacon is designed as the probe's
gene
identification site in addition to the ring sequence. When the probe and the target sequence of special complementary hybridization to form a double chain, Taq enzyme 5'-3' cut activity is activated, the probe 5' end of the connected fluorescent molecules cut off from the probe, so that the fluorescent molecules and the crushing molecules completely separated, fluorescence recovery. TagMan molecular beacons focus on the advantages of tagMan probes and molecular beacons, both of which produce fluorescent signals during hybridization and probe degradation, which makes TagMan probes more sensitive.
(4) Aptamer Beacon
Based on molecular beacon principles, Nobuko Hamaguchi and others designed aptamer beacons for direct detection of
protein
. They connected the 5' end of the anticoagulant enzyme fitting (Aptamer) to a piece of ssDNA, which complements the sequence of the 3' end of the body to form a stem ring structure. In a free state, the fluorescent molecule is close to the crushing molecule and the fluorescence is completely crushed. When the clotting enzyme exists, the suitable body folds into a certain structure, and the specific binding with the clotting enzyme occurs through three-dimensional structure. In this way, the stem ring structure of the Aptamer beacon is destroyed, the fluorescent molecule is separated from the annihilation molecule, and the fluorescence is restored. By changing the composition of the Aptamer beacon or the length of the stem area, the Aptamer beacon can be used for the determination of different proteins.
, the Aptamer beacon technology has the advantages of simple, direct, sensitive and time-saving compared with enzyme-linked immunosorption determination protein. A variety of Aptamer beacons can also be pinned to a chip for the detection and analysis of a single protein. The disadvantage of Aptamer beacon technology is that it cannot be used to detect nonse specific ssDNA binding proteins, and the composition of beacons is greatly affected by metal ions, the presence of some metal ions will interfere with the observation of fluorescent signals, special hairpin structure makes molecular beacons have a strong ability to identify target sequences, has become
molecular biology
and biotechnology in a powerful research tool.
.