Formation of G-quadruplex structure in supercoiled DNA under molecularly crowded conditions

Abstract

G-quadruplex is a secondary structure of nucleic acids that plays crucial roles in many significant biological processes. Potential G-quadruplex-forming sequences exist widely in various regions of the genome such as telomeres and gene promoters. In spite of the fact that G-quadruplex can be readily assembled from a single-stranded segment of DNA, its formation from duplex DNA is very difficult under physiological conditions because Watson-Crick interactions in guanine rich segments need to be weakened first. It is demonstrated in our studies that intrastrand G-quadruplex generated from a perfectly matched guanine-rich duplex in a circular DNA as a result of significant quadruplex stabilization and duplex destabilization created by the combined actions of negative DNA supercoiling and molecular crowding conditions.

Fig. 1. Diagrammatic illustration of G-quadruplex formation in supercoiled G-rich containing circular DNA.

Fig. 2. Examination of G-quadruplex formation in DNA topoisomers. (A) Electrophoretic analysis of DNA products. Lane 1: DNA-S alone; negative supercoiled DNA topoisomers (ΔLk = −4 to −6) were incubated in the solution containing 150 mM KCl and 4 mM NaCl in the absence (Lane 2–4) or presence (Lane 5–7) of 40% (w/v) PEG 200; Lane 8: DNA-C (no G-rich sequence containing) was treated with the same procedure as the one in Lane 7. (B) and (C) Structural confirmation of DNA-G and DNA-S using AFM. The DNA samples used for those AFM examinations were purified from the bands in Lane 7, scale bar 200 nm.

Fig. 3. Comparison of the length and height of DNA-S and DNA-G. (A) and (B) Frequency distributions of the lengths (nm) of DNA-S and DNA-G in their AFM images. The curves indicate the fitted Gaussian functions. (C) Section analyses of two duplex DNA strands in DNA-S. (D) Section analyses of a G-quadruplex in DNA-G. (E) Histogram of the difference between the height of G-quadruplex in DNA-G and duplex in DNA-S.

Fig. 4. Endonuclease digestion assays on DNA-G and DNA-S. (A) Lane 1: DNA-G alone; Lanes 2 to 5: T7 Endonuclease I-catalyzed reaction products obtained by incubating DNA-G with T7 Endonuclease I at 37 °C for 5 min (Lane 2), 10 min (Lane 3), 15 min (Lane 4), and 30 min (Lane 5). (B) The same procedures were performed except that DNA-G was replaced with DNA-S. (C) AFM images of linear DNA obtained from the reactions of DNA-G with T7 Endonuclease I. The DNA sample used for the AFM examination was purified from the lower band in Lane 5 in (A), scale bar 200 nm.