Elsevier

Biosensors and Bioelectronics

Volume 26, Issue 12, 15 August 2011, Pages 4733-4738
Biosensors and Bioelectronics

Fluorescent aptasensors based on conformational adaptability of abasic site-containing aptamers in combination with abasic site-binding ligands

https://doi.org/10.1016/j.bios.2011.05.051Get rights and content

Abstract

Aptamers are nucleic acids that can selectively bind to a variety of targets. Aptamers usually undergo conformational transitions from a flexible or disordered structure into a rigid or ordered structure upon target-binding. This study describes a detection method for l-argininamide (l-Arm) and adenosine based on the conformational adaptability of nucleic acid aptamers. An abasic site (AP site) was formed in the stem and close to the target-binding site of a stem-loop aptamer as an anchoring pocket for a fluorescent ligand. 3,5-Diamino-6-chloro-2-pyrazine carbonitrile (DCPC), which can bind to AP site-containing DNA duplexes by pseudo-base pairing, was utilized as a signaling reporter for the target-binding. The binding of a target to an aptamer induces the tight pairing of bases flanking the AP site, so that DCPC can effectively bind to the stem. The binding of DCPC is accompanied by a significant enhancement of its fluorescence. This new sensing method without an antisense DNA strand was demonstrated by using l-Arm and its aptamer as a model. It was confirmed that the method can sensitively detect l-Arm with a detection limit of 2.1 μM. The proposed method was also applied to adenosine detection, where the reported sequence of an adenosine aptamer was slightly modified. The method based on an AP site-containing aptamer and an AP site-binding ligand was applicable to detection of a target in horse serum.

Highlights

► A strategy for fluorescence assay of l-Arm and adenosine was developed. ► It is based on the conformational adaptability of stem-loop aptamers. ► The emissive response DCPC was used for the selective detection of targets. ► The method was applicable to detection of a target in horse serum.

Introduction

Aptamers are short oligonucleotides that have high affinity and specificity toward their targets, such as metal ions, organic molecules, proteins, drugs, and whole cells (Herr et al., 2006, Mairal et al., 2008, Tombelli et al., 2005, Torres-Chavolla and Alocilja, 2009). The dissociation constants of aptamers toward their targets usually lie in the micromolar to nanomolar range which is comparable to the affinity of antibodies to antigens (Jayasena, 1999, Jenison et al., 1994). Since aptamers possess considerable advantages over antibodies and enzymes, such as adaptability to various targets, versatility in labeling, and chemical stability, they have become increasingly important molecular tools for bioassays, diagnostics, and therapeutics. In particular, aptamers often undergo significant conformational transitions upon target binding, and this feature has opened a wide variety of signal transduction systems in aptasensors including detection by optical absorption, fluorescence, electrochemistry, electro-chemiluminescence, surface plasmon resonance, etc. (Li et al., 2010, Liu et al., 2009, Mairal et al., 2008, Navani and Li, 2006, Wang et al., 2010, Willner and Zayats, 2007). Among these detection methods, fluorescence detection has been widely adopted because of its simplicity, sensitivity, and quick response.

Many aptamers have a stem-loop and/or an internal-loop structure and the loop moiety offers a binding cavity to encapsulate targets (Hermann and Patel, 2000, Patel, 1997). Various design principles for fluorescence signaling of recognition events have been proposed based on conformational transitions. Fluorophores (Jhaveri et al., 2000) or fluorescent nucleotide analogues (Katilius et al., 2006) labeled onto the loop moiety exhibited a fluorescence increase due to changes in the microenvironment of the signaling molecules upon target-binding to the loop moiety. Chemical reactivity of an amino group substituted into 2′-ribose of cytidine was used for tethering fluorophores and the target-binding event was detected by the increase in the fluorescence intensity (Merino and Weeks, 2003, Merino and Weeks, 2005). One pair of a fluorophore and a quencher respectively labeled onto 5′- and 3′-ends exhibited fluorescence quenching upon target-binding due to the structural change from a single-stranded structure to the stem-loop structure (Ozaki et al., 2006). The increased fluorescence anisotropy of a fluorophore labeled onto one end of the aptamer sequence was also utilized as a detection method based on the formation of a stable target-binding structure (Ruta et al., 2009).

Related to the interactions of small molecules with DNAs, a series of aromatic fluorescent ligands that can selectively bind to a nucleobase opposite an abasic site (AP site) in DNA duplexes or opposite a gap in a ternary duplex have been reported (Dai et al., 2006, Ihara et al., 2009, Rajendar et al., 2008, Sankaran et al., 2006, Sato et al., 2009, Ye et al., 2008, Yoshimoto et al., 2003a, Yoshimoto et al., 2003b, Zhao et al., 2006). These ligands can recognize target nucleobases selectively by forming a pseudo-base pairing along the Watson-Crick edge of the intrahelical target nucleobases. Typing of single nucleotide polymorphisms has been successfully demonstrated using cytosine-selective naphthyridine derivatives (Sato et al., 2009, Yoshimoto et al., 2003a), guanine-selective pterin derivatives (Dai et al., 2006, Yoshimoto et al., 2003b), thymine-selective riboflavin (Sankaran et al., 2006) and amiloride (Zhao et al., 2006), and adenine-selective alloxazine (Rajendar et al., 2008) and lumazine (Ye et al., 2008).

Based on base-selective gap- or AP site-binding fluorescent ligands, aptasensors for adenosine or metal ions have been developed recently (Xiang et al., 2009, Xiang et al., 2010, Xu et al., 2009). However, the reported aptasensors based on an AP site required the presence of an antisense DNA strand containing an AP site to obtain fluorescence signaling of AP site-binding ligands. Binding of targets to aptamers caused the structure-switching from an aptamer/DNA linear duplex to an aptamer/target complex accompanied by releasing fluorescent ligands from the AP site of the aptamer/DNA duplex, resulting in the emissive response. Herein, we report on new simple aptasensors that do not require antisense DNA strands and the fluorescence signaling is based on the conformational adaptability of aptamers upon binding with a target. The aptasensors are comprised of an engineered aptamer and a non-covalently labeled fluorescent ligand. Although such target-induced adaptive transitions have been applied for biosensing systems (Jhaveri et al., 2000, Katilius et al., 2006, Merino and Weeks, 2003, Merino and Weeks, 2005, Ozaki et al., 2006, Ruta et al., 2009), it is usually necessary to attach fluorophores to the aptamers for fluorescence detection. In contrast, our method does not require covalent labeling of signal transduction units to aptamers. The present label-free aptasensors do not require the antisense DNA strand and emissive response can be obtained by choosing a suitable combination of a fluorescent ligand, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), and thymine bases flanking/opposite the AP site with enough response to detect targets in horse serum.

As shown in Scheme 1A, an AP site is situated within the stem and close to the loop in a stem-loop structure as a binding pocket for a fluorescent ligand. In the absence of an aptamer target, the loop is disordered, resulting in unpairing of bases around the AP site. The binding of a target to the aptamer induces conformational transitions in the loop structure, and then Watson-Crick and non-Watson-Crick base pairs are formed, which facilitates the stacking interactions in the stem part. The enhanced rigidity of the stem part around the AP site allows a ligand to bind to the AP site effectively with fluorescent signaling. In the reported aptasensors comprised of an aptamer, an antisense DNA strand containing an AP site, and an AP site-binding fluorescent ligand (Xiang et al., 2009, Xu et al., 2009), emissive response was obtained by releasing a ligand quenched at the AP site to a solution phase caused by the structure-switching from an aptamer/DNA duplex to an aptamer/target complex upon addition of the target. In the present study, signaling ligands are required to show enhanced fluorescence at the AP site upon addition of a target. Accordingly, DCPC was chosen as a signaling ligand, because DCPC shows emissive response at the AP site when the base opposite the AP site is thymine and bases flanking the AP site are thymine (Zhao et al., 2008). The signaling strategy based on the enhanced rigidity of the stem around the AP site was first demonstrated by using l-argininamide (l-Arm) and its aptamer as a model. The effects of salt concentration and the AP site position on the fluorescence signaling were investigated to obtain a sensitive response. The proposed method was also applied to the adenosine detection using modified adenosine aptamers with a stable stem-loop structure.

Section snippets

Reagents

DCPC was purchased from Aldrich Chemical Co. (Milwaukee, WI). l-Argininamide dihydrochloride and l-lysine were purchased from Sigma–Aldrich (Milwaukee, WI, USA). Adenosine was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). The other reagents were commercially available analytical grade and used without further purification. All of the DNA samples were custom synthesized and HPLC purified (>97%) by Nihon Gene Research Laboratories Inc. (Sendai, Japan). The concentrations of

Detection principle and l-Arm aptasensor

Many aptamers that bind small, organic targets such as l-Arm (Bishop et al., 2007, Harada and Frankel, 1995, Lin and Patel, 1996, Lin et al., 1998, Robertson et al., 2000), flavin mononucleotide (Fan et al., 1996), saccharide (Jiang et al., 1999, Wallis et al., 1995), and cocaine (Stojanovic et al., 2001), have been reported to contain a stem-loop region in their three-dimensional structures. Here we chose an l-Arm aptamer (Bishop et al., 2007, Harada and Frankel, 1995, Lin and Patel, 1996, Lin

Conclusions

A novel strategy for fluorescence assay of l-Arm and adenosine was developed based on the adaptive conformational transitions of stem-loop aptamers in combination with an AP site in the stem and an AP site-binding fluorescent ligand. Target-binding to stem-loop aptamers containing an AP site induces a tight structure around the AP site, which facilitates the base-pairing and the enhanced stacking interaction at the stem. The emissive response of a fluorescent ligand of DCPC can be used for the

Acknowledgements

This work was partially supported by Grants-in-Aid for Scientific Research (No. 17205009) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Z. Xu would like to acknowledge the financial support for young researchers provided from the Graduate School of Science, Tohoku University.

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