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: Similarly, H-Bi-G4 forms a bimolecular quadruplex in equilibrium with a monomer theoretically unfolded since it only contains two runs of guanines (Figure 7A). This is further verified by plotting the IDS of the monomer/dimer couple, calculated from the absorbance spectra under the pure areas of the peaks (see experimental section and Supplementary Figure S17C). Using ESI-MS at lower strand concentration (10 μM), the bimolecular quadruplex cannot be detected using 100–200 V capillary tensions, which suggests that this structure is relatively unstable, at least in ammonium conditions (Supplementary Figures S19 and S20). This is subsequently confirmed in the presence of potassium by UV-melting experiments (Tm10 μM = 34.5°C; Supplementary Figure S21A), which explains the large percentage of monomer detected by SE-HPLC. This is an interesting result as it shows that SE-HPLC can be a tool to measure relative stabilities. Additionally, UV-melting experiments further confirmed the intermolecular nature of H-Bi-G4 quadruplex because the melting temperature is concentration dependent (Tm5 μM = 30.5°C). Finally, since H-Bi-G4 forms a bimolecular structure, a decrease in strand concentration (from 250 to 100 μM) leads to the formation of a lower proportion of quadruplex, as expected from the law of mass action (Supplementary Figure S22). The kinetics of formation is also fairly slow, at room temperature, compared to a typical intramolecular folding.
Affiliation: ARNA Laboratory, University of Bordeaux, Bordeaux 33000, France INSERM, U869, IECB, Pessac 33600, France.