Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides
Introduction
Aptamers are single stranded nucleic acid selected in evolutionary process binding to targets with good specificity and binding affinity (Citartan et al., 2012, Feng et al., 2014, Juskowiak, 2011, Kim and Gu, 2014, Li et al., 2015, Liu et al., 2009, McKeague and Derosa, 2012). Since the discovery of aptamers in 1990s, aptamer-based assays for small molecules have drawn increasing attentions in environmental sensing, food safety, and clinical analysis due to the unique features of aptamers, such as easy generation, facile labeling, good thermal stability, small size, and target-binding induced structure change (Citartan et al., 2012, Feng et al., 2014, Juskowiak, 2011, Kim and Gu, 2014, Li et al., 2015, Liu et al., 2009, McKeague and Derosa, 2012). Taking advantage of fluorescence anisotropy (FA) or fluorescence polarization (FP) in sensitivity, reproducibility, and simplicity (Lea and Simeonov, 2011, Gradinaru et al., 2010, Le et al., 2002, Smith and Eremin, 2008), the aptamer-based FA/FP sensors are attractive (Gokulrangan et al., 2005, Liu et al., 2009, Ruta et al., 2009, Zhang et al., 2011).
In spite of ease of analyzing proteins (Gokulrangan et al., 2005, Zhang et al., 2011), the dye-labeled aptamer based direct FA sensors remain challenging for small molecule analysis because the binding of small target only brings negligible mass change of aptamer. Under a rational design, aptamers with dye labeled on terminal ends can be used to direct detection of small molecules due to structure-switch induced FA alteration (Kidd et al., 2011, Perrier et al., 2010, Ruta et al., 2009), but the produced FA changes are usually mild. To improve FA/FP signals in small molecule detection, large biomolecules (e.g. proteins and oligonucleotides) or nanomaterials (e.g. gold nanoparticles, graphene, and silica nanoparticles) have been introduced in assay development by increasing the binding-induced molecular weight change (Cruz-Aguado and Penner, 2008, Cui et al., 2012, Huang et al., 2012, Liu et al., 2013, Ye and Yin, 2008, Yu et al., 2013, Zhu et al., 2011, Zhu et al., 2012).
Recently, we have reported a simple and noncompetitive FA strategy for a small molecule, ochratoxin A, by using a TMR-labeled aptamer. (Zhao et al., 2014) It is based on the target-binding induced change of intramolecular interactions between TMR and the guanine (G) bases of the aptamer. The interaction significantly affects local rotation of TMR and FA of TMR. In this FA strategy, TMR-labeling position of the aptamers plays a crucial role. The intramolecular interaction between TMR and G bases is distance dependent, and TMR labeling may influence the binding affinity of aptamer depending on the labeling sites (Zhao et al., 2014). An ideal labeling position allows TMR-labeled aptamer showing FA change upon target without loss in binding affinity. Previously, only terminal ends and T base of aptamers could be examined for TMR labeling in FA analysis of ochratoxin A (Zhao et al., 2014). However, G base in aptamers may not be surrounded by T bases or terminal ends and some aptamers may have less T bases. In addition, the binding affinity of aptamer may be disrupted by introducing TMR on those limited labeling sites. Therefore, lack of labeling single TMR on other labeling sites may cause failure and limitation of FA sensors using TMR-labeled aptamer. Achieving conjugation of a single TMR on other bases like A and C in aptamer will allow a full mapping of labeling positions of aptamers and overcome the limitation.
Herein, we successfully built a TMR-labeled aptamer FA sensor for adenosine triphosphate (ATP), an important compound in cell biology and biochemistry (Knowles, 1980), by comprehensive mapping the labeling sites of 5’ terminal, 3ʹ terminal, and the A, C, and T bases of aptamers. We introduced a single TMR dye on various labeling sites of a 25-mer anti-ATP aptamer (Huizenga and Szostak, 1995, Lin and Patel, 1997), and screened FA response of each aptamer probe among 14 TMR-labeled aptamers on ATP. The aptamer with TMR labeled on the 16th base C showed remarkable FA-decreasing response to ATP, while the aptamer with TMR labeled on the 23rd base A showed complementary and significant FA-increasing response to ATP. These aptamer probes enabled FA sensing ATP with detection limit of 1 µM and detection of ATP in diluted serum samples. The achievement of conjugating TMR on A, C, and T bases of aptamers can greatly facilitate the construction of FA sensor with TMR-labeled aptamer and expand the application of TMR-labeled aptamer FA sensor relying on the intramolecular interaction of TMR and G base to the analysis of a variety of targets.
Section snippets
Chemical and reagents
Adenosine triphosphate (ATP) was ordered from Sigma. All the DNA oligonucleotides were synthesized and purified by Takara Biotechnology (Dalian, China) or Sangon Biotech (Shanghai, China). Guanosine, thymidine, and cytidine were purchased from Amresco. Guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) were ordered from Takara Biotechnology (Dalian, China). The 25-mer DNA aptamer against ATP had the following sequence: 5ʹ-CCT GGG GGA GTA TTG CGG AGG AAG
Comprehensive screening FA responses of aptamers with TMR on various labeling sites
Fig.1 shows the principle for FA sensing of ATP with TMR-labeled aptamers. The 25-nt anti-ATP aptamer contains 13G bases, 5A bases, 3C bases, and 4T bases (Huizenga and Szostak, 1995, Lin and Patel, 1997). Each G base is surrounded by different bases, experiencing distinct environment. The anti-ATP aptamer can form a hairpin structure when the aptamer binds to ATP, bringing a conformation change (Lin and Patel, 1997). The binding of ATP possibly alters the interaction between TMR and G bases,
Conclusion
We achieved comprehensive mapping the labeling sites (A, C, T bases, and terminals) of the anti-ATP aptamer to construct fluorescence anisotropy sensors for ATP detection with TMR-labeled aptamers. The FA sensing was based on the ATP-binding induced change of intramolecular interaction between TMR and G bases of aptamers and alteration of FA of labeled TMR. By screening 14 labeling positions, favorable aptamer probes with remarkable FA-increase or FA-decrease response to ATP were obtained when
Acknowledgment
The work was supported from the National Natural Science Foundation of China (Grant no. 21222503, 21435008), Outstanding Youth Talents Program of Shanxi Province, and the Key Project of Chinese Ministry of Education (Grant no. 212020).
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