G-Quadruplex and i-Motif Are Mutually Exclusive in ILPR Double-Stranded DNA (original) (raw)

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 0 -(ACA GGGG TGT GGGG) 2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K þ , and an i-motif at pH 5.5 with 100 mM Li þ . Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K þ ), a unique determination of change in the free energy of unfolding (DG unfold ) 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 R 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.