Limits...
Junctions between i-motif tetramers in supramolecular structures.

Guittet E, Renciuk D, Leroy JL - Nucleic Acids Res. (2012)

Bottom Line: The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms).We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms.The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie et Biologie Structurales, Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France.

ABSTRACT
The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.

Show MeSH

Related in: MedlinePlus

C7 association into multimers and sms. Left panel: GPC-100 chromatograms of 1.5 mM C7 solutions. The peak eluted at 8.3 min is that of thymidine, a marker used for normalization. (A) The solution incubated at 20°C shows two weak peaks. One corresponds to a tetramer (T) and the other to non-resolved (dimer+monomer) species. (B) The same sample injected in the column immediately after melting at 100°C shows an intense (dimer+monomer) peak. The comparison of both chromatograms reveals that in the non-melted sample, 95% of the oligonucleotide is incorporated in large sms that are retained on the chromatography column. Right panel: sms (red), tetramer (cyan) and (monomer + dimer) (green) equilibrium fractions in C7 solutions versus the concentration of the incubated samples. The sms proportion was determined by the comparison of chromatograms recorded before and after melting as in the left panel. The exclusion and permeation times are indicated by dashed lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3367196&req=5

gks161-F1: C7 association into multimers and sms. Left panel: GPC-100 chromatograms of 1.5 mM C7 solutions. The peak eluted at 8.3 min is that of thymidine, a marker used for normalization. (A) The solution incubated at 20°C shows two weak peaks. One corresponds to a tetramer (T) and the other to non-resolved (dimer+monomer) species. (B) The same sample injected in the column immediately after melting at 100°C shows an intense (dimer+monomer) peak. The comparison of both chromatograms reveals that in the non-melted sample, 95% of the oligonucleotide is incorporated in large sms that are retained on the chromatography column. Right panel: sms (red), tetramer (cyan) and (monomer + dimer) (green) equilibrium fractions in C7 solutions versus the concentration of the incubated samples. The sms proportion was determined by the comparison of chromatograms recorded before and after melting as in the left panel. The exclusion and permeation times are indicated by dashed lines.

Mentions: These two oligonucleotides have the same properties. A few minutes after melting at 100°C and fast cooling at 20°C, we observe that 1.5 mM C7 or C12 solutions become opalescent and precipitate. The chromatogram of a C7 solution displayed in Figure 1 shows two very weak components. According to the column calibration (Supplementary Figure S1), one is eluted as a tetramer; the other may be either the unstructured monomer, or/and an i-motif dimer formed by the association of two C7 hairpins. The same solution injected immediately after melting at 100°C shows an intense (monomer + dimer) peak. The comparison of both chromatograms establishes that a fraction of ∼95% of the sample is missing on the chromatogram of the non-melted sample and therefore that C7 associates into extremely large sms that are trapped on the column. It is remarkable that the sms of C7 are also trapped in a GPC-1000 column whose exclusion size corresponds to structures including about 104 nucleotides. The behavior of C7 depends on the NaCl concentration. We do not observe precipitation in a 1.5 mM C7 solution incubated in a buffer containing only 10 mM Na–acetate and 10 mM Na–phosphate, pH 4.6. In this buffer, the sms and tetramer fractions are 10% and 90% respectively at equilibrium, the sms half formation time is ∼30 min and the elution time of sms is centered on a position corresponding to the assembly of 20 tetramers.Figure 1.


Junctions between i-motif tetramers in supramolecular structures.

Guittet E, Renciuk D, Leroy JL - Nucleic Acids Res. (2012)

C7 association into multimers and sms. Left panel: GPC-100 chromatograms of 1.5 mM C7 solutions. The peak eluted at 8.3 min is that of thymidine, a marker used for normalization. (A) The solution incubated at 20°C shows two weak peaks. One corresponds to a tetramer (T) and the other to non-resolved (dimer+monomer) species. (B) The same sample injected in the column immediately after melting at 100°C shows an intense (dimer+monomer) peak. The comparison of both chromatograms reveals that in the non-melted sample, 95% of the oligonucleotide is incorporated in large sms that are retained on the chromatography column. Right panel: sms (red), tetramer (cyan) and (monomer + dimer) (green) equilibrium fractions in C7 solutions versus the concentration of the incubated samples. The sms proportion was determined by the comparison of chromatograms recorded before and after melting as in the left panel. The exclusion and permeation times are indicated by dashed lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367196&req=5

gks161-F1: C7 association into multimers and sms. Left panel: GPC-100 chromatograms of 1.5 mM C7 solutions. The peak eluted at 8.3 min is that of thymidine, a marker used for normalization. (A) The solution incubated at 20°C shows two weak peaks. One corresponds to a tetramer (T) and the other to non-resolved (dimer+monomer) species. (B) The same sample injected in the column immediately after melting at 100°C shows an intense (dimer+monomer) peak. The comparison of both chromatograms reveals that in the non-melted sample, 95% of the oligonucleotide is incorporated in large sms that are retained on the chromatography column. Right panel: sms (red), tetramer (cyan) and (monomer + dimer) (green) equilibrium fractions in C7 solutions versus the concentration of the incubated samples. The sms proportion was determined by the comparison of chromatograms recorded before and after melting as in the left panel. The exclusion and permeation times are indicated by dashed lines.
Mentions: These two oligonucleotides have the same properties. A few minutes after melting at 100°C and fast cooling at 20°C, we observe that 1.5 mM C7 or C12 solutions become opalescent and precipitate. The chromatogram of a C7 solution displayed in Figure 1 shows two very weak components. According to the column calibration (Supplementary Figure S1), one is eluted as a tetramer; the other may be either the unstructured monomer, or/and an i-motif dimer formed by the association of two C7 hairpins. The same solution injected immediately after melting at 100°C shows an intense (monomer + dimer) peak. The comparison of both chromatograms establishes that a fraction of ∼95% of the sample is missing on the chromatogram of the non-melted sample and therefore that C7 associates into extremely large sms that are trapped on the column. It is remarkable that the sms of C7 are also trapped in a GPC-1000 column whose exclusion size corresponds to structures including about 104 nucleotides. The behavior of C7 depends on the NaCl concentration. We do not observe precipitation in a 1.5 mM C7 solution incubated in a buffer containing only 10 mM Na–acetate and 10 mM Na–phosphate, pH 4.6. In this buffer, the sms and tetramer fractions are 10% and 90% respectively at equilibrium, the sms half formation time is ∼30 min and the elution time of sms is centered on a position corresponding to the assembly of 20 tetramers.Figure 1.

Bottom Line: The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms).We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms.The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie et Biologie Structurales, Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France.

ABSTRACT
The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.

Show MeSH
Related in: MedlinePlus