Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double-stranded DNA Whereas single-molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C)-rich region complementary to G quadruplex-forming sequences Here, we have used laser tweezers to investigate the structures formed in 5'-(TGTCCCCACACCCC)(2), a predominate variant in the insulin-linked polymorphic region (ILPR) We have observed two species with the change in contour length (Delta L) of 104 (+/-0.1) and 5 1 (+/-0.5) nm, respectively Since Delta L of 104 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br-2 footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23% to 1 3%) with pH 5.5 -> 7.0, which is a well-established behavior for i-motifs In contrast to that of the i-motif, the formation of the second species with Delta L of 5.1 nm remains unchanged (6.1 +/- 1 6%) in the same pH range, implying that pH-sensitive C.CH+ pairs may not contribute to the structure as significantly as those the i-motif. Compared to the Delta G(unfold) of an i-motif (16.0 +/- 0 8 kcal/mol), the decreased free energy in the partially folded structure (Delta G(unfold) 10 4 +/- 0 7 kcal/mol) may reflect a weakened structure with reduced C CH+ pairs Both Delta L and Delta G(unfold) argue for the intermediate nature of the partially folded structure in comparison to the i-motif In line with this argument, we have directly observed the unfolding of an i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.