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Structural and affinity analyses of g-quadruplex DNA aptamers for camptothecin derivatives.

Fujita H, Imaizumi Y, Kasahara Y, Kitadume S, Ozaki H, Kuwahara M, Sugimoto N - Pharmaceuticals (Basel) (2013)

Bottom Line: In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed.We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities.Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. mkuwa@gunma-u.ac.jp.

ABSTRACT
We recently selected DNA aptamers that bind to camptothecin (CPT) and CPT derivatives from a 70-mer oligodeoxyribonucleotide (ODN) library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method. The target-binding activity of the obtained 70-mer CPT-binding DNA aptamer, termed CA-70, which contains a 16-mer guanine (G)-core motif (G3TG3TG3T2G3) that forms a three-tiered G-quadruplex, was determined using fluorescence titration. In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed. We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities. Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers. Further analyses using circular dichroism (CD) spectroscopy and fluorescence polarization (FP) assay were conducted to investigate the structure and affinity of G-quadruplex DNA aptamers.

No MeSH data available.


Predicted secondary structures of CA-70 and -40 using mfold-DNA folding form. The G-quadruplex motif was highlighted with yellow.
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pharmaceuticals-06-01082-f006: Predicted secondary structures of CA-70 and -40 using mfold-DNA folding form. The G-quadruplex motif was highlighted with yellow.

Mentions: In our previous study, CA-70 was truncated into the 40-mer fragment CA-40 on the basis of secondary structure predictions by mfold performed at 37 °C in the presence of 100 mM Na+ [24,25] (Figure 6). As seen in the predicted structures, the 16-mer G-core sequence is located in a loop flanked by sequences that form a stem structure. Thus, we then further truncated CA-40 into CA-20 and -16, both of which contained the G-core sequence. The Kd values of CA-40 and -16 for CPT1 were 3.0 and 1.8 µM, respectively, which were comparable to that of non-truncated CA-70 (Kd = 1.8 µM), whereas CA-20 had little or no binding affinity for the target. First, we generated four new truncated fragments (CA-19, -18A, -18B, and -17) having the G-core sequence with a 3′-tail of 1–3 nucleotides and with or without a 5′-tail of a single nucleotide, and then compared their binding activities with each other.


Structural and affinity analyses of g-quadruplex DNA aptamers for camptothecin derivatives.

Fujita H, Imaizumi Y, Kasahara Y, Kitadume S, Ozaki H, Kuwahara M, Sugimoto N - Pharmaceuticals (Basel) (2013)

Predicted secondary structures of CA-70 and -40 using mfold-DNA folding form. The G-quadruplex motif was highlighted with yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818834&req=5

pharmaceuticals-06-01082-f006: Predicted secondary structures of CA-70 and -40 using mfold-DNA folding form. The G-quadruplex motif was highlighted with yellow.
Mentions: In our previous study, CA-70 was truncated into the 40-mer fragment CA-40 on the basis of secondary structure predictions by mfold performed at 37 °C in the presence of 100 mM Na+ [24,25] (Figure 6). As seen in the predicted structures, the 16-mer G-core sequence is located in a loop flanked by sequences that form a stem structure. Thus, we then further truncated CA-40 into CA-20 and -16, both of which contained the G-core sequence. The Kd values of CA-40 and -16 for CPT1 were 3.0 and 1.8 µM, respectively, which were comparable to that of non-truncated CA-70 (Kd = 1.8 µM), whereas CA-20 had little or no binding affinity for the target. First, we generated four new truncated fragments (CA-19, -18A, -18B, and -17) having the G-core sequence with a 3′-tail of 1–3 nucleotides and with or without a 5′-tail of a single nucleotide, and then compared their binding activities with each other.

Bottom Line: In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed.We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities.Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. mkuwa@gunma-u.ac.jp.

ABSTRACT
We recently selected DNA aptamers that bind to camptothecin (CPT) and CPT derivatives from a 70-mer oligodeoxyribonucleotide (ODN) library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method. The target-binding activity of the obtained 70-mer CPT-binding DNA aptamer, termed CA-70, which contains a 16-mer guanine (G)-core motif (G3TG3TG3T2G3) that forms a three-tiered G-quadruplex, was determined using fluorescence titration. In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed. We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities. Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers. Further analyses using circular dichroism (CD) spectroscopy and fluorescence polarization (FP) assay were conducted to investigate the structure and affinity of G-quadruplex DNA aptamers.

No MeSH data available.