Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Sep 18;43(16):7961-70.
doi: 10.1093/nar/gkv749. Epub 2015 Jul 21.

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

Alex Kreig  1 , Jacob Calvert  1 , Janet Sanoica  1 , Emily Cullum  1 , Ramreddy Tipanna  1 , Sua Myong  2
Affiliations

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

Alex Kreig et al. Nucleic Acids Res. .

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.

PubMed Disclaimer

Figures

Figure 1.
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.
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.
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.
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.
Figure 5.
Summary of GQ-forming propensity. (A, B) The loop composition governs the GQ-forming potential differently in ssDNA and dsDNA.
See this image and copyright information in PMC

References

    1. Todd A.K., Johnston M., Neidle S. Highly prevalent putative quadruplex sequence motifs in human DNA. Nucleic Acids Res. 2005;33:2901–2907. - PMC - PubMed
    1. Hud N.V., Smith F.W., Anet F.A., Feigon J. The selectivity for K +versus Na+ in DNA quadruplexes is dominated by relative free energies of hydration: a thermodynamic analysis by 1H NMR. Biochemistry. 1996;35:15383–15390. - PubMed
    1. Schultze P., Hud N.V., Smith F.W., Feigon J. The effect of sodium, potassium and ammonium ions on the conformation of the dimeric quadruplex formed by the Oxytricha nova telomere repeat oligonucleotide d(G(4)T(4)G(4)) Nucleic Acids Res. 1999;27:3018–3028. - PMC - PubMed
    1. Crnugelj M., Sket P., Plavec J. Small change in a G-rich sequence, a dramatic change in topology: new dimeric G-quadruplex folding motif with unique loop orientations. J. Am. Chem. Soc. 2003;125:7866–7871. - PubMed
    1. Hazel P., Huppert J., Balasubramanian S., Neidle S. Loop-length-dependent folding of G-quadruplexes. J. Am. Chem. Soc. 2004;126:16405–16415. - PubMed

Publication types

LinkOut - more resources