Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism.
Bottom Line: The knowledge of the structure(s) formed by oligonucleotides is thus critical to correctly interpret the results, and gain insight into the biological role of these particular sequences.Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure analysis.Finally, this manuscript features a number of important observations contributing to a better understanding of nucleic acid structural polymorphism.
Affiliation: ARNA Laboratory, University of Bordeaux, Bordeaux 33000, France INSERM, U869, IECB, Pessac 33600, France.Show MeSH
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Mentions: The formation of i-motifs requires a partial protonation of the cytosines. This pH dependence can be easily monitored by SE-HPLC. At near physiological pH (7.5), a single peak is observable for both Py22 and 21CC, corresponding to the unstructured single strands (Figure 8 and Supplementary Figure S31A, respectively). However, at pH 6.0, the strands can fold into a more compact intramolecular i-motif, and the corresponding delayed peak appears. The second peaks observed at pH 6.0 do not strictly elute at the same elution volume than the unfolded controls. The typical i-motif signatures inferred from the IDS analysis of that couple of peaks, together with the elution volumes, reveal that these species are i-motif dimers (Supplementary Figure S31B) (79).
Affiliation: ARNA Laboratory, University of Bordeaux, Bordeaux 33000, France INSERM, U869, IECB, Pessac 33600, France.