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Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism.

Largy E, Mergny JL - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: ARNA Laboratory, University of Bordeaux, Bordeaux 33000, France INSERM, U869, IECB, Pessac 33600, France.

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(A) Normalized chromatograms of some auto-complementary oligonucleotides (plain lines), and the hairpin-forming ds15-h (dashed line). Dimeric species are indicated with a d, and hairpins with a h. Examples of data treatment are given in Supplementary Figure S2. Other chromatograms can be found in Supplementary Figures S7 and S8. (B) Predictive plot of the log10(MW) against Ve/V0 for duplex-forming oligonucleotides structured in intermolecular antiparallel duplex (red squares) and hairpin (orange circles). The two sets are linearly fitted independently (solid lines; dashed lines: 80% prediction bands). Parallel duplexes are shown as brown diamonds. The linear fit obtained for dTn oligonucleotides is depicted as a green dotted line.
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Figure 3: (A) Normalized chromatograms of some auto-complementary oligonucleotides (plain lines), and the hairpin-forming ds15-h (dashed line). Dimeric species are indicated with a d, and hairpins with a h. Examples of data treatment are given in Supplementary Figure S2. Other chromatograms can be found in Supplementary Figures S7 and S8. (B) Predictive plot of the log10(MW) against Ve/V0 for duplex-forming oligonucleotides structured in intermolecular antiparallel duplex (red squares) and hairpin (orange circles). The two sets are linearly fitted independently (solid lines; dashed lines: 80% prediction bands). Parallel duplexes are shown as brown diamonds. The linear fit obtained for dTn oligonucleotides is depicted as a green dotted line.

Mentions: Next, duplex-forming oligonucleotides, varying in length (10–26 nt) and base composition, are analyzed (Figure 3A). Auto-complementary sequences were chosen as they can be structured in the homo-duplex and/or the hairpin-duplex form, depending on the annealing conditions (nucleic acid concentration, cation concentration, cooling rate). Additionally to the widely studied ds26 and dslac (lac operon) sequences, four sequences are based on the so-called Drew-Dickerson model (ds10, ds14 and the mirror-image sequences ds12-GC and ds12-CG) (35–37). Finally, in order to obtain a reference peak for hairpin structures, the synthetic construct ds15-h, which is not fully auto-complementary, is also injected (Table 1). Indeed, the three central nucleotides forming the loop in the hairpin mismatch in the case of the homo-duplex, rendering the latter significantly less stable than the former (38).


Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism.

Largy E, Mergny JL - Nucleic Acids Res. (2014)

(A) Normalized chromatograms of some auto-complementary oligonucleotides (plain lines), and the hairpin-forming ds15-h (dashed line). Dimeric species are indicated with a d, and hairpins with a h. Examples of data treatment are given in Supplementary Figure S2. Other chromatograms can be found in Supplementary Figures S7 and S8. (B) Predictive plot of the log10(MW) against Ve/V0 for duplex-forming oligonucleotides structured in intermolecular antiparallel duplex (red squares) and hairpin (orange circles). The two sets are linearly fitted independently (solid lines; dashed lines: 80% prediction bands). Parallel duplexes are shown as brown diamonds. The linear fit obtained for dTn oligonucleotides is depicted as a green dotted line.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231728&req=5

Figure 3: (A) Normalized chromatograms of some auto-complementary oligonucleotides (plain lines), and the hairpin-forming ds15-h (dashed line). Dimeric species are indicated with a d, and hairpins with a h. Examples of data treatment are given in Supplementary Figure S2. Other chromatograms can be found in Supplementary Figures S7 and S8. (B) Predictive plot of the log10(MW) against Ve/V0 for duplex-forming oligonucleotides structured in intermolecular antiparallel duplex (red squares) and hairpin (orange circles). The two sets are linearly fitted independently (solid lines; dashed lines: 80% prediction bands). Parallel duplexes are shown as brown diamonds. The linear fit obtained for dTn oligonucleotides is depicted as a green dotted line.
Mentions: Next, duplex-forming oligonucleotides, varying in length (10–26 nt) and base composition, are analyzed (Figure 3A). Auto-complementary sequences were chosen as they can be structured in the homo-duplex and/or the hairpin-duplex form, depending on the annealing conditions (nucleic acid concentration, cation concentration, cooling rate). Additionally to the widely studied ds26 and dslac (lac operon) sequences, four sequences are based on the so-called Drew-Dickerson model (ds10, ds14 and the mirror-image sequences ds12-GC and ds12-CG) (35–37). Finally, in order to obtain a reference peak for hairpin structures, the synthetic construct ds15-h, which is not fully auto-complementary, is also injected (Table 1). Indeed, the three central nucleotides forming the loop in the hairpin mismatch in the case of the homo-duplex, rendering the latter significantly less stable than the former (38).

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.

View Article: PubMed Central - PubMed

Affiliation: ARNA Laboratory, University of Bordeaux, Bordeaux 33000, France INSERM, U869, IECB, Pessac 33600, France.

Show MeSH
Related in: MedlinePlus