New insights into a classic aptamer: binding sites, cooperativity and more sensitive adenosine detection

Abstract

The DNA aptamer for adenosine (also for AMP and ATP) is a highly conserved sequence that has recurred in a few selections. It it a widely used model aptamer for biosensor development, and its nuclear magnetic resonance structure shows that each aptamer binds two AMP molecules. In this work, each binding site was individually removed by rational sequence design, while the remaining site still retained a similar binding affinity and specificity as confirmed by isothermal titration calorimetry. The thermodynamic parameters of binding are presented, and its biochemical implications are discussed. The number of binding sites can also be increased, and up to four sites are introduced in a single DNA sequence. Finally, the different sequences are made into fluorescent biosensors based on the structure-switching signaling aptamer design. The one-site aptamer has 3.8-fold higher sensitivity at lower adenosine concentration with a limit of detection of 9.1 μM adenosine, but weaker fluorescence signal at higher adenosine concentrations, consistent with a moderate cooperativity in the original aptamer. This work has offered insights into a classic aptamer for the relationship between the number of binding sites and sensitivity, and a shorter aptamer for improved biosensor design.

Figure 1.

The secondary structures of (A) the wild-type adenosine aptamers (Apt2a), and the mutants (B–G). The wild-type aptamer has two binding sites (1 and 2). (B) Site 1 is removed; (C) site 2 removed; (D) three base pairs deleted from the wild-type; (E) three base pairs deleted from Apt1a. Extended aptamers with (F) 3 and (G) 4 adenosine binding sites. The red color ‘A’ represents the bound adenosine. The nucleotides in pink color are the mutated to remove adenosine binding sites. The number in the name of each sequence indicates the number of adenosine binding sites.

Figure 2.

ITC traces and integrated heat of (A) the wild-type aptamer Atp2a, and (B) the Apt1a and (C) Apt1b mutants binding adenosine, cytidine and guanosine. ITC titrations of the shortened aptamers (D) Apt2b and (E) Apt1c binding adenosine.

Figure 3.

ITC trace and integrated heat of 10 μM of wild-type aptamer (Apt2a) binding to adenosine at different temperatures: (A) 20°C, (B) 15°C and (C) 10°C. Related thermodynamic parameters are shown in Supplementary Table S1. (D) The enthalpy changes (ΔH) of 10 μM of Apt2a and Apt1a binding adenosine (0.5 mM) as a function of temperature. ΔC_p (unit: kcal mol⁻¹ K⁻¹) was obtained from the linearly fitted slope (ΔH/ΔT).

Figure 4.

ITC traces and integrated heat of the engineered aptamers with (A) 3 and (B) 4 adenosine binding sites. The thermodynamic constants are listed in Table 1. (C) The total enthalpy change (ΔH) of different aptamers as a function of added adenosine. (D) The Hill coefficients, h, fitted from (C) using Equation (1); h > 1 represents positively cooperative and h = 1 represents non-cooperative behavior between binding sites.

Figure 5.

(A) A scheme of structure-switching signal aptamer. Binding of adenosine releases the quencher-labeled fragment and produces enhanced fluorescence. The scheme describes the wild-type aptamer binding two adenosine molecules. For the Apt1a mutant, only one adenosine is bound. (B) The DNA sequences of the two sensors tested.

Figure 6.

Biosensor performance. The sensor fluorescence kinetics with (A) the wild-type Apt2a aptamer and (B) the one site mutant Apt1a as a function of adenosine concentration (0.01–5 mM). The fluorescence intensity of the two sensors after 30 min as a function of (C) high (0.3–5 mM) and (D) low (10–100 μM) adenosine concentration. The fluorescence kinetics of (E) the Apt2a aptamer and (F) the Apt1a aptamer contained sensors after adding other nucleosides: guanosine (G), cytidine (C) and thymidine (T) (5 mM) with the background fluorescence subtracted.