Intramolecular Folding in Human ILPR Fragment with Three C-Rich Repeats

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br2 footprinting experiments on a mixture of the ILPR-I3 and a 5′-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures

CD experiments of ILPR-I3 at different pH and temperature in a 10 mM sodium phosphate buffer with 100 mM KCl and 5 µM DNA concentration.

<p>(A) CD spectra of the ILPR-I3 in pH 4.5–8.0. (B) Peak wavelength <i>vs</i> pH for the ILPR-I3 (obtained from (A)) and ILPR-I4 (obtained from the CD spectra of the ILPR-I4 at pH 4.5–8.0, data not shown). (C) CD spectra acquired at 23–68°C (pH 5.5). (D) Peak wavelength <i>vs</i> temperature (obtained from (C)). The transition points in B) and D) are determined by sigmoidal fitting (solid curves).</p

Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.

<p>Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.</p

Formation of an intermolecular i-motif.

<p>(A) Schematic of the formation of an intermolecular i-motif. The proposed structure in the ILPR-I3 is shown on the left. Each C:CH⁺ pair is represented by two opposite rectangles. (B) PAGE gel image of the Br₂ footprinting experiment. Lane 1, the ILPR-I3/ILPR-I1 (I₃+I₁) mixture at pH 7.0. Lane 2, the I₃+I₁ sample at pH 5.5. Lane 3, the ILPR-I3 (I₃) at pH 5.5. Lane 4, the ILPR-I4 (I₄) at pH 5.5. The band intensity for lane 2 is shown to the left of the gel. The fold protection for the I₃+I₁ sample at pH 5.5 is shown to the right. The dotted vertical lines indicate the average fold protection for each C4 tract. The blue arrows indicate the loop cytosines. Error bar represents the standard deviation of three independent experiments. The blue arrows indicate the cytosines in the ACA section of each fragment. Note that the fold protection for adenines at 3'-end (indicated by asterisk *) is not accurate since they are close to the uncut oligo. (C) Normalized rupture force histogram for the I₃+I₁ sample at pH 5.5. The solid black curve represents a two-peak Gaussian function. The dotted curve is the Gaussian fit for the rupture force histogram of the ILPR-I3 at pH 5.5.</p

Mutation analysis in a 10 mM sodium phosphate buffer (pH 5.5) with 100 mM KCl.

<p>(A) 295 nm UV melting curves of the ILPR-I3 (“Wild Type”) and the mutants at 10 µM concentration. (B) Top panel, <i>T</i>_1/2-melt of the mutants and the ILPR-I3. “W” depicts the wild type ILPR-I3. Bottom panel, CD peak shift of the mutants and the scrambled sequence (ILPR-S3) with respect to the 285 nm peak in the ILPR-I3. The horizontal dotted lines (green) represent the average value for each C4 tract. Statistical treatment is represented by the <i>P</i> values in the bottom panel. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039271#pone-0039271-t001" target="_blank">Table 1</a> for DNA sequences.</p