Fast detection of quadruplex structure in DNA by the intrinsic fluorescence of a single‐stranded DNA binding protein

Abstract

Single-stranded guanine-rich (G-rich) DNA can fold into a four-stranded G-quadruplex structure and such structures are implicated in important biological processes and therapeutic applications. So far, bioinformatic analysis has identified up to several hundred thousand of putative quadruplex sequences in the genome of human and other animal. Given such a large number of sequences, a fast assay would be desired to experimentally verify the structure of these sequences. Here we describe a method that identifies the quadruplex structure by a single-stranded DNA binding protein from a thermoautotrophic archaeon. This protein binds single-stranded DNA in the unfolded, but not in the folded form. Upon binding to DNA, its fluorescence can be quenched by up to 70%. Formation of quadruplex greatly reduces fluorescence quenching in a K+-dependent manner. This structure-dependent quenching provides simple and fast detection of quadruplex in DNA at low concentration without DNA labelling.

Design, synthesis and biological evaluation of a novel class of anticancer agents: Anthracenylisoxazole lexitropsin conjugates

“…The microarray fluorescence experiment for the mixture of Calf Thymus (CT) DNA with 29 indicates a quenching of the fluorescence but shows no bathochromic shift which would be consistent with an anthracene intercalation mechanism (Figure 2). 54,55 The Hurley-Oligonucleotide experiment with 29 shows considerable quenching of the fluorescence which is consistent with a π-stacking interaction with the G-tetrad of the quadruplex structure that forms in situ under the experimental conditions 56,57…”

G-Quadruplexes in the Archaea Domain

“…Interestingly, it was found in several Haloarchaea species that a specific (TGGGGGGG) motif occurs in one of the two origins of replication (oriC1) [ 77 ]. This long G-rich motif was shown to be necessary for efficient replication initiation in Haloarcula hispanica [ 78 , 79 ] and predicted to be prone to inter-molecular quadruplex formation.…”

“…Besides protein actively unfolding G4 structures, other peptides may actually bind to single-strand G-rich sequences and passively contribute to G4 unfolding by conformational selection. This is the case for a single-strand binding protein isolated from Methanococcus jannaschii , which was used to design an assay to detect G4 formation [ 79 ]. Apart from proteins that actively or passively unfold quadruplexes, others may bind to and sometimes promote G4 formation.…”

Conformation Effects of CpG Methylation on Single-Stranded DNA Oligonucleotides: Analysis of the Opioid Peptide Dynorphin-Coding Sequences

“…Oligonucleotides that form non-canonical structures such as DNA hairpins and G-quadruplexes differ in mobility on the native PAGE from unstructured oligonucleotides [38]–[44], and even single-nucleotide substitutions may induce conformational changes that can be detected with PAGE analysis [16].…”