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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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Fluorescence melting profiles for the quadruplex-forming oligonucleotides. The reactions were performed in 10 mM lithium phosphate pH 7.4 containing either 20 mM KCl (left hand panel) or 200 mM NaCl (right hand panel). The temperature was changed at 0.2°C.min−1. The curves show the fraction folded (α) as a function of temperature, calculated as described in the Methods section. G3T, black; G3T-T4-T, red; G3T4-T-T, blue; G3T4-T-T4, pink; G3T4-T4-T, green; G3T4, cyan.
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Figure 4: Fluorescence melting profiles for the quadruplex-forming oligonucleotides. The reactions were performed in 10 mM lithium phosphate pH 7.4 containing either 20 mM KCl (left hand panel) or 200 mM NaCl (right hand panel). The temperature was changed at 0.2°C.min−1. The curves show the fraction folded (α) as a function of temperature, calculated as described in the Methods section. G3T, black; G3T-T4-T, red; G3T4-T-T, blue; G3T4-T-T4, pink; G3T4-T4-T, green; G3T4, cyan.

Mentions: Representative fluorescent melting curves for these sequences are shown in Figure 4 in the presence of potassium and sodium ions. The Tm values at different ionic strengths, along with the calculated values for ΔH, are shown in Table 2. The samples were melted and annealed at 0.2°C.min−1; no hysteresis was observed at this rate of temperature change (except for G3T4 at low ionic strengths). The melting temperatures were all independent of concentration (between 0.1 and 10 µM; Supplementary material Figure 1) confirming that these oligonucleotide sequences form intramolecular (not intermolecular) complexes. As expected, all the complexes are more stable in potassium than sodium ions. G3T is the most stable and, in the presence of potassium, substituting a T4 loop instead of a loop with a single T decreases the Tm by about 20°C, irrespective of whether the replacement is in a central (G3T-T4-T) or peripheral loop (G3T4-T-T), though G3T-T4-T is about 2–3°C more stable than G3T4-T-T. Replacing a second T loop with T4 causes a further 20°C decrease in Tm and G3T4-T4-T is about 2–4°C more stable than G3T4-T-T4. In each case, the sequence with a single T in the central loop is slightly less stable than the equivalent sequence with T4 in the same position. Replacing all three single T loops with T4 decreases the Tm by a further 10°C, though the melting and annealing curves with this sequence show hysteresis at low ionic strengths and the melting (but not the annealing) profiles are biphasic.Figure 4.


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)

Fluorescence melting profiles for the quadruplex-forming oligonucleotides. The reactions were performed in 10 mM lithium phosphate pH 7.4 containing either 20 mM KCl (left hand panel) or 200 mM NaCl (right hand panel). The temperature was changed at 0.2°C.min−1. The curves show the fraction folded (α) as a function of temperature, calculated as described in the Methods section. G3T, black; G3T-T4-T, red; G3T4-T-T, blue; G3T4-T-T4, pink; G3T4-T4-T, green; G3T4, cyan.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919480&req=5

Figure 4: Fluorescence melting profiles for the quadruplex-forming oligonucleotides. The reactions were performed in 10 mM lithium phosphate pH 7.4 containing either 20 mM KCl (left hand panel) or 200 mM NaCl (right hand panel). The temperature was changed at 0.2°C.min−1. The curves show the fraction folded (α) as a function of temperature, calculated as described in the Methods section. G3T, black; G3T-T4-T, red; G3T4-T-T, blue; G3T4-T-T4, pink; G3T4-T4-T, green; G3T4, cyan.
Mentions: Representative fluorescent melting curves for these sequences are shown in Figure 4 in the presence of potassium and sodium ions. The Tm values at different ionic strengths, along with the calculated values for ΔH, are shown in Table 2. The samples were melted and annealed at 0.2°C.min−1; no hysteresis was observed at this rate of temperature change (except for G3T4 at low ionic strengths). The melting temperatures were all independent of concentration (between 0.1 and 10 µM; Supplementary material Figure 1) confirming that these oligonucleotide sequences form intramolecular (not intermolecular) complexes. As expected, all the complexes are more stable in potassium than sodium ions. G3T is the most stable and, in the presence of potassium, substituting a T4 loop instead of a loop with a single T decreases the Tm by about 20°C, irrespective of whether the replacement is in a central (G3T-T4-T) or peripheral loop (G3T4-T-T), though G3T-T4-T is about 2–3°C more stable than G3T4-T-T. Replacing a second T loop with T4 causes a further 20°C decrease in Tm and G3T4-T4-T is about 2–4°C more stable than G3T4-T-T4. In each case, the sequence with a single T in the central loop is slightly less stable than the equivalent sequence with T4 in the same position. Replacing all three single T loops with T4 decreases the Tm by a further 10°C, though the melting and annealing curves with this sequence show hysteresis at low ionic strengths and the melting (but not the annealing) profiles are biphasic.Figure 4.

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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