In 1996, Tyagi and Kramer established the molecular beacon probe for the first time. The original purpose was to quantitatively measure the amount of target in the liquid phase. Molecular beacon technology is not only widely used in biological research, but also in disease gene detection and diagnosis due to its simple operation, high sensitivity, strong specificity, real-time quantitative determination of nucleic acids, and even in vivo analysis. It will also play an important role in biomedical basic and clinical research. Recently, people have designed many new molecular beacons by changing the structure of classic molecular beacons, such as RNA-DNA chimeric molecular beacons using ssDNA strands as loops and RNA-DNA double strands as stems, and replaced with PNA strands PNA molecular beacons formed by ssDNA. The emergence of new molecular beacons has broadened the field for the further application of molecular beacons.
Molecular beacons are cleverly designed fluorescent probes. A stem region complementary to the 5-8mer sequence is added to both ends of the 15-30mer oligonucleotide probe respectively. In the free state, the probe molecules form a hairpin-like structure due to the combination of complementary sequences in the stem region, so they are also called hairpin probes. The 5 and 3 ends of the probe are combined with fluorescein molecules and fragmentor molecules, respectively. It is a classic molecular beacon structure, in which 1-aminocai-8-betanoic acid (EDANS) is fluorescein and dimethylaminoazophenol (DABSYI.) Is a quencher. In the free state, the two ends of the hairpin structure are close, so that the fluorescent molecule and the fragmentation molecule are close (about 7-1Onm). At this time, fluorescence resonance energy transfer occurs, so that the fluorescence emitted by the fluorescent molecules is absorbed by the fragmented molecules and emitted in the form of heat. The fluorescence is almost completely quenched, and the fluorescence background is extremely low. For the working principle of molecular beacons, when molecular beacons and target molecules with completely complementary sequences combine to form a double-stranded hybrid, the complementary region of the beacon stem is pulled apart, and the distance between the fluorescent molecules and the broken molecules increases. According to Foerster theory, the central fluorescence energy transfer efficiency is inversely proportional to the sixth power of the distance between the two. After hybridization, the fluorescence of the beacon molecule recovered almost 100%. And the detected fluorescence intensity is proportional to the amount of target in the solution.
The most commonly used quencher in molecular beacons is DABSYL, which has a strong fluorescence quenching efficiency for a variety of fluoresceins. Recently, Dubertret et al. Used gold nanoparticle clusters instead of DABSYL as quenchers. One can also obtain different quenchers by adjusting the shape, size and composition of metal nanoclusters. Because gold nanoclusters have a higher quenching efficiency for fluorescent reagents, the replacement of DABSYL with gold nanoparticles greatly improves the sensitivity and specificity of molecular beacons
Molecular beacon, a fluorescent signal transmission mechanism, is based on fluorescence energy transfer. There may be two forms of energy transfer: direct energy transfer and fluorescence resonance energy transfer. When the fluorescent group and the quenching group are very close, direct energy transfer can occur due to the collision of the two group molecules. When the distance between the two groups is large and the emission spectrum of the energy donor (fluorescent group) and the absorption spectrum of the energy acceptor (quenching group) overlap to a large extent, fluorescence resonance energy transfer can occur. Since it has been found that (dimethylaminophenyl) azobenzoic acid can be used as a general quenching group for molecular beacons, it has a good quenching effect on a variety of fluorescent groups with different emission spectra. Therefore, direct energy transfer may be a major fluorescent energy transfer mechanism.
Molecular beacons were originally used as fluorescent probes for polymerase chain reaction (PCR). The working principle of molecular beacons can not only quantitatively detect the amplification products, but also monitor the amplification process in real time. Etc. designed TagMan molecular beacon, which is also a kind of PCR fluorescent probe with good performance.
After inventing the traditional molecular beacon, Tyagi et al. Designed a fluorescent wavelength transfer type molecular beacon, that is, two different fluorescent groups are connected at one end of the molecular beacon: a fluorescent collection group and a fluorescent emission group , The other end of the molecular beacon is connected to the quenching group. When the target molecule is not bound, it is the same as the traditional molecular beacon. The energy absorbed by the fluorescence collection group is transferred to the quenching group, which is released in the form of heat without fluorescence; when the conformation changes with the target molecule, the fluorescence The fluorescence of the collecting group is not recovered, but the energy is converted to fluorescence resonance energy
The form of quantity transfer is transferred to the fluorescent emitting group, and the fluorescent emitting group releases energy in the form of fluorescence. In this way, by selecting fluorescent emitting groups of different wavelengths, molecular beacons can emit different colors of fluorescence according to the design requirements. At the same time, this new type of molecular beacon has a large Stoke shift, which solves the small difference in excitation wavelength and emission wavelength in traditional molecular beacons, so that part of the excitation light reaches the detector through reflection and scattering, which affects the sensitivity. problem.
The TagMan molecular beacon still retains the stem-loop structure of the classic molecular beacon. The difference is that in addition to the loop sequence, the 5th-end stem sequence of the TagMan molecular beacon is also designed as the gene recognition site of the probe. When the probe specifically hybridizes with the target sequence to form a double strand, the 5-3 exonuclease activity of the Taq enzyme is activated, cleaving the fluorescent molecule attached to the 5 end of the probe from the probe, thereby making the fluorescent molecule and fragment The molecules are completely separated and the fluorescence is restored. The TagMan molecular beacon combines the advantages of the TagMan probe and molecular beacon technology. During the hybridization and probe degradation process, the TagMan molecular beacon can generate a fluorescent signal, which makes the TagMan probe have higher sensitivity.
Based on the principle of molecular beacons, Nobuko Hamaguchi et al. Designed Aptamer beacons for direct protein detection. They connected the 5th end of the antithrombin aptamer (Aptamer) to a piece of ssDNA to make it complementary to the partial sequence of the 3rd end of the aptamer to form a stem-loop structure. In the free state, the fluorescent molecules are close to the broken molecules, and the fluorescence is completely broken. When thrombin is present, the aptamer folds into a certain conformation, and specifically binds to thrombin through the three-dimensional structure. In this way, the stem-loop structure of the Aptamer beacon is destroyed, the fluorescent molecules are separated from the quencher molecules, and the fluorescence is restored. By changing the composition of Aptamer beacons or the length of the stalk region, Aptamer beacons can be used for the determination of different proteins. Compared with ELISA for protein detection, Aptamer beacon technology has the advantages of simplicity, directness, sensitivity and time saving. People can also fix multiple Aptamer beacons on the chip for single protein detection and analysis. The disadvantage of Aptamer beacon technology is that it cannot be used to detect non-specific ssDNA binding proteins, and the conformation of the beacon is greatly affected by metal ions. The presence of some metal ions will interfere with the observation of the fluorescent signal. The special hairpin structure makes the molecular beacon have The strong ability to specifically recognize target sequences has now become a powerful research tool in molecular biology and biotechnology.
In recent years, molecular beacon technology has developed rapidly, and has penetrated into various aspects of structural and molecular biology, genes and biotechnology. There is no doubt that molecular beacon technology has a broad application space. Chance and others have synthesized a molecular beacon that can diagnose breast cancer, which indicates that molecular beacon technology will bring a major breakthrough in the diagnosis and treatment of cancer and other diseases. Molecular beacon technology that can be directly used to detect non-amplified target sequences has emerged and attracted much attention. However, improving detection sensitivity is still the key to the development of this technology. With the development of micro-detection technology and spectroscopy technology and nucleic acids Enzymes and inorganic nanoparticles are used in the design of molecular beacon structures. The emergence of various new molecular beacons has made it possible to further improve the sensitivity of molecular beacons. In addition, molecular beacons will continue to play their advantages in the fields of studying small molecule-DNA interactions, protein-DNA interactions, and the development of biosensors.
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