Crystal violet-G-quadruplex complexes as fluorescent sensors for homogeneous detection of potassium ion.

A novel K(+) detection method was reported using a label-free G-quadruplex-forming oligonucleotide and a triphenylmethane fluorescent dye crystal violet (CV). This method is based on the fluorescence difference of some CV/G-quadruplex complexes in the presence of K(+) or Na(+), and the fluorescence change with the variation of K(+) concentration. According to the nature of the fluorescence change of CV as a function of ionic conditions, two K(+) detection modes can be developed. One is a fluorescence-decreasing mode, in which T(3)TT(3) (5'-GGGTTTGGGTGGGTTTGGG) is used, and the fluorescence of CV decreases with an increased concentration of K(+). The other is a fluorescence-increasing mode, in which Hum21 (5'-GGGTTAGGGTTAGGGTTAGGG) is used, and the fluorescence of CV increases with an increased concentration of K(+). Compared with some published K(+) detection methods, this method has some important characteristics, such as lower cost of the test, higher concentrations of Na(+) that can be tolerated, adjustable linear detection range and longer excitation and emission wavelengths. Preliminary results demonstrated that the method might be used in biological systems, for example in urine.

Fluorescent sensor for monitoring structural changes of G-quadruplexes and detection of potassium ion.

G-rich sequences with the potential for quadruplex formation are common in genomic DNA. Considering that the biological functions of G-quadruplexes may well depend on their structures, the development of a sensitive structural probe for distinguishing different types of quadruplexes has received great attention. Crystal violet (CV) is a triphenylmethane dye, which can stack onto the two external G-quartets of a G-quadruplex. The ability of CV to discriminate G-quadruplexes from duplex and single-stranded DNAs has been reported by us. Herein, the ability of CV to discriminate parallel from antiparallel structures of a G-quadruplex was studied. The binding of CV to an antiparallel G-quadruplex can make its fluorescence intensity increase to a high level because of the protection of bound CV from the solvent by quadruplex end loops. The presence of side loops in parallel G-quadruplexes cannot provide bound CV such protection, causing the fluorescence intensity of CV/G-quadruplex mixture to be obviously weaker when the G-quadruplex adopts a parallel structure than that when the G-quadruplex adopts an antiparallel structure. Therefore, CV can be developed as a sensitive fluorescent biosensor for the discrimination of antiparallel G-quadruplexes from parallel G-quadruplexes and for monitoring the structural interconversion of G-quadruplexes. In addition, considering that some G-rich DNA sequences can adopt different G-quadruplex structures under Na(+) or K(+) ion conditions, a novel, cheap and simple K(+) ion detection method was developed. This method displays a high K(+) ion selectivity against Na(+) ion, the change of 200 mM in Na(+) ion concentration only causes a similar fluorescent signal change to 0.3 mM K(+) ion.

Discrimination of G-quadruplexes from duplex and single-stranded DNAs with fluorescence and energy-transfer fluorescence spectra of crystal violet.

G-rich nucleic acid sequences with the potential to form G-quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G-quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G-quadruplex, duplex, or single-stranded DNAs were studied by fluorescence spectroscopy and energy-transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single-stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy-transfer fluorescence spectra of CV and MG. In addition, by using energy-transfer fluorescence titrations of CV with G-quadruplexes, the binding-stoichiometry ratios of CV to G-quadruplexes can be determined. By using the fluorescence titrations of G-quadruplex-CV complexes with C-rich complementary strands, the fraction of G-rich oligonucleotide that engages in G-quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single-stranded DNAs and for measuring G-quadruplex percentages in the presence of the complementary C-rich sequences.

A new method for the study of G-quadruplex ligands.

A new method for the study of G-quadruplex ligands was developed, in which the interaction of G-quadruplexes with ligands can be judged by the naked eye, eliminating the need for any expensive machines.