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DNA triplex formation with 5-dimethylaminopropargyl deoxyuridine.

Rusling DA, Peng G, Srinivasan N, Fox KR, Brown T - Nucleic Acids Res. (2009)

Bottom Line: The results were compared with those for oligonucleotides containing 5-aminopropargyl-dU (APdU), 5-guanidinopropargyl-dU (GPdU) and 5-propynyl dU (PdU).We find that DMAPdU enhances triplex stability relative to T, though slightly less than the other analogues that bear positive charges (T < PdU < DMAPdU < APdU < GPdU).For oligonucleotides that contain multiple substitutions with DMAPdU dispersed residues are more effective than clustered combinations.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

ABSTRACT
We have prepared triplex-forming oligonucleotides containing the nucleotide analogue 5-dimethylaminopropargyl deoxyuridine (DMAPdU) in place of thymidine and examined their ability to form intermolecular triple helices by thermal melting and DNase I footprinting studies. The results were compared with those for oligonucleotides containing 5-aminopropargyl-dU (APdU), 5-guanidinopropargyl-dU (GPdU) and 5-propynyl dU (PdU). We find that DMAPdU enhances triplex stability relative to T, though slightly less than the other analogues that bear positive charges (T < PdU < DMAPdU < APdU < GPdU). For oligonucleotides that contain multiple substitutions with DMAPdU dispersed residues are more effective than clustered combinations. DMAPdU will be especially useful as a nucleotide analogue as, unlike APdU and GPdU, the base does not require protection during oligonucleotide synthesis and it can therefore be used with other derivatives that require mild deprotection conditions.

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Representative annealing (filled symbols) and heating profiles (open symbols) for the interaction of the singly substituted APdU (A) and DMAPdU (B) containing-oligonucleotides with their intended AT-containing duplex target. The profiles were obtained at a rate of 6°Cmin−1. Fraction folded plots for each triplex are included as insets. The experiments were performed in 50 mM sodium acetate pH 5.0, containing 200 mM NaCl. (C) Representative Arrhenius plots for the association (k1; open symbols) and dissociation (k−1; filled symbols) constants for the APdU (squares) and DMAPdU (circles) oligonucleotides.
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Figure 5: Representative annealing (filled symbols) and heating profiles (open symbols) for the interaction of the singly substituted APdU (A) and DMAPdU (B) containing-oligonucleotides with their intended AT-containing duplex target. The profiles were obtained at a rate of 6°Cmin−1. Fraction folded plots for each triplex are included as insets. The experiments were performed in 50 mM sodium acetate pH 5.0, containing 200 mM NaCl. (C) Representative Arrhenius plots for the association (k1; open symbols) and dissociation (k−1; filled symbols) constants for the APdU (squares) and DMAPdU (circles) oligonucleotides.

Mentions: Hysteresis between melting and annealing profiles occurs when the reaction is not at thermodynamic equilibrium and indicates the presence of slow steps in the association or dissociation reactions. This hysteresis can be used to estimate individual association and dissociation rate constants as previously described (8,24,25). Initially we examined the singly modified fluorescently labelled triplexes shown in Figure 1Bi for which heating and cooling these complexes at a rate of 6°C min−1 generated a suitable degree of hysteresis for kinetic analysis. Annealing and melting profiles for each of the triplexes are shown in Figure 5A. Each analogue generates a similar degree of hysteresis, suggesting similar slow steps in the reaction of each of these analogues with an AT base pair. Arrhenius plots derived from these curves are shown in Figure 5B and are typical of those seen with other triplexes; a negative slope is seen for the dissociation reaction along with a positive slope for the association reaction. The apparent negative activation energy of the association reaction indicates that we are not observing the primary kinetic event and is consistent with the suggestion that triplex formation occurs via a nucleation-zipper mechanism (28). Examining these Arrhenius plots reveals that the association rate constants for each analogue are similar but the dissociation rate constants are in different regions of the plot. By extrapolating these lines to 37°C we estimate half-lives of 3.6 × 106 and 3.7 × 107 s for the DMAPdU and APdU triplexes, respectively. This suggests that the difference in stability between the two triplexes arises from difference in their dissociation rates.Figure 5.


DNA triplex formation with 5-dimethylaminopropargyl deoxyuridine.

Rusling DA, Peng G, Srinivasan N, Fox KR, Brown T - Nucleic Acids Res. (2009)

Representative annealing (filled symbols) and heating profiles (open symbols) for the interaction of the singly substituted APdU (A) and DMAPdU (B) containing-oligonucleotides with their intended AT-containing duplex target. The profiles were obtained at a rate of 6°Cmin−1. Fraction folded plots for each triplex are included as insets. The experiments were performed in 50 mM sodium acetate pH 5.0, containing 200 mM NaCl. (C) Representative Arrhenius plots for the association (k1; open symbols) and dissociation (k−1; filled symbols) constants for the APdU (squares) and DMAPdU (circles) oligonucleotides.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Representative annealing (filled symbols) and heating profiles (open symbols) for the interaction of the singly substituted APdU (A) and DMAPdU (B) containing-oligonucleotides with their intended AT-containing duplex target. The profiles were obtained at a rate of 6°Cmin−1. Fraction folded plots for each triplex are included as insets. The experiments were performed in 50 mM sodium acetate pH 5.0, containing 200 mM NaCl. (C) Representative Arrhenius plots for the association (k1; open symbols) and dissociation (k−1; filled symbols) constants for the APdU (squares) and DMAPdU (circles) oligonucleotides.
Mentions: Hysteresis between melting and annealing profiles occurs when the reaction is not at thermodynamic equilibrium and indicates the presence of slow steps in the association or dissociation reactions. This hysteresis can be used to estimate individual association and dissociation rate constants as previously described (8,24,25). Initially we examined the singly modified fluorescently labelled triplexes shown in Figure 1Bi for which heating and cooling these complexes at a rate of 6°C min−1 generated a suitable degree of hysteresis for kinetic analysis. Annealing and melting profiles for each of the triplexes are shown in Figure 5A. Each analogue generates a similar degree of hysteresis, suggesting similar slow steps in the reaction of each of these analogues with an AT base pair. Arrhenius plots derived from these curves are shown in Figure 5B and are typical of those seen with other triplexes; a negative slope is seen for the dissociation reaction along with a positive slope for the association reaction. The apparent negative activation energy of the association reaction indicates that we are not observing the primary kinetic event and is consistent with the suggestion that triplex formation occurs via a nucleation-zipper mechanism (28). Examining these Arrhenius plots reveals that the association rate constants for each analogue are similar but the dissociation rate constants are in different regions of the plot. By extrapolating these lines to 37°C we estimate half-lives of 3.6 × 106 and 3.7 × 107 s for the DMAPdU and APdU triplexes, respectively. This suggests that the difference in stability between the two triplexes arises from difference in their dissociation rates.Figure 5.

Bottom Line: The results were compared with those for oligonucleotides containing 5-aminopropargyl-dU (APdU), 5-guanidinopropargyl-dU (GPdU) and 5-propynyl dU (PdU).We find that DMAPdU enhances triplex stability relative to T, though slightly less than the other analogues that bear positive charges (T < PdU < DMAPdU < APdU < GPdU).For oligonucleotides that contain multiple substitutions with DMAPdU dispersed residues are more effective than clustered combinations.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

ABSTRACT
We have prepared triplex-forming oligonucleotides containing the nucleotide analogue 5-dimethylaminopropargyl deoxyuridine (DMAPdU) in place of thymidine and examined their ability to form intermolecular triple helices by thermal melting and DNase I footprinting studies. The results were compared with those for oligonucleotides containing 5-aminopropargyl-dU (APdU), 5-guanidinopropargyl-dU (GPdU) and 5-propynyl dU (PdU). We find that DMAPdU enhances triplex stability relative to T, though slightly less than the other analogues that bear positive charges (T < PdU < DMAPdU < APdU < GPdU). For oligonucleotides that contain multiple substitutions with DMAPdU dispersed residues are more effective than clustered combinations. DMAPdU will be especially useful as a nucleotide analogue as, unlike APdU and GPdU, the base does not require protection during oligonucleotide synthesis and it can therefore be used with other derivatives that require mild deprotection conditions.

Show MeSH
Related in: MedlinePlus