G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence

Abstract

G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

A practical guide to studying G-quadruplex structures using single-molecule FRET

“…Force spectroscopy measurements have shown that the end to end distance change upon unfolding of a GQ is in the ~ 1 nm range (You et al 2015). Therefore, it is possible to monitor GQ dynamics by smFRET in isolation (Ying et al 2003; Lee et al 2005; Shirude and Balasubramanian 2008; Okumus and Ha 2010; Kruger and Birkedal 2013; Long and Stone 2013; Tippana et al 2014) or while it interacts with single stranded DNA binding proteins (Kruger et al 2010; Hwang et al 2012; Qureshi et al 2012; Ray et al 2013; Hwang et al 2014; Ray et al 2014; Zhou et al 2014), helicases (Budhathoki et al 2014; Chatterjee et al 2014; Zhou et al 2014; Budhathoki et al 2015), or GQ-stabilizing small molecules (Jena et al 2009; Kreig et al 2015). Furthermore, monitoring more subtle features, such as different conformations of GQ, is also possible with carefully designed DNA constructs (Long and Stone 2013; Budhathoki et al 2015).…”

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

“…Thus, investigation of thermodynamics and kinetics of the equilibrium between G4 and duplex DNA is important because the double helix is the native context for genomic DNA. The fluores cence assay in a combination with G4 recognizing low molecular weight ligands was used to measure the G4 folding propensity in both single stranded and double stranded DNAs [116]. As expected, the ability of G4 motif to form G4 was substantially diminished in the DNA duplexes due to the competition from the Watson-Crick base pairing.…”

“…THERMODYNAMICS AND KINETICS OF QUADRUPLEX FOLDING-UNFOLDING To characterize G quadruplexes formed in a certain genome locus, each identified G4 motif is investigated in vitro under different experimental conditions close, to a lesser or greater degree, to the intracellular ones. The methods commonly used for the description of G4 topol ogy and determination of thermodynamic and kinetic parameters of G quadruplex folding and unfolding include CD [106,107] and UV spectroscopy [108,109], differential scanning calorimetry [110,111], gel elec trophoresis [29,52], X ray crystallography [25,112], chemical and enzymatic probing [101,113,114], fluores cence spectroscopy, fluorescence resonance energy trans fer (FRET) [34,46,115,116], including single molecule BIOCHEMISTRY (Moscow) Vol. 81 No.…”

“…It was shown in several studies that G4-DNA duplex equi librium depended on many factors affecting relative sta bility of both secondary structures: temperature, avail ability of fragments flanking G4, primary structure of G motif, cationic composition, and other factors. One such factor is the length of loops in the quadruplex; the short er they are, the more stable is the G4 and, consequently, the less stable is the duplex formed in the presence of complementary strand due to the shorter hybridization site [116]. Thus, oligonucleotide with human telomeric repeats d(TTAGGG) 4 (the length of loops in the G4 structure are 3 : 3 : 3) forms duplex under physiological conditions in the presence of complementary C rich strand, while the sequence of the C myc promoter d(GGGTGGGGAGGGTGGG) predominantly assumes G4 conformation with 1 : 2 : 1 loops.…”

“…It was shown that the G4 population increased from 2.4 to 23% with increase in the density of negative supercoiling to -0.05; moreover, the G4 formation efficiency correlated with the readiness of the DNA double helix melting [175]. In addition to supercoiling, the structure of chromatin, binding of pro teins specifically recognizing quadruplex structures [4], and the effect of molecular crowding [116] can mediate a shift of equilibrium from DNA duplex to G4 in vivo.…”