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 experimental setup is first calibrated with 10 d(Tn) sequences of various lengths (n = 5–70), which do not form any defined secondary structure. As expected, the relative elution volume Ve/V0 decreased with increasing oligonucleotide length, ranging from 1.79 to 1.41 (Figure 2A). A near-perfect linear relationship between the decimal logarithm of the molecular weight and Ve/V0 was observed (R2 = 0.9996), as commonly found for other type of linear polymers (Figure 2B). This plot could be considered as a compactness baseline since the d(Tn) oligonucleotides are unstructured. To ensure that there is no bias caused by the use of polypyrimidine sequences, it is verified that polypurine sequences give similar results. Five dAn sequences are injected (n = 10–40), and the plot of log10(MW) against Ve/V0 of the main peak is linearly fitted (R2 = 0.9994) with a similar slope (−2.98 versus −3.08). However, a small portion of the strands fold into non-canonical secondary structures via A•A base-pairing, resulting in secondary peaks (Supplementary Figure S6).
Affiliation: ARNA Laboratory, University of Bordeaux, Bordeaux 33000, France INSERM, U869, IECB, Pessac 33600, France.