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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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Related in: MedlinePlus

Representative fluorescence melting curves showing the interaction of the singly substituted TFOs with duplex target sites containing a variable central base pair, generating X.AT, X.TA, X.GC or X.CG base triplets where X is T (squares), DMAPdU (circles), APdU (triangles) or GPdU (diamonds). The experiments were performed in 50 mM sodium acetate pH 6.0, containing 200 mM NaCl. The complexes were heated and cooled at a rate of 0.2°C min−1.
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Figure 2: Representative fluorescence melting curves showing the interaction of the singly substituted TFOs with duplex target sites containing a variable central base pair, generating X.AT, X.TA, X.GC or X.CG base triplets where X is T (squares), DMAPdU (circles), APdU (triangles) or GPdU (diamonds). The experiments were performed in 50 mM sodium acetate pH 6.0, containing 200 mM NaCl. The complexes were heated and cooled at a rate of 0.2°C min−1.

Mentions: Representative melting curves for the complexes at pH 6.0 are shown in Figure 2 and Tm values derived from these and for the same complexes at pH 5.0 and 5.5 are summarized in Table 1. Examination of these data reveals that as expected all the deoxyuridine analogues increased triplex stability when positioned opposite an AT base pair in the target (top left panel). The magnitude of the stabilization was dependent on the analogue; the dimethylamino derivative DMAPdU increased the Tm by about 2.5°C, while the primary amine (APdU) and guanidine (GPdU) variants increased the Tm by about 4°C relative to T. The stabilization was also greater at higher pHs with ΔTm values nearly twice as large at pH 6.0 compared to pH 5.0. Interestingly, the pH also affected the kinetics of the modified complexes since there was a small amount of hysteresis between the melting and annealing curves at pH 5.0 but not at pH 5.5 or 6.0. In contrast, no hysteresis was observed with the T-containing TFO. This suggests that there are some slow association and/or dissociation kinetics with these modified triplexes, which are examined in greater detail below.Figure 2.


DNA triplex formation with 5-dimethylaminopropargyl deoxyuridine.

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

Representative fluorescence melting curves showing the interaction of the singly substituted TFOs with duplex target sites containing a variable central base pair, generating X.AT, X.TA, X.GC or X.CG base triplets where X is T (squares), DMAPdU (circles), APdU (triangles) or GPdU (diamonds). The experiments were performed in 50 mM sodium acetate pH 6.0, containing 200 mM NaCl. The complexes were heated and cooled at a rate of 0.2°C min−1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651792&req=5

Figure 2: Representative fluorescence melting curves showing the interaction of the singly substituted TFOs with duplex target sites containing a variable central base pair, generating X.AT, X.TA, X.GC or X.CG base triplets where X is T (squares), DMAPdU (circles), APdU (triangles) or GPdU (diamonds). The experiments were performed in 50 mM sodium acetate pH 6.0, containing 200 mM NaCl. The complexes were heated and cooled at a rate of 0.2°C min−1.
Mentions: Representative melting curves for the complexes at pH 6.0 are shown in Figure 2 and Tm values derived from these and for the same complexes at pH 5.0 and 5.5 are summarized in Table 1. Examination of these data reveals that as expected all the deoxyuridine analogues increased triplex stability when positioned opposite an AT base pair in the target (top left panel). The magnitude of the stabilization was dependent on the analogue; the dimethylamino derivative DMAPdU increased the Tm by about 2.5°C, while the primary amine (APdU) and guanidine (GPdU) variants increased the Tm by about 4°C relative to T. The stabilization was also greater at higher pHs with ΔTm values nearly twice as large at pH 6.0 compared to pH 5.0. Interestingly, the pH also affected the kinetics of the modified complexes since there was a small amount of hysteresis between the melting and annealing curves at pH 5.0 but not at pH 5.5 or 6.0. In contrast, no hysteresis was observed with the T-containing TFO. This suggests that there are some slow association and/or dissociation kinetics with these modified triplexes, which are examined in greater detail below.Figure 2.

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