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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.


Continuous variation binding analysis (Job plot) of CPT1 using UV-vis spectroscopy (left) and fluorescence spectroscopy (right). Total concentration of CPT1 and CA-16 was maintained at 10 μM.
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pharmaceuticals-06-01082-f002: Continuous variation binding analysis (Job plot) of CPT1 using UV-vis spectroscopy (left) and fluorescence spectroscopy (right). Total concentration of CPT1 and CA-16 was maintained at 10 μM.

Mentions: To determine the binding stoichiometry between the aptamer and target, a continuous variation binding analysis (Job plot) was performed [17,18]. First, CA-16 was dissolved in 10 mM PBS buffer to make a final concentration of 10 µM, and refolded by denaturing at 94 °C for 0.5 min, followed by cooling to 25 °C at a rate of 0.5 °C/min, while CPT1 was dissolved in 10 mM PBS buffer to make a final concentration of 10 µM. These solutions were mixed to maintain the total molar concentration (CA-16 and CPT1) at 10 µM, whereas the CA-16 mole fraction was varied at regular intervals from 0 to 1.0. After incubation at 25 °C for 30 min, the spectra of 11 sample solutions were measured using UV-vis spectroscopy and fluorescence spectroscopy to generate the Job plots (Figure 2).


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)

Continuous variation binding analysis (Job plot) of CPT1 using UV-vis spectroscopy (left) and fluorescence spectroscopy (right). Total concentration of CPT1 and CA-16 was maintained at 10 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

pharmaceuticals-06-01082-f002: Continuous variation binding analysis (Job plot) of CPT1 using UV-vis spectroscopy (left) and fluorescence spectroscopy (right). Total concentration of CPT1 and CA-16 was maintained at 10 μM.
Mentions: To determine the binding stoichiometry between the aptamer and target, a continuous variation binding analysis (Job plot) was performed [17,18]. First, CA-16 was dissolved in 10 mM PBS buffer to make a final concentration of 10 µM, and refolded by denaturing at 94 °C for 0.5 min, followed by cooling to 25 °C at a rate of 0.5 °C/min, while CPT1 was dissolved in 10 mM PBS buffer to make a final concentration of 10 µM. These solutions were mixed to maintain the total molar concentration (CA-16 and CPT1) at 10 µM, whereas the CA-16 mole fraction was varied at regular intervals from 0 to 1.0. After incubation at 25 °C for 30 min, the spectra of 11 sample solutions were measured using UV-vis spectroscopy and fluorescence spectroscopy to generate the Job plots (Figure 2).

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.