Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

Abstract

a b s t r a c tWe previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMRlabeled aptamers enabled direct FA sensing ATP with a detection limit of 1 mM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets.

Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer

“…The linear detection range with FA measurement was from 0.5 μM to 20 μM (y = 0.220-0.004x, R 2 = 0.990; y is FA value). The sensitivity of this competitive aptamer FA assay is comparable to those of aptamer FA assays [10,13,15,[29][30][31], and it is also comparable to that of some other aptamer-based assays for ATP with different detection methods [21,32,33]. The generated maximum FA/FP change in our method is much higher than that of some aptamer FA assays [15,[29][30][31].…”

“…In presence of 5 μM AD25, with addition of MgCl 2 , γ-TMR-ATP showed increasing FA until the concentration of MgCl 2 exceeded to 50 mM. These results show that MgCl 2 significantly affects the FA increase of γ-TMR-ATP caused by affinity binding of AD25 [13,24]. In buffer containing NaCl, no remarkable FA change of γ-TMR-ATP was observed in presence of 5 μM AD25.…”

“…Y. Li et al Talanta 174 (2017) [7][8][9][10][11][12][13] in complex sample matrix, the aptamer-based competitive FA assay was used to detect ATP spiked in diluted human urine or in diluted human serum sample ( When ATP spiked in 100-fold-diluted human serum sample was analyzed by the aptamer competitive FA assays, the initial FA signal in absence of ATP increased in the diluted serum sample solution due to possible nonspecific binding between aptamer and some proteins in serum sample or the binding between dye-labeled ATP and some proteins in serum sample. The addition of ATP in diluted serum sample still caused remarkable FA decrease with the linear detection range from 0.5 μM to 20 μM (y = 0.243-0.004x, R 2 = 0.986; y is FA value), suggesting the assay is applicable for ATP spiked in the diluted serum sample.…”

“…Many efforts have been made to develop aptamer-based FA assays for small molecules taking advantage of aptamer and FA [9][10][11][12][13][14]. Direct FA assays for small molecules using dye-labeled aptamers usually rely on the binding-induced structure change [11] or change of interaction between labeled dye and bases of aptamer [12,13]. To increase change in molecular size during affinity binding for FA signal generation and amplification, large biomolecules (e.g., proteins and oligonucleotides) or nanomaterials (e.g., silica nanoparticles and graphene) have been used in indirect aptamer-based FA assays for small molecules [9,10,[15][16][17][18].…”

Fluorescence anisotropy (polarization): from drug screening to precision medicine

“…82 Reflecting the high potential for aptamers as therapeutics, several anisotropy assays for aptamer screening have been developed. 83–85 One such approach is schematically illustrated in Figure 4.…”