Competitive assay for theophylline based on an abasic site-containing DNA duplex aptamer and a fluorescent ligand

Yusuke Sato, Yushuang Zhang, Seiichi Nishizawa, Takehiro Seino, Kodai Nakamura, Minjie Li, Norio Teramae

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)

Abstract

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site-containing DNA duplex aptamer (AP aptamer) in combination with an abasic site-binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23-mer AP aptamer (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/5'-GTG TGC GTT GCC CTG GAC GCA GA-3'; X: the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM. Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin. Lighting up the competition: A fluorescence assay is reported for the detection of theophylline by using an abasic site (AP site)-containing DNA duplex aptamer (AP aptamer) in combination with an abasic site-binding fluorescent ligand, namely riboflavin. The assay is based on the competitive binding of theophylline or riboflavin at the AP site of the AP aptamer (see scheme). Fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.

Original languageEnglish
Pages (from-to)12719-12724
Number of pages6
JournalChemistry - A European Journal
Volume18
Issue number40
DOIs
Publication statusPublished - 2012 Oct 1

Keywords

  • DNA binding
  • abasic sites
  • aptamers
  • fluorescent probes
  • theophylline

ASJC Scopus subject areas

  • Chemistry(all)

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