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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.

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Evolution during incubation at 20°C of the composition of a 0.3 mM C5TC2 solution initially melted. Left panel: The GPC-100 chromatograms are normalized to the same integrated area. The vertical scale of the chromatograms drawn in heavy lines are multiplied by a factor of five. On the chromatogram recorded at t = 0, the oligonucleotide is eluted as a monomer (M) and as a dimer (D). The anomalous elution order of D and M reflects probably the hydrodynamic radius difference between the unstructured monomer and the compact i-motif (Supplementary Figure S1). The chromatograms recorded versus time show the early formation of a tetramer (Te), the transient apparition of species including 2, 3 and 4 tetramers and the accumulation of non-resolved sms. Right panel: Evolution of the sms (red), tetramer (blue) and (monomer + dimer) (green) fractions as a function of the incubation time. The dotted line shows the exclusion time.
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gks161-F3: Evolution during incubation at 20°C of the composition of a 0.3 mM C5TC2 solution initially melted. Left panel: The GPC-100 chromatograms are normalized to the same integrated area. The vertical scale of the chromatograms drawn in heavy lines are multiplied by a factor of five. On the chromatogram recorded at t = 0, the oligonucleotide is eluted as a monomer (M) and as a dimer (D). The anomalous elution order of D and M reflects probably the hydrodynamic radius difference between the unstructured monomer and the compact i-motif (Supplementary Figure S1). The chromatograms recorded versus time show the early formation of a tetramer (Te), the transient apparition of species including 2, 3 and 4 tetramers and the accumulation of non-resolved sms. Right panel: Evolution of the sms (red), tetramer (blue) and (monomer + dimer) (green) fractions as a function of the incubation time. The dotted line shows the exclusion time.

Mentions: The evolution during incubation at 20°C of the composition of a C5TC2 solution is displayed in Figure 3. The chromatogram recorded immediately after melting shows two poorly resolved components labeled M and D. The measure of the equilibrium concentration of M and D as a function of the oligonucleotide concentration establishes that peak D increases as the square of peak M and thus identifies M to a monomer and D to a dimer (Supplementary Figure S4). The calibration curves of Supplementary Figure S1 show that the anomalously long elution time of the dimer, by comparison to that of the unstructured monomer, seems to be a common feature in relation with the compact character of i-motif. The dimer reduced dissociation constant estimated from Supplementary Figure S4 is 8 × 10−8 M at 20°C and 9 × 10−6 M at 42°C. The chromatograms recorded after several hours show the slow apparition of sms peaks whose elution times correspond to the assemblies of 2, 3 and 4 tetramers. Lastly the chromatograms show larger sms and the decrease of the species including a small number of tetramers. C5TC2 association into sms was also followed by NMR (Supplementary Figure S5). It must be reminded that the imino proton peak of non-paired cytidines is broadened out by exchange with water at pH 4.6 (16). Hence, the narrow imino proton peaks (∼15.3 ppm) of the first spectrum recorded after melting demonstrate the early formation of a structured hemiprotonated species corresponding to that detected as a dimer by chromatography. The spectrum recorded at equilibrium shows extremely broad NMR lines indicative of large sms. The proportion of short sms is always too small to be detected by NMR. Supplementary Figure S6 show that the sms half formation time decreases approximately as the power of −1 of the oligonucleotide concentration in contrast to the tetramer half formation time that decreases as the power of −2. The half formation times of C5TC2 and C3TC3 sms decrease when the temperature is raised with comparable activation energies of −190 kJ/M (Supplementary Figure S7).Figure 3.


Junctions between i-motif tetramers in supramolecular structures.

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

Evolution during incubation at 20°C of the composition of a 0.3 mM C5TC2 solution initially melted. Left panel: The GPC-100 chromatograms are normalized to the same integrated area. The vertical scale of the chromatograms drawn in heavy lines are multiplied by a factor of five. On the chromatogram recorded at t = 0, the oligonucleotide is eluted as a monomer (M) and as a dimer (D). The anomalous elution order of D and M reflects probably the hydrodynamic radius difference between the unstructured monomer and the compact i-motif (Supplementary Figure S1). The chromatograms recorded versus time show the early formation of a tetramer (Te), the transient apparition of species including 2, 3 and 4 tetramers and the accumulation of non-resolved sms. Right panel: Evolution of the sms (red), tetramer (blue) and (monomer + dimer) (green) fractions as a function of the incubation time. The dotted line shows the exclusion time.
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Related In: Results  -  Collection

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gks161-F3: Evolution during incubation at 20°C of the composition of a 0.3 mM C5TC2 solution initially melted. Left panel: The GPC-100 chromatograms are normalized to the same integrated area. The vertical scale of the chromatograms drawn in heavy lines are multiplied by a factor of five. On the chromatogram recorded at t = 0, the oligonucleotide is eluted as a monomer (M) and as a dimer (D). The anomalous elution order of D and M reflects probably the hydrodynamic radius difference between the unstructured monomer and the compact i-motif (Supplementary Figure S1). The chromatograms recorded versus time show the early formation of a tetramer (Te), the transient apparition of species including 2, 3 and 4 tetramers and the accumulation of non-resolved sms. Right panel: Evolution of the sms (red), tetramer (blue) and (monomer + dimer) (green) fractions as a function of the incubation time. The dotted line shows the exclusion time.
Mentions: The evolution during incubation at 20°C of the composition of a C5TC2 solution is displayed in Figure 3. The chromatogram recorded immediately after melting shows two poorly resolved components labeled M and D. The measure of the equilibrium concentration of M and D as a function of the oligonucleotide concentration establishes that peak D increases as the square of peak M and thus identifies M to a monomer and D to a dimer (Supplementary Figure S4). The calibration curves of Supplementary Figure S1 show that the anomalously long elution time of the dimer, by comparison to that of the unstructured monomer, seems to be a common feature in relation with the compact character of i-motif. The dimer reduced dissociation constant estimated from Supplementary Figure S4 is 8 × 10−8 M at 20°C and 9 × 10−6 M at 42°C. The chromatograms recorded after several hours show the slow apparition of sms peaks whose elution times correspond to the assemblies of 2, 3 and 4 tetramers. Lastly the chromatograms show larger sms and the decrease of the species including a small number of tetramers. C5TC2 association into sms was also followed by NMR (Supplementary Figure S5). It must be reminded that the imino proton peak of non-paired cytidines is broadened out by exchange with water at pH 4.6 (16). Hence, the narrow imino proton peaks (∼15.3 ppm) of the first spectrum recorded after melting demonstrate the early formation of a structured hemiprotonated species corresponding to that detected as a dimer by chromatography. The spectrum recorded at equilibrium shows extremely broad NMR lines indicative of large sms. The proportion of short sms is always too small to be detected by NMR. Supplementary Figure S6 show that the sms half formation time decreases approximately as the power of −1 of the oligonucleotide concentration in contrast to the tetramer half formation time that decreases as the power of −2. The half formation times of C5TC2 and C3TC3 sms decrease when the temperature is raised with comparable activation energies of −190 kJ/M (Supplementary Figure S7).Figure 3.

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