Coexistence of an ILPR i-Motif and a Partially Folded Structure with Comparable Mechanical Stability Revealed at the Single-Molecule Level

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• 作者：Soma Dhakal ; Joseph D. Schonhoft ; Deepak Koirala ; Zhongbo Yu ; Soumitra Basu ; Hanbin Mao
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：July 7, 2010
• 年：2010
• 卷：132
• 期：26
• 页码：8991-8997
• 全文大小：306K
• 年卷期：v.132,no.26(July 7, 2010)
• ISSN：1520-5126

文摘

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 (ΔL) of 10.4 (±0.1) and 5.1 (±0.5) nm, respectively. Since ΔL of 10.4 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 Br₂ 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 ΔL 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 to the i-motif. Compared to the ΔG_unfold of an i-motif (16.0 ± 0.8 kcal/mol), the decreased free energy in the partially folded structure (ΔG_unfold 10.4 ± 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH⁺ pairs. Both ΔL and ΔG_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.