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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 multiply substituted dispersed (A) or clustered (B) TFOs with their intended duplex target site. The TFOs contained T (squares), DMAPdU (circles), APdU (triangles), GPdU (diamonds) or PdU (hexagons). 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 3: Representative fluorescence melting curves showing the interaction of the multiply substituted dispersed (A) or clustered (B) TFOs with their intended duplex target site. The TFOs contained T (squares), DMAPdU (circles), APdU (triangles), GPdU (diamonds) or PdU (hexagons). 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 these complexes at pH 6.0 are shown in Figure 3 and the Tm values determined from the melting and annealing curves (shown in parenthesis) are presented in Table 2. It can be seen that the TFO containing dispersed substitutions of DMAPdU (left panel; circles) produces a ΔTm of 11°C relative to the triplex formed with the unmodified TFO (left panel; squares). The incorporation of more than one substitution gives greater stabilization with a ΔTm of about 2°C per substitution. Comparison with the other charged analogues reveals that the amine and guanidine modifications are more stabilizing, leading to increases in Tm of 19°C and 21°C, respectively. The oligonucleotides containing the uncharged propynyl derivative (PdU) also generate stable triplexes, though the ΔTm values are less that with DMAPdU or APdU; the difference between DMAPdU and PdU is greatest at pH 5.0. The triplex containing contiguous substitutions of DMAPdU in the TFO (right panel; circles) is stabilized less than when these residues are dispersed; this difference is greater at higher pHs. In contrast, the stabilization produced with the three other analogues is not affected by the arrangement of the modified bases and similar Tm values are produce with TFOs in which these residues are dispersed or contiguous, suggesting that steric factors may be involved in limiting the stability of triplexes with contiguous DMAPDU residues.Figure 3.


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 multiply substituted dispersed (A) or clustered (B) TFOs with their intended duplex target site. The TFOs contained T (squares), DMAPdU (circles), APdU (triangles), GPdU (diamonds) or PdU (hexagons). 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 3: Representative fluorescence melting curves showing the interaction of the multiply substituted dispersed (A) or clustered (B) TFOs with their intended duplex target site. The TFOs contained T (squares), DMAPdU (circles), APdU (triangles), GPdU (diamonds) or PdU (hexagons). 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 these complexes at pH 6.0 are shown in Figure 3 and the Tm values determined from the melting and annealing curves (shown in parenthesis) are presented in Table 2. It can be seen that the TFO containing dispersed substitutions of DMAPdU (left panel; circles) produces a ΔTm of 11°C relative to the triplex formed with the unmodified TFO (left panel; squares). The incorporation of more than one substitution gives greater stabilization with a ΔTm of about 2°C per substitution. Comparison with the other charged analogues reveals that the amine and guanidine modifications are more stabilizing, leading to increases in Tm of 19°C and 21°C, respectively. The oligonucleotides containing the uncharged propynyl derivative (PdU) also generate stable triplexes, though the ΔTm values are less that with DMAPdU or APdU; the difference between DMAPdU and PdU is greatest at pH 5.0. The triplex containing contiguous substitutions of DMAPdU in the TFO (right panel; circles) is stabilized less than when these residues are dispersed; this difference is greater at higher pHs. In contrast, the stabilization produced with the three other analogues is not affected by the arrangement of the modified bases and similar Tm values are produce with TFOs in which these residues are dispersed or contiguous, suggesting that steric factors may be involved in limiting the stability of triplexes with contiguous DMAPDU residues.Figure 3.

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