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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.


Related in: MedlinePlus

Titration curves for relative fluorescence intensity at 456 nm versus fragment concentration: CA-70 (open squares), CA-40 (closed squares), CA-23A (crosses), CA-23B (x-marks), CA-20 (open triangles), CA-19 (closed triangles), CA-18A (open diamonds), CA-18B (closed diamonds), CA-17 (open circles), and CA-16 (closed circles). CPT1 fluorescence without fragment was set at 100%.
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pharmaceuticals-06-01082-f003: Titration curves for relative fluorescence intensity at 456 nm versus fragment concentration: CA-70 (open squares), CA-40 (closed squares), CA-23A (crosses), CA-23B (x-marks), CA-20 (open triangles), CA-19 (closed triangles), CA-18A (open diamonds), CA-18B (closed diamonds), CA-17 (open circles), and CA-16 (closed circles). CPT1 fluorescence without fragment was set at 100%.

Mentions: Fluorescence titration of target (CPT1, CPT2, and CPT; final conc. 1.0 µM) with increasing concentrations of CA-70 and its fragments (final conc. 0–10 µM) was performed at 25 °C using LS-55. CA-70, -40, -23A, -23B, -20, -19, -18A, -18B, -17, and -16 were dissolved in 10 mM PBS buffer at appropriate concentrations (0–20 µM), and refolded by denaturing at 94 °C for 0.5 min and cooling to 25 °C at a rate of 0.5 °C/min. These solutions (50 µL each) were mixed with 50 µL solutions of target (CPT1, CPT2, and CPT; 2 µM) in 10 mM PBS buffer, respectively. Emission spectra of CPTs were obtained by exciting at 370 nm and monitoring fluorescence between 400 and 530 nm. In Figure 3, Y-axis indicates the relative fluorescence intensity (%) at 456 nm for CPT1 as an emission peak, where the intensity in the absence of fragments was set at 100%, and X-axis indicates fragment concentration. The relative fluorescence intensity (%) at 443 nm for CPT and at 456 nm for CPT2 was used to obtain the titration curves, respectively. The Kd values determined from the titration curves were listed on Table 1. To assess the binding specificity of CA-70, fluorescence titrations were also performed using the protocol described above and the following fluorophores: FMN, 9(10H)-acridone, 2-(carboxymethyl)-9(10H)acridone, and fluorescein (Figure 1).


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)

Titration curves for relative fluorescence intensity at 456 nm versus fragment concentration: CA-70 (open squares), CA-40 (closed squares), CA-23A (crosses), CA-23B (x-marks), CA-20 (open triangles), CA-19 (closed triangles), CA-18A (open diamonds), CA-18B (closed diamonds), CA-17 (open circles), and CA-16 (closed circles). CPT1 fluorescence without fragment was set at 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

pharmaceuticals-06-01082-f003: Titration curves for relative fluorescence intensity at 456 nm versus fragment concentration: CA-70 (open squares), CA-40 (closed squares), CA-23A (crosses), CA-23B (x-marks), CA-20 (open triangles), CA-19 (closed triangles), CA-18A (open diamonds), CA-18B (closed diamonds), CA-17 (open circles), and CA-16 (closed circles). CPT1 fluorescence without fragment was set at 100%.
Mentions: Fluorescence titration of target (CPT1, CPT2, and CPT; final conc. 1.0 µM) with increasing concentrations of CA-70 and its fragments (final conc. 0–10 µM) was performed at 25 °C using LS-55. CA-70, -40, -23A, -23B, -20, -19, -18A, -18B, -17, and -16 were dissolved in 10 mM PBS buffer at appropriate concentrations (0–20 µM), and refolded by denaturing at 94 °C for 0.5 min and cooling to 25 °C at a rate of 0.5 °C/min. These solutions (50 µL each) were mixed with 50 µL solutions of target (CPT1, CPT2, and CPT; 2 µM) in 10 mM PBS buffer, respectively. Emission spectra of CPTs were obtained by exciting at 370 nm and monitoring fluorescence between 400 and 530 nm. In Figure 3, Y-axis indicates the relative fluorescence intensity (%) at 456 nm for CPT1 as an emission peak, where the intensity in the absence of fragments was set at 100%, and X-axis indicates fragment concentration. The relative fluorescence intensity (%) at 443 nm for CPT and at 456 nm for CPT2 was used to obtain the titration curves, respectively. The Kd values determined from the titration curves were listed on Table 1. To assess the binding specificity of CA-70, fluorescence titrations were also performed using the protocol described above and the following fluorophores: FMN, 9(10H)-acridone, 2-(carboxymethyl)-9(10H)acridone, and fluorescein (Figure 1).

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.


Related in: MedlinePlus