Table 1.
Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.
Figure 1.
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.
(A) CD spectra of the ILPR-I3 in pH 4.5–8.0. (B) Peak wavelength vs 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 vs temperature (obtained from (C)). The transition points in B) and D) are determined by sigmoidal fitting (solid curves).
Figure 2.
Electrophoretic mobility shift assays (EMSA) and thermal melting/reannealing of the ILPR-I3.
(A) EMSA of the ILPR-S3 (scrambled DNA, lane 1) and the ILPR-I3 (lane 2) at 1 µM strand concentration. Lane 3 shows the DNA marker (M). Left panel, a native gel at pH 5.5. Right panel, a denatured gel (10% PAGE, 7×M urea). (B) Melting (T1/2-melting), reannealing (T1/2-reannealing), and equilibrium (Tm) temperatures of the structure in the ILPR-I3 with 5 to 100 µM concentration. The melting and reannealing temperatures were determined by 295 nm UV-melting and UV-reannealing, respectively, in a 10 mM sodium phosphate buffer (pH 5.5) with 100 mM KCl. The equilibrium melting temperature (Tm) of the ILPR-I3 was determined based on the non-equilibrated melting and reannealing curves (see Materials and Methods).
Figure 3.
Single-molecule study of the ILPR-I3 at different pH using laser tweezers.
(A) Change in contour length (ΔL) (left panel) and rupture force histograms (right panel) of the ILPR-I3 and the Mut-C19T at different pH (23°C). The histograms are fitted with Gaussians (solid and dotted curves). N depicts the number of experiments. (B) Percentage formation of the ILPR-I3 at pH 5.5 and 7.0 (“pH 5.5″ and “pH 7.0″), the Mut-C19T at pH 5.5 (“Mut-pH 5.5″), and a DNA construct that does not contain the C-rich sequence at 23°C (“Control”). (C) Comparison of ΔL (triangles linked by the red dotted line at the bottom) and rupture force (filled circles linked by the black dotted line at the top) between the ILPR-I3 and the Mut-C19T fragments. Notations are the same as described in (B).
Figure 4.
Mutation analysis in a 10 mM sodium phosphate buffer (pH 5.5) with 100 mM KCl.
(A) 295 nm UV melting curves of the ILPR-I3 (“Wild Type”) and the mutants at 10 µM concentration. (B) Top panel, T1/2-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 P values in the bottom panel. Please refer to Table 1 for DNA sequences.
Figure 5.
Formation of an intermolecular i-motif.
(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.