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A minimal i-motif stabilized by minor groove G:T:G:T tetrads.

Escaja N, Viladoms J, Garavís M, Villasante A, Pedroso E, González C - Nucleic Acids Res. (2012)

Bottom Line: The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes.This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines.Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

View Article: PubMed Central - PubMed

Affiliation: Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.

ABSTRACT
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

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Top: series of CD spectra of d<pTCGTTTCGTT> at different pH (left) and pH titration (right), [oligonucleotide] = 20 µM, T = 5°C. Middle: CD melting curves of d<pTCGTTTCGTT> at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Bottom: CD melting curves of d(TCGTTTCGT) at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Buffer conditions: 20 mM AcONa, pH 4.5, 200 mM NaCl.
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gks911-F2: Top: series of CD spectra of d<pTCGTTTCGTT> at different pH (left) and pH titration (right), [oligonucleotide] = 20 µM, T = 5°C. Middle: CD melting curves of d<pTCGTTTCGTT> at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Bottom: CD melting curves of d(TCGTTTCGT) at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Buffer conditions: 20 mM AcONa, pH 4.5, 200 mM NaCl.

Mentions: CD spectra of d(TCGTTTCGT) and d<pTCGTTTCGTT> exhibit strong dependence on temperature and oligonucleotide concentration. For d<pTCGTTTCGTT> (Figure 2, middle) increasing oligonucleotide concentration from 20 µM to 50 µM raises the Tm value from 38°C to 42°C, indicating a concentration dependent melting behavior, characteristic of multimeric structures. In addition, the CD spectra also show a strong dependence on pH (Figure 2, top). The CD spectrum at pH 4.5 and low temperature exhibits a small negative band at 240 nm, a large positive band at 262 nm and a small positive band at 302 nm. The thermal unfolding transition followed by CD shows a bathochromic shift of the band at 262 nm, accompanied by a progressive disappearance of all the three bands. Similar changes in the CD spectra are observed upon pH titration, indicating a pH dependent denaturation process. The midpoint pH of this transition provides an apparent pKa value for the overall structure of 5.9. Different counter-ions (Na+, Mg2+) do not affect significantly the global shape of the CD spectra at 5°C, indicating a similar degree of structuration (data not shown), although increasing ionic strenght (i.e. by addition of Mg2+) enhances the thermal stability of the dimeric species. An analog behavior is observed in the CD spectra of d(TCGTTTCGT) (Figure 2, bottom). As expected, the CD spectra indicate a low degree of structuration under very low salt concentration (data not shown).Figure 2.


A minimal i-motif stabilized by minor groove G:T:G:T tetrads.

Escaja N, Viladoms J, Garavís M, Villasante A, Pedroso E, González C - Nucleic Acids Res. (2012)

Top: series of CD spectra of d<pTCGTTTCGTT> at different pH (left) and pH titration (right), [oligonucleotide] = 20 µM, T = 5°C. Middle: CD melting curves of d<pTCGTTTCGTT> at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Bottom: CD melting curves of d(TCGTTTCGT) at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Buffer conditions: 20 mM AcONa, pH 4.5, 200 mM NaCl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526289&req=5

gks911-F2: Top: series of CD spectra of d<pTCGTTTCGTT> at different pH (left) and pH titration (right), [oligonucleotide] = 20 µM, T = 5°C. Middle: CD melting curves of d<pTCGTTTCGTT> at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Bottom: CD melting curves of d(TCGTTTCGT) at different oligonucleotide concentration (left) and series of CD spectra at different temperature, [oligonucleotide] = 50 µM (right). Buffer conditions: 20 mM AcONa, pH 4.5, 200 mM NaCl.
Mentions: CD spectra of d(TCGTTTCGT) and d<pTCGTTTCGTT> exhibit strong dependence on temperature and oligonucleotide concentration. For d<pTCGTTTCGTT> (Figure 2, middle) increasing oligonucleotide concentration from 20 µM to 50 µM raises the Tm value from 38°C to 42°C, indicating a concentration dependent melting behavior, characteristic of multimeric structures. In addition, the CD spectra also show a strong dependence on pH (Figure 2, top). The CD spectrum at pH 4.5 and low temperature exhibits a small negative band at 240 nm, a large positive band at 262 nm and a small positive band at 302 nm. The thermal unfolding transition followed by CD shows a bathochromic shift of the band at 262 nm, accompanied by a progressive disappearance of all the three bands. Similar changes in the CD spectra are observed upon pH titration, indicating a pH dependent denaturation process. The midpoint pH of this transition provides an apparent pKa value for the overall structure of 5.9. Different counter-ions (Na+, Mg2+) do not affect significantly the global shape of the CD spectra at 5°C, indicating a similar degree of structuration (data not shown), although increasing ionic strenght (i.e. by addition of Mg2+) enhances the thermal stability of the dimeric species. An analog behavior is observed in the CD spectra of d(TCGTTTCGT) (Figure 2, bottom). As expected, the CD spectra indicate a low degree of structuration under very low salt concentration (data not shown).Figure 2.

Bottom Line: The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes.This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines.Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

View Article: PubMed Central - PubMed

Affiliation: Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.

ABSTRACT
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

Show MeSH
Related in: MedlinePlus