Tseng, T-Y, Chien C-H, Chu J-F, Huang W-C, Lin M-Y, Chang C-C, Chang T-C.
2013.
A specific fluorescent probe for visualizing G-quadruplex DNA by fluorescence lifetime imaging microscopy. J Biomed Opt.. 18(10):101309.
AbstractABSTRACT. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8 ns and that of interaction with the duplex structure of a calf thymus is ∼1.2 ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.
Lin, CT, Tseng TY, Wang ZF, Chang TC.
2011.
Structural Conversion of Intramolecular and Intermolecular G-Quadruplexes of bcl2mid: The Effect of Potassium Concentration and Ion Exchange, Mar 17. Journal of Physical Chemistry B. 115:2360-2370., Number 10
AbstractThe gel assay, circular dichroism, and differential scanning calorimetry results all demonstrate that a major monomer component of bcl2mid exists at low [K(+)] and an additional dimer component appears at high [K(+)]. This implies that bcl2mid is a good candidate for elucidating the mechanisms of structural conversion between different G-quadruplexes. We further discovered that the conversion between the monomer and dimer forms of bcl2mid does not occur at room temperature but is detected when heated above the melting point. In addition, the use of the lithium cation to keep the same ionic strength in a K(+) solution favors the formation of the bcl2mid dimer. We also found that the bcl2mid dimer is more stable than the monomer. However, after the bcl2mid monomer is formed in a K(+) solution, there is no appreciable structural conversion from the monomer to the dimer detected with addition of Li(+) at room temperature. Furthermore, the spectral changes of bcl2mid when transitioning from sodium form to potassium form take place upon K(+) titration. The absence of the dimer form for bcl2mid after the direct addition of 150 mM [K(+)] at room temperature suggests that the spectral changes are not due to rapid unfolding and refolding. In addition, this work reveals the conditions that would be useful for NMR studies of G-quadruplexes.
Chang, TC, Chu JF, Tsai YL, Wang ZF.
2012.
Structure conversion and structure separation of G-quadruplexes investigated by carbazole derivatives. Curr Pharm Des. 18:2002-13., Number 14
AbstractThe challenge of G-quadruplexes is that the G-rich sequences can adopt various G4 structures and possibly interconvert among them, particularly under the change of environmental conditions. Both NMR and circular dichroism (CD) show the spectral conversion of d[AG3(T2AG3)3] (HT22) from Na-form to K-form after Na+/K+ ion exchange. No appreciable change on the induced CD spectra of BMVC molecule and the single molecule tethered particle motion of HT22 in Na+ solution upon K+ titration suggests that the spectral conversion is unlikely due to the structural conversion via fully unfolded intermediate. Although a number of mechanisms were proposed for the spectral change induced by the Na+/K+ ion exchange, determining the precise structures of HT22 in K+ solution may be essential to unravel the mechanism of the structural conversion. Thus, development of a new method for separating different structures is of critical importance for further individual verification. In the second part of this review, we describe a new approach based on "micelle-enhanced ultrafiltration" method for DNA structural separation. The BMVC, a G-quadruplex ligand, is first modified and then forms a large size of emulsion after ultrasonic emulsification, together with its different binding affinities to various DNA structures; for the first time, we are able to separate different DNA structures after membrane filtration. Verification of the possible structural conversion and investigation of structural diversity among various G4 structures are essential for exploring their potential biological roles and for developing new anticancer drugs.