1. . Structural and Thermodynamic Studies of the i-Motif of DNA: Sequence Context Effects Mikeal McKim, Alexander Buxton, Amanda Metz, Courtney Johnson, Mayra Alvarado, William Stone and Richard D. Sheardy Department of Chemistry and Biochemistry Abstract Acknowledgements This work was funded in part by the National Science Foundation, the Robert A. Welch Foundation, and the TWU Chancellor’s Research Scholars Fund. The Big Picture Conclusions 1)(CCCXXX) 4 oligomers undergo a transition from a single strand to the i-motif upon lowering the pH in K + buffer solutions. 2)The transition begins below pH 6.2 3)The presence of a sharp isoelliptic point indicates a two state transition. 4)The transition does appear to be cooperative. 5)Both the T m and pH mp are sequence dependent... The Questions 1. How will the sequence context of the DNA oligomer influence the structure and stability? 2. How does pH influence the structure and stability? The i-Motif Whereas G-rich DNA sequences can form quadruplexes in the presence of monovalent cations, C- rich DNA sequences can form the so called i-motif under acidic conditions (1). This structure is stabilized by C:C+ base pairs as shown below on the left. A schematic of the i-motif is shown below on the left and the solution structure of (CCCTAA) 3 CCCT (the complement to the human telomere G rich strand) is shown below on the right (2). We have begun an investigation of the effect of pH and temperature on the structure of related sequences, (CCCXXX) 4, where X = A and/or T. The CD spectra to the left indicates that this sequence is single stranded at pH > 7.0 but quickly undergoes the transition to the i-motif at pH < 6.20. (1) Choi, J.; Kim, S.; Tachikawa, T.; Fujitsuka, M.; Majima, T. “pH-induced intramolecular folding dynamics of i-motif DNA.” J. Am. Chem. Soc. 133, 16146-16153 (2011) and reference therein. (2) Phan, A. T.; Gueron, M; Leroy, J. L. “The Solution Structure and Internal Motions of a Fragment of the Cytidine-rich Strand of the Human Telomere.” J. Mol. Biol. 299, 123-144 (2000). The telomere, located at the end of eukaryotic chromosomes, consists of tandem repeating nucleic acid bases. The G rich strand, with a (TTAGGG) repeat, of the human telomere has a C-rich complimentary stand (a CCCTAA repeat). Former investigations performed on a DNA oligomer possessing the G-rich repeat indicated that it folds into what is known as the G-quadruplex, where the GGG segments form tetrads, and the TTA segments are loops that join the tetrads together. While under acidic conditions, the C-rich strand has also been shown to form a unique structure known as the i-motif. Investigated here is the conformations formed from synthetic DNA oligomers of general sequence (CCCXXX)4, where X= A and/or T, when in the presence of K+ buffer ranging in pH from 7.0 to 5.0. Circular dichroism (CD) spectra were determined at different solution pH and temperatures to investigate the pH and temperature dependence of the folding of these sequences into i-motifs. Typical CD Spectra: (CCCAAA) 4 A C:C + Base Pair References The DNA i-Motif SequenceT m ( o C)pH MP (CCCAAA) 4 55.85.9 (CCCAAT) 4 (CCCATA) 4 (CCCATT) 4 72.4 (CCCTAA) 4 67.35.9 (CCCTAT) 4 75.56.2 (CCCTTA) 4 (CCCTTT) 4 > 80 Each DNA sequence was prepared in standard phosphate buffer (10 mM phosphate, 115 mM K + ) with a final pH between 5.0 and 7.0 and [DNA] ranged from 1e-5 M to 4e-5 M in bases. The pH of each DNA solution was determined before each scan using a microelectrode. The panel to the right displays a typical pH titration whereby the DNA undergoes the single strand to i-motif transition at 25 o C. The panel to the left displays a typical optical melt whereby the DNA undergoes the i-motif to single strand transition at pH 5.0. Both sets of data indicate the presence of an isoelliptic point suggesting a two state transition. Using the ellipticity values at 292 nm, we constructed plots of the Normalized Spectral Response (NSR) as a function of either pH (left panel) or T (right panel). As can be seen on the right, the transition from the single strand to the i-motif appears to be cooperative and results in a midpoint at pH 5.9 for (CCCAAA) 4. From the optical melting profile seen on the left, we can determine the T m for (CCCAAA) 4 to be about 55.8 o C. For those oligomers that have been studied to date, the T m and pH mp are tabulated below.