Discrimination of G‐Quadruplexes from Duplex and Single‐Stranded DNAs with Fluorescence and Energy‐Transfer Fluorescence Spectra of Crystal Violet

Abstract

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 benzindole substituted carbazole cyanine dye: a novel targeting fluorescent probe for parallel c-myc G-quadruplexes

“…1,2 A variety of G-rich sequences are found in locations widely prevalent at eukaryotic telomeres in the human genome, in proliferation, in RNA sequences and in oncogene promoters. [19][20][21][22][23][24][25][26][27][28][29][30][31] The c-myc gene promoter G4 structure was selected as the target in view of its important role linked to human cancers, such as breast cancer, cervical cancer, leukemia, etc. [3][4][5][6][7][8][9][10] Because their formation is mostly related to the processes of transcription, replication, and recombination, they are highly associated with human diseases 11 (e.g., cancer, 12,13 cardiovascular diseases 14 and diabetes 15 ).…”

Sensitive and selective detection of uracil-DNA glycosylase activity with a new pyridinium luminescent switch-on molecular probe

“…To overcome the drawbacks of the traditional assays, fluorescence-based assay techniques performing in homogeneous phases have been developed to realize rapid assay of UDG activity under simple conditions. [24][25][26] The double quadruplex DNA has also been found applications in biology and nanotechnology under specific conditions; 27 however, the G-quadruplex DNA represents a more versatile sensing platform for bioanalytical assays because the specific recognition can be done by luminescent sensing probes. 21 The G-quadruplex motif is a DNA secondary structure consisting of square-planar arrangements of guanine nucleobases stabilized by Hoogsteen hydrogen bondings and monovalent cations.…”

Simple and Convenient G-Quadruplex-Based Turn-On Fluorescence Assay for 3′ → 5′ Exonuclease Activity

“…The circular dichroism (CD) spectrum of the annealed oligonucleotide confirmed the presence of duplex DNA (see below). We used the human telomeric G-quadruplex sequence [5′-AG 3 (T 2 AG 3 ) 3 -3′] due to the strong fluorescent response of crystal violet (CV) to this G-quadruplex 6. CV is an inexpensive, commonly available triphenylmethane dye that has been demonstrated to display significant selectivity for the G-quadruplex secondary structure over single-stranded and double-stranded DNA 6.…”

“…We used the human telomeric G-quadruplex sequence [5′-AG 3 (T 2 AG 3 ) 3 -3′] due to the strong fluorescent response of crystal violet (CV) to this G-quadruplex 6. CV is an inexpensive, commonly available triphenylmethane dye that has been demonstrated to display significant selectivity for the G-quadruplex secondary structure over single-stranded and double-stranded DNA 6. Celada and coworkers have measured the 3′ → 5′ exonucleolytic activity of TREX1 employing SYBR Green as a probe to monitor the double-stranded to single-stranded DNA transition of a short duplex 4a.…”