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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) Chemical structures of the LNA modified guanine residue (left) and UNA modified cytosine residue (right). (b) Sequences and the corresponding 1H NMR spectra of G-rich oligonucleotides V7t1 (top), RNV66 (middle) and RNV70 (bottom) in the presence of 100 mM concentration of K+ ions. 600 MHz 1H NMR spectra of ca. 1 mM oligonucleotide concentration per strand were acquired at 25°C in 10% 2H2O, 10 mM KPi buffer with pH 7. LNA and UNA nucleotides are designated with letter L and letter uC, respectively. Numbering of the residues is indicated.
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gkt697-F1: (a) Chemical structures of the LNA modified guanine residue (left) and UNA modified cytosine residue (right). (b) Sequences and the corresponding 1H NMR spectra of G-rich oligonucleotides V7t1 (top), RNV66 (middle) and RNV70 (bottom) in the presence of 100 mM concentration of K+ ions. 600 MHz 1H NMR spectra of ca. 1 mM oligonucleotide concentration per strand were acquired at 25°C in 10% 2H2O, 10 mM KPi buffer with pH 7. LNA and UNA nucleotides are designated with letter L and letter uC, respectively. Numbering of the residues is indicated.

Mentions: Aptamers are short nucleic acids with high affinities toward target molecules (14). Their generation involves several cycles of selection and enrichment using the so-called systematic evolution of ligands by exponential enrichment (SELEX) procedures starting from a random oligonucleotide sequence library (14,15). Aptamers display several advantages in comparison with antibodies, such as low toxicity, low immunogenicity, small size, ease of production and amenability to structural modification. Modifications introduced during or after the selection process are generally needed to optimize synthesis, stability, resistance toward nucleases and blood stream circulation time. Amongst numerous existing aptamer modifications, locked nucleic acids (LNA) (Figure 1a) contribute to increased binding affinity and higher nuclease resistance (16,17). LNA are widely exploited as a building block in therapeutic oligonucleotides (18,19). Sugar moieties of LNA nucleotides with their O2′-C4′-methylene bridge are locked in an N-type sugar conformation, mimicking that of RNA nucleotides. In Watson–Crick base-paired helices, LNA residues were shown to induce local reorganization of the sugar-phosphate backbone and to drive N↔S pseudorotational equilibria in neighboring residues toward N-type conformations. As a consequence, inclusion of LNA residues drives nucleic acid double helices toward an A form (20,21). On the other hand, a search for correlations between target binding and the propensity of aptamers to adopt secondary and tertiary structures has stimulated the introduction of other unnatural modified residues. Unlocked nucleic acid (UNA) in many ways represents an antipode to LNA (Figure 1a). The missing bond between the C2′ and C3′ atoms in the sugar moiety induces a high flexibility of UNA residues, which is reflected in destabilization of helices on UNA modifications (22). UNA modification was shown to improve nuclease resistance and to be valuable toward increasing target specificity (23–25). The applicability of UNA nucleotides in aptamer development was recently studied by systematically incorporating them in a known DNA-based thrombin-binding aptamer, whereby it was demonstrated that introduction of UNA residues at specific positions significantly increased the thrombin-binding affinity (26,27).Figure 1.


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) Chemical structures of the LNA modified guanine residue (left) and UNA modified cytosine residue (right). (b) Sequences and the corresponding 1H NMR spectra of G-rich oligonucleotides V7t1 (top), RNV66 (middle) and RNV70 (bottom) in the presence of 100 mM concentration of K+ ions. 600 MHz 1H NMR spectra of ca. 1 mM oligonucleotide concentration per strand were acquired at 25°C in 10% 2H2O, 10 mM KPi buffer with pH 7. LNA and UNA nucleotides are designated with letter L and letter uC, respectively. Numbering of the residues is indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814366&req=5

gkt697-F1: (a) Chemical structures of the LNA modified guanine residue (left) and UNA modified cytosine residue (right). (b) Sequences and the corresponding 1H NMR spectra of G-rich oligonucleotides V7t1 (top), RNV66 (middle) and RNV70 (bottom) in the presence of 100 mM concentration of K+ ions. 600 MHz 1H NMR spectra of ca. 1 mM oligonucleotide concentration per strand were acquired at 25°C in 10% 2H2O, 10 mM KPi buffer with pH 7. LNA and UNA nucleotides are designated with letter L and letter uC, respectively. Numbering of the residues is indicated.
Mentions: Aptamers are short nucleic acids with high affinities toward target molecules (14). Their generation involves several cycles of selection and enrichment using the so-called systematic evolution of ligands by exponential enrichment (SELEX) procedures starting from a random oligonucleotide sequence library (14,15). Aptamers display several advantages in comparison with antibodies, such as low toxicity, low immunogenicity, small size, ease of production and amenability to structural modification. Modifications introduced during or after the selection process are generally needed to optimize synthesis, stability, resistance toward nucleases and blood stream circulation time. Amongst numerous existing aptamer modifications, locked nucleic acids (LNA) (Figure 1a) contribute to increased binding affinity and higher nuclease resistance (16,17). LNA are widely exploited as a building block in therapeutic oligonucleotides (18,19). Sugar moieties of LNA nucleotides with their O2′-C4′-methylene bridge are locked in an N-type sugar conformation, mimicking that of RNA nucleotides. In Watson–Crick base-paired helices, LNA residues were shown to induce local reorganization of the sugar-phosphate backbone and to drive N↔S pseudorotational equilibria in neighboring residues toward N-type conformations. As a consequence, inclusion of LNA residues drives nucleic acid double helices toward an A form (20,21). On the other hand, a search for correlations between target binding and the propensity of aptamers to adopt secondary and tertiary structures has stimulated the introduction of other unnatural modified residues. Unlocked nucleic acid (UNA) in many ways represents an antipode to LNA (Figure 1a). The missing bond between the C2′ and C3′ atoms in the sugar moiety induces a high flexibility of UNA residues, which is reflected in destabilization of helices on UNA modifications (22). UNA modification was shown to improve nuclease resistance and to be valuable toward increasing target specificity (23–25). The applicability of UNA nucleotides in aptamer development was recently studied by systematically incorporating them in a known DNA-based thrombin-binding aptamer, whereby it was demonstrated that introduction of UNA residues at specific positions significantly increased the thrombin-binding affinity (26,27).Figure 1.

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