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Tetramolecular G-quadruplex formation pathways studied by electrospray mass spectrometry.

Rosu F, Gabelica V, Poncelet H, De Pauw E - Nucleic Acids Res. (2010)

Bottom Line: The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified.The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant.We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry B6c, University of Liège, Liège, Belgium. f.rosu@ulg.ac.be

ABSTRACT
Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG(5)T, in ammonium acetate. The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified. The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant. However, these tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few, and ion mobility spectrometry shows that their conformation is more extended. (iv) At 4 degrees C, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slow (not complete after 4 months). We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly.

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Mechanism of tetramolecular G-quadruplex formation by dTGnT. The strands are represented by black lines and the cations are represented by grey circles. M stands for monomer, D for dimer, T for trimer, Q for tetramer and P for pentamer.
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Figure 6: Mechanism of tetramolecular G-quadruplex formation by dTGnT. The strands are represented by black lines and the cations are represented by grey circles. M stands for monomer, D for dimer, T for trimer, Q for tetramer and P for pentamer.

Mentions: ESI-MS experiments have therefore unambiguously confirmed the presence of several stoichiometries (dimers, trimers, tetramers with incomplete ammonium ion incorporation), and revealed others (pentamers). The presence of ammonium ions is related to the number of stacked G-quartets in the structures. Ion mobility spectrometry experiments have shown that incomplete ammonium incorporation in the tetramers was associated with more extended conformations. Moreover, the temporal evolution of the composition of the solution can be characterized. These results help to refine the mechanism of tetramolecular G-quadruplex formation (Figure 6).Figure 6.


Tetramolecular G-quadruplex formation pathways studied by electrospray mass spectrometry.

Rosu F, Gabelica V, Poncelet H, De Pauw E - Nucleic Acids Res. (2010)

Mechanism of tetramolecular G-quadruplex formation by dTGnT. The strands are represented by black lines and the cations are represented by grey circles. M stands for monomer, D for dimer, T for trimer, Q for tetramer and P for pentamer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926595&req=5

Figure 6: Mechanism of tetramolecular G-quadruplex formation by dTGnT. The strands are represented by black lines and the cations are represented by grey circles. M stands for monomer, D for dimer, T for trimer, Q for tetramer and P for pentamer.
Mentions: ESI-MS experiments have therefore unambiguously confirmed the presence of several stoichiometries (dimers, trimers, tetramers with incomplete ammonium ion incorporation), and revealed others (pentamers). The presence of ammonium ions is related to the number of stacked G-quartets in the structures. Ion mobility spectrometry experiments have shown that incomplete ammonium incorporation in the tetramers was associated with more extended conformations. Moreover, the temporal evolution of the composition of the solution can be characterized. These results help to refine the mechanism of tetramolecular G-quadruplex formation (Figure 6).Figure 6.

Bottom Line: The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified.The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant.We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry B6c, University of Liège, Liège, Belgium. f.rosu@ulg.ac.be

ABSTRACT
Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG(5)T, in ammonium acetate. The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified. The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant. However, these tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few, and ion mobility spectrometry shows that their conformation is more extended. (iv) At 4 degrees C, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slow (not complete after 4 months). We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly.

Show MeSH
Related in: MedlinePlus