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.
Highlights
Nucleic acid aptamers with high binding affinity and specificity for various molecular targets have been created using Systematic Evolution of Ligands by EXponential enrichment (SELEX) methods [1,2,3], many of them, DNA aptamers in particular, are known to contain G-quadruplex structures [4,5,6,7]
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]
CA-18A bound to CPT1 with a Kd value of 6.0 μM, which was comparable to that of CA-17
Summary
Nucleic acid aptamers with high binding affinity and specificity for various molecular targets have been created using SELEX methods [1,2,3], many of them, DNA aptamers in particular, are known to contain G-quadruplex structures [4,5,6,7]. It is generally considered that DNA aptamers with G-quadruplexes can target a broad range of molecular structures while retaining specificity. Studying the effects of tail strands on the binding activity of G-quadruplex DNA aptamers for small molecules may be of great importance for researches of G-quadruplex formation in genomic DNA, because the clinical potential of G-quadruplex binding with small molecules in down regulating gene transcription has recently been demonstrated [14,15]. Here we determined which nucleotide residues in tail strands are critical for CPT-binding of DNA aptamers and verified structural differences between active and inactive fragments
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