Rapid and Highly Efficient Separation of i‑Motif DNA Species by CEUVand Multivariate Curve Resolution

Abstract

The i-motif is a class of nonstandard DNA structure with potential biological implications. A novel capillaryelectrophoresis with an ultraviolet absorption spectrophotometric detection (CE-UV) method has been developed for the rapidanalysis of the i-motif folding equilibrium as a function of pH and temperature. The electrophoretic analyses are performed inreverse polarity of the separation voltage with 32 cm long fused silica capillaries permanently coated with hydroxypropyl cellulose(HPC), after an appropriate conditioning procedure was used to achieve good repeatability. However, the electrophoretic separationbetween the folded and unfolded conformers of the studied cytosine-rich i-motif sequences (i.e., TT, Py39WT, and nmy01) iscompromised, especially for Py39WT and nmy01, which result in completely overlapped peaks. Therefore, deconvolution withmultivariate curve resolution-alternating least-squares (MCR-ALS) has been required for the efficient separation of the folded andunfolded species found at different concentration levels at pH 6.5 and between 12 and 40 °C, taking advantage of the smalldissimilarities in the electrophoretic mobilities and UV spectra levels. MCR-ALS has also provided quantitative information that hasbeen used to estimate melting temperatures (Tm), which are similar to those determined by UV and circular dichroism (CD)spectroscopies. The obtained results demonstrate that CE-UV assisted by MCR-ALS may become a very useful tool to get novelinsight into the folding of i-motifs and other complex DNA structures.