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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

Melting curves for CA-16 in the presence (red) and absence (black) of CPT1, which were obtained by plotting the ellipticity at 265 nm against the temperature.
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pharmaceuticals-06-01082-f007: Melting curves for CA-16 in the presence (red) and absence (black) of CPT1, which were obtained by plotting the ellipticity at 265 nm against the temperature.

Mentions: However, if this is the case, it remains unknown why CA-70 and -40 that have long 3′-tails retain the binding activity. Before addressing this issue, we had to ascertain the differences between active and inactive fragments in the binding activity, because the abovementioned observations were made using fluorescence quenching titration; the degree of quenching is not necessary consistent with the target-binding ratio. Therefore, we analyzed CA-16 and -19 by the FP assay and confirmed that the former has evident CPT1-binding activity but the latter does not (Figure 4). Then, we speculated that the extra 3′-terminus G37T38A39 may interfere with G-quadruplex formation; however, CD spectra showed that both CA-16 and -19 formed a parallel G-quadruplex in either buffer solution [27,28] (Figure 5). Also, the K+ dependency on G-quadruplex formation was confirmed (Figure 4b). A similar G-core sequence (AG3TG4AG3TG4) in a promoter region of c-Myc gene is known to form a three-tiered parallel G-quadruplex [29,30]. Therefore, it is conceivable that CAs can form parallel G-quadruplexes, although CAs have short loops consisting of 1–2 nucleotide residues. These parallel G-quadruplex cores are extremely stable. The Tm value of CA-16 was observed to be 73 °C in 10 mM PBS buffer (Figure 7). The Tm value was elevated by 80 °C when CPT1 associated with CA-16, indicating a significant contribution to the thermodynamic stability owing to π–π stacking interaction between the G-quartet plane and heterocyclic rings of the target.


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)

Melting curves for CA-16 in the presence (red) and absence (black) of CPT1, which were obtained by plotting the ellipticity at 265 nm against the temperature.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

pharmaceuticals-06-01082-f007: Melting curves for CA-16 in the presence (red) and absence (black) of CPT1, which were obtained by plotting the ellipticity at 265 nm against the temperature.
Mentions: However, if this is the case, it remains unknown why CA-70 and -40 that have long 3′-tails retain the binding activity. Before addressing this issue, we had to ascertain the differences between active and inactive fragments in the binding activity, because the abovementioned observations were made using fluorescence quenching titration; the degree of quenching is not necessary consistent with the target-binding ratio. Therefore, we analyzed CA-16 and -19 by the FP assay and confirmed that the former has evident CPT1-binding activity but the latter does not (Figure 4). Then, we speculated that the extra 3′-terminus G37T38A39 may interfere with G-quadruplex formation; however, CD spectra showed that both CA-16 and -19 formed a parallel G-quadruplex in either buffer solution [27,28] (Figure 5). Also, the K+ dependency on G-quadruplex formation was confirmed (Figure 4b). A similar G-core sequence (AG3TG4AG3TG4) in a promoter region of c-Myc gene is known to form a three-tiered parallel G-quadruplex [29,30]. Therefore, it is conceivable that CAs can form parallel G-quadruplexes, although CAs have short loops consisting of 1–2 nucleotide residues. These parallel G-quadruplex cores are extremely stable. The Tm value of CA-16 was observed to be 73 °C in 10 mM PBS buffer (Figure 7). The Tm value was elevated by 80 °C when CPT1 associated with CA-16, indicating a significant contribution to the thermodynamic stability owing to π–π stacking interaction between the G-quartet plane and heterocyclic rings of the target.

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