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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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Ion mobility spectrometry of the tetramer formed from 80 µM TG5T in 20% MeOH and 150 mM NH4OAc after (a) 10 min, (b) 2 h and (c) 4 months. Red triangles, arrival time distribution of − (m/z = 1762–1765). Green squares, arrival time distribution of − (m/z = 1765–1768). Black circles, arrival time distribution of the sum of all G-quadruplex adduct forms (m/z = 1762–1775). Shorter arrival times indicate more compact conformation.
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Figure 5: Ion mobility spectrometry of the tetramer formed from 80 µM TG5T in 20% MeOH and 150 mM NH4OAc after (a) 10 min, (b) 2 h and (c) 4 months. Red triangles, arrival time distribution of − (m/z = 1762–1765). Green squares, arrival time distribution of − (m/z = 1765–1768). Black circles, arrival time distribution of the sum of all G-quadruplex adduct forms (m/z = 1762–1775). Shorter arrival times indicate more compact conformation.

Mentions: Based on the number of ammonium ions incorporated, we can conclude that the tetramers are present in several forms. The presence of non-canonical, slipped G-quadruplexes has been suggested by the modeling studies by Stefl et al. (26), and by the NMR studies of Bardin and Leroy (25). The − tetramers observed by ESI-MS could correspond to these slipped G-quadruplexes, because less than five G-quartets are formed (Figure 1c). To verify this interpretation, we performed ion mobility spectrometry (IMS) experiments (Figure 5). Ion mobility allows separating ions of a given mass-to-charge ratio based on their ion mobility, which for a given charge is directly related to the ion’s collision cross section. The regular G-quadruplexes being more compact, they will travel faster in the ion mobility cell than the slipped G-quadruplex structures. The experiments not only confirm that the − tetramers (red triangles) have indeed a more extended conformation than the − tetramers (green squares), but also reveal that the degree of compactness of the − tetramers increases with formation time. Each stoichiometry therefore corresponds to an ensemble of conformations. The arrival time distribution of the whole-tetramer adduct distribution (black circles) is shifting to shorter times (more compact conformations) as the formation time increases from 2 min (Figure 5a) to 120 min (Figure 5b) and 165 days (Figure 5c).Figure 5.


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

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

Ion mobility spectrometry of the tetramer formed from 80 µM TG5T in 20% MeOH and 150 mM NH4OAc after (a) 10 min, (b) 2 h and (c) 4 months. Red triangles, arrival time distribution of − (m/z = 1762–1765). Green squares, arrival time distribution of − (m/z = 1765–1768). Black circles, arrival time distribution of the sum of all G-quadruplex adduct forms (m/z = 1762–1775). Shorter arrival times indicate more compact conformation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Ion mobility spectrometry of the tetramer formed from 80 µM TG5T in 20% MeOH and 150 mM NH4OAc after (a) 10 min, (b) 2 h and (c) 4 months. Red triangles, arrival time distribution of − (m/z = 1762–1765). Green squares, arrival time distribution of − (m/z = 1765–1768). Black circles, arrival time distribution of the sum of all G-quadruplex adduct forms (m/z = 1762–1775). Shorter arrival times indicate more compact conformation.
Mentions: Based on the number of ammonium ions incorporated, we can conclude that the tetramers are present in several forms. The presence of non-canonical, slipped G-quadruplexes has been suggested by the modeling studies by Stefl et al. (26), and by the NMR studies of Bardin and Leroy (25). The − tetramers observed by ESI-MS could correspond to these slipped G-quadruplexes, because less than five G-quartets are formed (Figure 1c). To verify this interpretation, we performed ion mobility spectrometry (IMS) experiments (Figure 5). Ion mobility allows separating ions of a given mass-to-charge ratio based on their ion mobility, which for a given charge is directly related to the ion’s collision cross section. The regular G-quadruplexes being more compact, they will travel faster in the ion mobility cell than the slipped G-quadruplex structures. The experiments not only confirm that the − tetramers (red triangles) have indeed a more extended conformation than the − tetramers (green squares), but also reveal that the degree of compactness of the − tetramers increases with formation time. Each stoichiometry therefore corresponds to an ensemble of conformations. The arrival time distribution of the whole-tetramer adduct distribution (black circles) is shifting to shorter times (more compact conformations) as the formation time increases from 2 min (Figure 5a) to 120 min (Figure 5b) and 165 days (Figure 5c).Figure 5.

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