Existence of G-Quadruplex Structure within the Human Chromosomes

The functions and conformations behind the G-quadruplexes have increased interests about considerable amounts of researches since there has been the revelation of G-quadruplexes and G-tetraplexes telomeric DNA (Chen et al. 2016). This is because, the structure of these types is actually the subtstrates for telomerase and the stabilizers of quadruplex also have a potential of serving as antitumor. Therefore, it a huge help in the field of designing the cancer drugs as well as the diagnosis of various diseases are helped by the competent recognition of the G-quadruplexes with the factor of the G-quadruplex structures and its stabilization. Tandem G-rich duplication units, as for example, the TTAGGG containing telomeres originate where the final ends of chromosomes reside in vertebrate animals. The stabilization and formation of the G-quadruplex forms the need for the monovalent cations, especially, potassium and sodium. In presence of various cations, there is a chance of adopting different structures in the G-rich sequences. There are also some researchers that would like to try and investigate about the real G-quadraplex using the different types of quadruplex-binding fluorophores. However, most of the structures that are described about above can be completed with the help of the spectral methods with the help of an mock sequence of DNA in vitro. In addition, there has been scattered discussion over the issue about whether or not these structures have their literal existence in vivo.

The TTAGGG sequence can be showcased with the help of the Fluorescenecs in situ Hybridization or FISH. However, there has been no presence of the qudruplex structure within a cell as per reports as well as there are no evidence in the chromosomes (Wang et al. 2018). Only after the in situ immunostaining, occurrence of G-quadruplexes has been pragmatic directly. There has been a strong signal identified only within the antibodies that are specific in the anti parallel G-quadruplexes. However, it is still a questionable issue about every single G-quadruplex structure and their existence that has been mapped within the human chromosome or telomere. A much more direct confirmation about quadruplex structures being in an existing form, fluorescent signal detection having the potential of targeting the G-quadruplex fluorophores can be used to formulate the surety of the constructions that are specifically able to be seen beneath fluorescence microscopy. The key hub is within the method for fluorescent image detection since it is convenient and visible. Taking this method into consideration, it is imperative that a good stabilizer for G-quadruplex or recognizer having fluorescent emission within a region that is visible to be present.

Fluorescence Resonance Energy Transfer (FRET) and Quadruplex-Binding Fluorophores

The studies in this research has found out that the G-quadruplex arrangement of the human telomere can be stabilized with the help of the natural minute molecule BMVC and the (TTAGGG)₄ (Kotar et al. 2016). The G-quadruplex arrangement of the human telomere acts as the antitumor agents. In addition, fluorescence acquiesce and the discrete fluorescence chattels of BMVC intertwined with various DNA structures has been increased as a result since it has been allowing the mapping of the G-quadruplex structure within the choromosomes of human metaphase. The distinguishable properties have also permitted the observation of the dazzling fluorescence spots from BMVC in the cancer cell nucleus in comparison to the weak fluorescence as observed within the normal cell BMVC. In addition, a simple handheld device that helps in the incorporation of the BMVC molecule has its design done in accordance with a inexpensive point-of-care screening for the oncogenic cells even if BMVC is a good quadruplex distinguishing fluorophore, but it still fails to construct the fluorescent signal to be noticeable with the help of microscopy techniques as there is a tendency that a large amount of excess duplex structures has the potential to interfere with the monitoring of quadruplexes within the chromosome. Additionally, the wavelength for the emissions comes to a difference in nature of the BMVC when it binds with duplex and as a result the quadruplex is observed to be less than 20 nm (Lee, Park and Park 2015). It determines that assortment of the fluorescent color sandwiched between quadruple and duplex is imperceptible when BMVC has its stains over the entire chromosome. Therefore, a strategy has been developed for making increments within the contrast in the coloration of the image with the utilization of FRET. The reason for choosing Hoechst and propidium iodide (PI) as duplex-binding fluorophores and puts in attendance for the conventional consequences with suitable staining modus operandi is clearly stated in this assignment.

The ideal strategy that are used as a result of this is the utilization of the FRET in sandwiched amid a duplex binding ligand along with the G-quadruplex stabilizer taken into consideration about the increasing and decreasing of the signals of the fluorescent quadruplexes. This in turn gradually increases the contrast colors between the duplexes and quadruplexes (Li et al. 2018). Hoechst takes action as the duplex-binding ligand that helps in the binding of most of the duplex structure in the chromosome. BMVC dupes as a stabilizer for G-quadruplex. The spectrum criteria is based on the blue emissions of Hoechst that revolves around the quadruplex is most likely to be absorbed by BMVC once they come really close enough to proceed with FRET. This is why; the blue fluorescence of the duplex in the region of the quadruplex comes to a decrease and the green and yellow fluorescence of the quadruplex increases. It is expected that this model has the ability to modify the color contrast of the image to show the quadruplex signal with the help of a microscope. In another way, in case B, the FRET donor is the BMVC molecule, which makes the PI molecule to act as an acceptor. It is expected that red emissions are emitted from the quadruplex moiety of the chromosomes to exhibit the emission bound with the quadruplex has been terminated with the help of the PI molecule (Zuffo et al. 2016). The other moieties however, transfer signals that are either yellow or orange fluorescence. Summarizing the entire FRET mechanism, the binding modes of quadruplex-binding molecules are regarded as the binding mechanism of stacking or groove. Hence, it would be proposed that the duplex structures are spread everywhere around the quadruplex structure unit within the chromosome. It has been proposed that the FRET of quadruplex binding molecule might occur with Hoechst, which binds to the same quadruplex structure unit with different binding modes and the free Hoeschst that bind around the quadruplex structures. The entire stain orders and incubation times for the unit can be depicted in the order of these probes. Thus, the research proves that the experiment results help in increasing the contrast of the fluorescent colors.

Detection of G-Quadruplex Structures within Human Chromosomes

Conclusion

The entire research done to make the fluorophores and their structures be much more visible to the fluorescence microscope, unswerving confirmation from the existence that the quadruplex structure has within the human chromosomes. Therefore, a proper G-quadruplex identifier having fluorescent emission is important. Therefore, the above report consists of the evaluation of the spectral energies of BMVC, PI and Hoechst that helps in establishing the FRET model. Following this, the binding preferences and abilities that are consisted of these three molecules to duplex and quadruplex had been investigated. It has been pragmatic, that the enhancement induced by FRET of fluorescent signal contrast sandwiched amid quadruplex and duplex was accomplished with the use of a G-quadruplex recognizer and a duplex-binding fluorophore following an incubation procedure that is appropriate. It has thus been observed that the enhancement as proposed by FRET increases the fluorescence contrast between that of the quadruplex and duplex as per investigated. This has been achieved as per the influence of the G-quadruplex recognizer and a duplex-binding fluorophore following an appropriate incubation procedure. The research has shown how the green-yellow color fluorescence increases. The results have been doubly confirmed with the PI/BMVC methods. The strategy has also been differentiated with the help of other methods like that of the FISH method and the antibody labeling method and it has been found that this may result in the overcoming of the detection problems. This results in the existence of increasingly low amount of the G-quadruplex structures when there is a distinguishable amount of duplex DNA found within the chromosomes.

References

Chen, S.B., Hu, M.H., Liu, G.C., Wang, J., Ou, T.M., Gu, L.Q., Huang, Z.S. and Tan, J.H., 2016. Visualization of NRAS RNA G-quadruplex structures in cells with an engineered fluorogenic hybridization probe. Journal of the American Chemical Society, 138(33), pp.10382-10385.

Kotar, A., Wang, B., Shivalingam, A., Gonzalez?Garcia, J., Vilar, R. and Plavec, J., 2016. NMR structure of a triangulenium?based long?lived fluorescence probe bound to a G?quadruplex. Angewandte Chemie, 128(40), pp.12696-12699.

Zuffo, M., Doria, F., Botti, S., Bergamaschi, G. and Freccero, M., 2017. G-quadruplex fluorescence sensing by core-extended naphthalene diimides. Biochimica et Biophysica Acta (BBA)-General Subjects, 1861(5), pp.1303-1311.

Lee, C.Y., Park, K.S. and Park, H.G., 2015. A fluorescent G-quadruplex probe for the assay of base excision repair enzyme activity. Chemical Communications, 51(72), pp.13744-13747.

Wang, M.Q., Ren, G.Y., Zhao, S., Lian, G.C., Chen, T.T., Ci, Y. and Li, H.Y., 2018. Development of a carbazole-based fluorescence probe for G-quadruplex DNA: The importance of side-group effect on binding specificity. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 199, pp.441-447.

Li, S., Liu, C., Gong, H., Chen, C., Chen, X. and Cai, C., 2018. Simple G-quadruplex-based 2-aminopurine fluorescence probe for highly sensitive and amplified detection of microRNA-21. Talanta, 178, pp.974-979.