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Intramolecular DNA quadruplexes with different arrangements of short and long loops.

Rachwal PA, Findlow IS, Werner JM, Brown T, Fox KR - Nucleic Acids Res. (2007)

Bottom Line: The stability increases with the number of single T loops, though the arrangement of different length loops has little effect.In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure.Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

ABSTRACT
We have examined the folding, stability and kinetics of intramolecular quadruplexes formed by DNA sequences containing four G3 tracts separated by either single T or T4 loops. All these sequences fold to form intramolecular quadruplexes and 1D-NMR spectra suggest that they each adopt unique structures (with the exception of the sequence with all three loops containing T4, which is polymorphic). The stability increases with the number of single T loops, though the arrangement of different length loops has little effect. In the presence of potassium ions, the oligonucleotides that contain at least one single T loop exhibit similar CD spectra, which are indicative of a parallel topology. In contrast, when all three loops are substituted with T4 the CD spectrum is typical of an antiparallel arrangement. In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure. Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

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1D imino proton NMR spectra of the quadruplex-forming oligonucleotides. The samples (100 µM) were prepared in 200 mM potassium phosphate pH 7.4. The top panel shows the 1D-NMR spectrum for G3T between 5 and 15 p.p.m., while the other panels show the imino proton region for each oliogonucleotide. The individual peaks are indicated.
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Figure 3: 1D imino proton NMR spectra of the quadruplex-forming oligonucleotides. The samples (100 µM) were prepared in 200 mM potassium phosphate pH 7.4. The top panel shows the 1D-NMR spectrum for G3T between 5 and 15 p.p.m., while the other panels show the imino proton region for each oliogonucleotide. The individual peaks are indicated.

Mentions: One of the defining features of structures that contain G-quartets is the appearance of imino proton resonances between 10.5 and 12.0 p.p.m. in NMR spectra (56). Examination of this region of NMR spectra has often been used to assess whether the sequence adopts a unique structure (20,34,36,56) and the presence of multiple or ill-defined peaks is evidence for the existence of multiple structures. The imino proton spectra for each of these sequences are shown in Figure 3. It can be seen that the spectra of G3T, G3T-T4-T, G3T4-T-T and G3T4-T-T4 display between 10 and 12 well-resolved and sharp peaks, indicative of well-defined structures. In the cases where 10 or 11 peaks are resolved, the intensities indicate that one or two imino protons have degenerate chemical shifts. Hence, the number of hydrogen-bonded imino protons is 12, as expected for three stacked G-quartets. G3T4-T4-T also shows 12 major peaks, though the spectrum contains some minor peaks, which might indicate the presence of a small amount of a second structure. In contrast, the imino proton spectrum of G3T4 shows multiple peaks confirming that it adopts more than one stable conformation.Figure 3.


Intramolecular DNA quadruplexes with different arrangements of short and long loops.

Rachwal PA, Findlow IS, Werner JM, Brown T, Fox KR - Nucleic Acids Res. (2007)

1D imino proton NMR spectra of the quadruplex-forming oligonucleotides. The samples (100 µM) were prepared in 200 mM potassium phosphate pH 7.4. The top panel shows the 1D-NMR spectrum for G3T between 5 and 15 p.p.m., while the other panels show the imino proton region for each oliogonucleotide. The individual peaks are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919480&req=5

Figure 3: 1D imino proton NMR spectra of the quadruplex-forming oligonucleotides. The samples (100 µM) were prepared in 200 mM potassium phosphate pH 7.4. The top panel shows the 1D-NMR spectrum for G3T between 5 and 15 p.p.m., while the other panels show the imino proton region for each oliogonucleotide. The individual peaks are indicated.
Mentions: One of the defining features of structures that contain G-quartets is the appearance of imino proton resonances between 10.5 and 12.0 p.p.m. in NMR spectra (56). Examination of this region of NMR spectra has often been used to assess whether the sequence adopts a unique structure (20,34,36,56) and the presence of multiple or ill-defined peaks is evidence for the existence of multiple structures. The imino proton spectra for each of these sequences are shown in Figure 3. It can be seen that the spectra of G3T, G3T-T4-T, G3T4-T-T and G3T4-T-T4 display between 10 and 12 well-resolved and sharp peaks, indicative of well-defined structures. In the cases where 10 or 11 peaks are resolved, the intensities indicate that one or two imino protons have degenerate chemical shifts. Hence, the number of hydrogen-bonded imino protons is 12, as expected for three stacked G-quartets. G3T4-T4-T also shows 12 major peaks, though the spectrum contains some minor peaks, which might indicate the presence of a small amount of a second structure. In contrast, the imino proton spectrum of G3T4 shows multiple peaks confirming that it adopts more than one stable conformation.Figure 3.

Bottom Line: The stability increases with the number of single T loops, though the arrangement of different length loops has little effect.In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure.Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

ABSTRACT
We have examined the folding, stability and kinetics of intramolecular quadruplexes formed by DNA sequences containing four G3 tracts separated by either single T or T4 loops. All these sequences fold to form intramolecular quadruplexes and 1D-NMR spectra suggest that they each adopt unique structures (with the exception of the sequence with all three loops containing T4, which is polymorphic). The stability increases with the number of single T loops, though the arrangement of different length loops has little effect. In the presence of potassium ions, the oligonucleotides that contain at least one single T loop exhibit similar CD spectra, which are indicative of a parallel topology. In contrast, when all three loops are substituted with T4 the CD spectrum is typical of an antiparallel arrangement. In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure. Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

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