<p> G-quadruplex is the most widely known four-stranded nucleic acid structure which has shown to alter gene regulation both in vitro and in vivo. Under certain conditions, another four-stranded structure, i-motif, is also formed in the strand complementary to the G-quadruplex forming sequence. Recent studies suggest gene regulatory roles for the i-motif structure as well. Although there is substantial understanding on the folding topology of G-quadruplex and i-motif structures, their mechanical stability which determines the interaction with motor proteins, such as DNA/RNA polymerases, are poorly studied. Since DNA exists as a double stranded form in vivo, the investigation of i-motif becomes highly important to fully understand the biological functions of G-quadruplexes. Using laser tweezers based single-molecule study, we investigated the mechanical stability of an i-motif structure in the predominant variant of human ILPR fragment (5'-TGTC4ACAC4TGTC4ACAC4TGT). In addition, we have shown that a partially folded structure composed of only three tandem C-rich repeats coexists with the i-motif. Both structures share similar unfolding forces of 22-26 pN. Discovery of stable structures in less than four C-rich repeats suggested that the structure can serve as an intermediate during the i-motif folding/unfolding pathway. Using chemical footprinting and single-molecule approaches, we show that a dsDNA fragment in ILPR, 5'-(ACAG4TGTG4ACAG4TGTG4ACA), can fold into G-quadruplex or i-motif structure under specific conditions. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif, changes in free energy of unfolding provided compelling evidence that only one species is present in each dsDNA. Based on this observation, we propose that G-quadruplex and i-motif are mutually exclusive in human ILPR. Furthermore, we show that these two species have an unfolding force >17 pN. From mechanical perspective, this could justify the regulatory role a DNA tetraplex may play in the expression of human insulin inside cells in which dsDNA is the predominate form.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:3618902 |
| Date | 13 June 2014 |
| Creators | Dhakal, Soma |
| Publisher | Kent State University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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