G-quadruplex and i-motif are mutually exclusive in ILPR double-stranded DNA.
G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin.
Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio, USA.
Soma
S
Dhakal
Zhongbo
Z
Yu
Ryan
R
Konik
Yunxi
Y
Cui
Deepak
D
Koirala
Hanbin
H
Mao
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D018983:DNA Footprinting; D054856:G-Quadruplexes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D059372:Nucleotide Motifs / Q000235:genetics*; D052898:Optical Tweezers; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D013816:Thermodynamics
D007328:Insulin; D004247:DNA