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G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold.

Marušič M, Veedu RN, Wengel J, Plavec J - Nucleic Acids Res. (2013)

Bottom Line: Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology.Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex.Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

View Article: PubMed Central - PubMed

Affiliation: Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia, School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Brisbane, 4072 Australia, Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark, EN-FIST Center of Excellence, SI-1000 Ljubljana, Slovenia and Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia.

ABSTRACT
The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

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(a) Imino–aromatic and imino–imino regions of 2D NOESY spectrum (τm = 250 ms) of RNV66 with resonance assignment indicated along 1D traces. Cross-peaks labeled with red, blue and orange rectangles correspond to the G6-G8-G16-L21, L5-G7-G15-G20 and G4-G11-G14-G19 quartets, respectively. Cross-peaks labeled with black rectangles correspond to correlations between sequential residues within a G-quadruplex core. Underlined assignments correspond to the correlations of residues involved in loop and flanking regions. Spectrum was recorded at 800 MHz, 25°C in 10% 2H2O, 50 mM K+, 10 mM KPi buffer with pH 7 and oligonucleotide concentration of 0.5 mM. Schematic presentations of observed NOE connections for imino–aromatic and imino–imino regions are shown in (b) and (c), respectively. Colors of the arrows correspond to the color of the NOE cross-peaks in panel (a).
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gkt697-F3: (a) Imino–aromatic and imino–imino regions of 2D NOESY spectrum (τm = 250 ms) of RNV66 with resonance assignment indicated along 1D traces. Cross-peaks labeled with red, blue and orange rectangles correspond to the G6-G8-G16-L21, L5-G7-G15-G20 and G4-G11-G14-G19 quartets, respectively. Cross-peaks labeled with black rectangles correspond to correlations between sequential residues within a G-quadruplex core. Underlined assignments correspond to the correlations of residues involved in loop and flanking regions. Spectrum was recorded at 800 MHz, 25°C in 10% 2H2O, 50 mM K+, 10 mM KPi buffer with pH 7 and oligonucleotide concentration of 0.5 mM. Schematic presentations of observed NOE connections for imino–aromatic and imino–imino regions are shown in (b) and (c), respectively. Colors of the arrows correspond to the color of the NOE cross-peaks in panel (a).

Mentions: A model of a G-quadruplex core was built through careful analysis of the NOE correlations between imino protons. Our analysis of several structures deposited in PDB has shown that the expected distance between imino protons of sequential residues in ‘anti-anti’ steps is below 4.0 Å, the distance between neighboring imino protons within a G-quartet is between 4.0 and 4.5 Å, and the distance between non-sequential imino protons of neighboring G-quartets is also between 4.0 and 4.5 Å. Cross-peaks of medium intensity in the imino–imino region of 2D NOESY spectra were therefore assigned to correlations between sequential G-quartets forming residues. Distinction between weak and medium cross-peaks was facilitated by a 2D NOESY spectrum (τm of 250 ms) recorded at 0°C, which enabled observation of medium intensity cross-peaks (Supplementary Figure S4). Residues involved in a G-quadruplex core were identified through NOE correlations of imino and aromatic protons (Figure 3a) that connected cross-peaks in the imino region with assignment of residues in the aromatic region (Figure 3b and c). Identification of G4-G6 and G19-L21 tracts, which were unambiguously identified through a sequential walk, formed basis to build a topology model. NOE correlations in the imino–aromatic region have proved unequivocally that G4-G6 and G19-L21 tracts are neighboring strands in a G-quadruplex structure. Subsequently, G14-G16 was recognized as the neighboring strand to G19-L21. As G2, G10 and L24 exhibited no NOE correlations in the imino–aromatic region, and as their imino protons were involved in fast exchange, it was concluded that these residues do not participate in the formation of G-quartets. Consequently, one of the DNA strands in the G-quadruplex core had to comprise residues G7, G8 and G11. Their relative positions were established with help of a crucial albeit very weak G8 H1′-T9 H7 cross-peak, which identified G8 as member of the G6-G8-G16-L21 quartet. Furthermore, G8 H1-G16 H8 and G8 H1-G16 H1 cross-peaks affirmed hydrogen-bond directionalities within the G6-G8-G16-L21 quartet. Even though G11–G7 is not a sequential step, classical H1′-H8 and H2′/H2′′-H8 NOE connectivities could be observed for these two residues (Figure 2). G7 and G11 were determined to be positioned in the L5-G7-G15-G20 and G4-G11-G14-G19 quartets, respectively, on the basis of the intra-quartet imino–imino and inter-quartet imino–imino and imino–aromatic contacts (Figure 3).Figure 3.


G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold.

Marušič M, Veedu RN, Wengel J, Plavec J - Nucleic Acids Res. (2013)

(a) Imino–aromatic and imino–imino regions of 2D NOESY spectrum (τm = 250 ms) of RNV66 with resonance assignment indicated along 1D traces. Cross-peaks labeled with red, blue and orange rectangles correspond to the G6-G8-G16-L21, L5-G7-G15-G20 and G4-G11-G14-G19 quartets, respectively. Cross-peaks labeled with black rectangles correspond to correlations between sequential residues within a G-quadruplex core. Underlined assignments correspond to the correlations of residues involved in loop and flanking regions. Spectrum was recorded at 800 MHz, 25°C in 10% 2H2O, 50 mM K+, 10 mM KPi buffer with pH 7 and oligonucleotide concentration of 0.5 mM. Schematic presentations of observed NOE connections for imino–aromatic and imino–imino regions are shown in (b) and (c), respectively. Colors of the arrows correspond to the color of the NOE cross-peaks in panel (a).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814366&req=5

gkt697-F3: (a) Imino–aromatic and imino–imino regions of 2D NOESY spectrum (τm = 250 ms) of RNV66 with resonance assignment indicated along 1D traces. Cross-peaks labeled with red, blue and orange rectangles correspond to the G6-G8-G16-L21, L5-G7-G15-G20 and G4-G11-G14-G19 quartets, respectively. Cross-peaks labeled with black rectangles correspond to correlations between sequential residues within a G-quadruplex core. Underlined assignments correspond to the correlations of residues involved in loop and flanking regions. Spectrum was recorded at 800 MHz, 25°C in 10% 2H2O, 50 mM K+, 10 mM KPi buffer with pH 7 and oligonucleotide concentration of 0.5 mM. Schematic presentations of observed NOE connections for imino–aromatic and imino–imino regions are shown in (b) and (c), respectively. Colors of the arrows correspond to the color of the NOE cross-peaks in panel (a).
Mentions: A model of a G-quadruplex core was built through careful analysis of the NOE correlations between imino protons. Our analysis of several structures deposited in PDB has shown that the expected distance between imino protons of sequential residues in ‘anti-anti’ steps is below 4.0 Å, the distance between neighboring imino protons within a G-quartet is between 4.0 and 4.5 Å, and the distance between non-sequential imino protons of neighboring G-quartets is also between 4.0 and 4.5 Å. Cross-peaks of medium intensity in the imino–imino region of 2D NOESY spectra were therefore assigned to correlations between sequential G-quartets forming residues. Distinction between weak and medium cross-peaks was facilitated by a 2D NOESY spectrum (τm of 250 ms) recorded at 0°C, which enabled observation of medium intensity cross-peaks (Supplementary Figure S4). Residues involved in a G-quadruplex core were identified through NOE correlations of imino and aromatic protons (Figure 3a) that connected cross-peaks in the imino region with assignment of residues in the aromatic region (Figure 3b and c). Identification of G4-G6 and G19-L21 tracts, which were unambiguously identified through a sequential walk, formed basis to build a topology model. NOE correlations in the imino–aromatic region have proved unequivocally that G4-G6 and G19-L21 tracts are neighboring strands in a G-quadruplex structure. Subsequently, G14-G16 was recognized as the neighboring strand to G19-L21. As G2, G10 and L24 exhibited no NOE correlations in the imino–aromatic region, and as their imino protons were involved in fast exchange, it was concluded that these residues do not participate in the formation of G-quartets. Consequently, one of the DNA strands in the G-quadruplex core had to comprise residues G7, G8 and G11. Their relative positions were established with help of a crucial albeit very weak G8 H1′-T9 H7 cross-peak, which identified G8 as member of the G6-G8-G16-L21 quartet. Furthermore, G8 H1-G16 H8 and G8 H1-G16 H1 cross-peaks affirmed hydrogen-bond directionalities within the G6-G8-G16-L21 quartet. Even though G11–G7 is not a sequential step, classical H1′-H8 and H2′/H2′′-H8 NOE connectivities could be observed for these two residues (Figure 2). G7 and G11 were determined to be positioned in the L5-G7-G15-G20 and G4-G11-G14-G19 quartets, respectively, on the basis of the intra-quartet imino–imino and inter-quartet imino–imino and imino–aromatic contacts (Figure 3).Figure 3.

Bottom Line: Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology.Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex.Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

View Article: PubMed Central - PubMed

Affiliation: Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia, School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Brisbane, 4072 Australia, Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark, EN-FIST Center of Excellence, SI-1000 Ljubljana, Slovenia and Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia.

ABSTRACT
The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

Show MeSH
Related in: MedlinePlus