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.

Figure 1.

NMM and CV binding to GQ DNA. (A) Chemical structure of NMM and CV. (B) Schematic of fluorescence assay by using NMM and CV. (C) DNA sequence of three constructs used here. (D, E) Scanned emission spectrum of NMM (D) and CV (E) bound to four DNAs. (F) Circular dichroism spectrum for three GQ-forming DNAs.

Figure 2.

NMM and CV fluorescence induced by GQ-forming DNAs. (A) Comparison of NMM quenching (previously published) and NMM fluorescence. (B) Scatter plot of NMM and CV fluorescence for all DNAs show three clusters corresponding to parallel, antiparallel and unfolded groups. (C) All DNA sequences used for NMM and CV fluorescence measurement.

Figure 3.

NMM and CV fluorescence assays on GQ formed on double strand DNA. (A) Schematic of NMM and CV binding to GQ on dsDNA. (B, C) Scanned emission spectrum of NMM (B) and CV (C) for the four DNAs. (D) All DNA sequences used for GQ formation in dsDNA. (E) Scatter plot of NMM versus CV fluorescence.

Figure 4.

Single molecule FRET assay to visualize GQ formation in dsDNA. (A) Schematic of DNA substrate with two dyes, Cy3 (green) and Cy5 (red) located on either side of GQ-forming sequence in dsDNA. (B) FRET histograms from five GQ-forming DNA annealed presence of molecular crowding reagent, PEG. (C) Quantitation of GQ folding by smFRET (black) and NMM fluorescence (red). (D) Representative single molecule trace obtained for five DNAs tested here. (E) smFRET traces of 1–4–4 and 1–5–5 exhibiting dynamic FRET fluctuations. (F) Quantitation of percent of molecules that display dynamic FRET.

Figure 5.

Summary of GQ-forming propensity. (A, B) The loop composition governs the GQ-forming potential differently in ssDNA and dsDNA.