In 1995, scientists first discovered an adenosine aptamer with a length of 27 nt. So far, this aptamer has been widely used in biophysical research and the development of biosensors. NMR results show that the aptamer has two identical binding sites, while most small molecule aptamers only have one binding site. Adjusting the number of binding sites is of great significance in affecting the sensitivity of the biosensor. Therefore, Liu et al. hope to experimentally explore whether the two adenosine binding sites are independent and cooperative, and measure the thermodynamic parameters of the binding process through isothermal titration calorimetry ITC to improve the detection performance of the biosensor.

The secondary structure of the initially selected adenosine aptamer is shown in Figure 1A, named Apt2a. It can bind to two adenosine molecules, and the two binding sites are "Site1" and "Site2" respectively. According to its NMR results, each adenosine can interact with two nearby nucleotides through hydrogen bonds and overlap with guanine through reverse mismatching, and these two sites have the same binding effect. Subsequently, the research team used isothermal titration calorimetry ITC to characterize the thermodynamic properties of its binding process. Using this label-free technology, the Kd value of the wild-type aptamer was found to be 16.4 μM, and each aptamer was able to bind 2.1±0.2 adenosine molecules (Figure 2A).

Figure 1 Schematic diagram of secondary structures and binding sites of wild-type and mutant aptamers.

Figure 2 Relevant thermodynamic parameters and specific characterization of wild-type and mutant aptamers

After confirming the binding thermodynamic properties, the research team wanted to verify whether one of the binding sites could be removed while the binding affinity and specificity of the other site were still retained. Therefore, a G5T mutant was introduced in the study, in which a C base was inserted to pair with G22, resulting in an increase of two base pairs. As shown in Figure 1B, this mutant is called Apt1a, and it is speculated that the base adjustment in it will eliminate the presence of binding site 1. The ITC results showed that the Kd value of mutant Apt1a was 12.0 μM, and each aptamer was able to bind 1.1±0.1 adenosine molecules, which was in line with the expected design. Both wild-type and mutant aptamers bind to the target with specificity, and there is no thermal response when adding cytidine or guanosine (Figure 2A and B, red and blue lines). Therefore, the research team successfully eliminated adenosine binding site 1 and retained binding site 2, experimentally proving for the first time that the classic adenosine wild-type aptamer can be transformed into a single-site aptamer.

Subsequently, the research team experimentally verified the thermodynamic equivalence of the two binding sites. They used the same approach to design Apt1b, retaining adenosine binding site 1 and blocking binding site 2 (Figure 1C). The Kd value was determined to be 14.1 μM through ITC experiments, and each aptamer was able to bind 0.8±0.2 adenosine molecules, proving that the two binding sites have similar binding affinity. Although there are certain differences in the entropy and enthalpy of the binding reaction, the two binding sites are basically equivalent.

In the above experiments, the research team blocked the response binding site by introducing new base pairs and replacing base pairs, but the stable structure of the blocked binding site was still retained. Next, the research team tried to destroy the stable structure of its binding site by deleting specific base pairs, thereby removing the binding site. For example, they deleted the first three base pairs of the wild-type Apt2a aptamer, which also disrupted the target-binding ability of the Apt2b mutant (Figure 1D and Figure 2D). This experiment demonstrates that disrupting the structure of one junction site causes the second binding site to also be blocked, even though the second binding site is structurally intact. Therefore, for wild-type aptamers, any binding site can bind adenosine first, and the binding of any site can also stabilize the other site. Due to similar Kd values, it is unlikely that the two binding sites have a preference in binding order. In addition, the research team optimized the aptamer concentration and reaction temperature through ITC results. The optimal conditions for the reaction were 10 μM aptamer binding at 10°C (Figure 3).

Figure 3 Temperature optimization

In previous studies, this adenosine aptamer has been widely used in the development of biosensors. However, through the above studies, one binding site can be eliminated and shorter aptamers can be used for biosensing. For example, although the wild-type aptamer cannot be truncated, three base pairs can be deleted from the Apt1a mutant to generate Apt1c (Figure 1E), and ITC results indicate that the aptamer still retains the binding effect of the corresponding region (Figure 2E ), have similar Kd values ​​and bind only one adenosine. This mutant has only 21 nt compared to the 27 nt wild-type aptamer.

In addition, as shown in Figure 4, by directly fitting the above ITC data, it was found that the Hill coefficients of the three mutants were approximately 1, while that of the wild type was approximately 1.2. Therefore, it is shown that the two binding sites have a very weak cooperative relationship. In addition, the structures of Apt3 and Apt4 mutants are shown in Figure 1F-G. The Hill coefficients of the two are close to 2. It is speculated that the proximity of the two sets of binding sites may lead to enhanced cooperativity.

Figure 4 Relevant thermodynamic parameters of wild-type and mutant aptamers

Biosensors built using multi-binding site aptamers have low sensitivity when analyzing low concentrations. Therefore, this study is the first attempt to use single-binding site adenosine aptamers for biosensors to build a more sensitive biosensor by reducing the number of binding sites. As shown in Figure 5A, the Apt2a and Apt1a aptamer sequences were extended respectively, and the extended sequence was hybridized with a fluorescent group-labeled fragment (F-DNA). Part of the extended sequence and part of the aptamer sequence were hybridized with a quenching group-labeled fragment (F-DNA). Q-DNA) hybridization. In the presence of target adenosine, the nucleic acid aptamer folds and releases the quenching chain, resulting in enhanced fluorescence, thereby achieving quantitative detection.

Figure 5 Schematic diagram of biosensor principle

As shown in Figure 6A-B, the fluorescence intensity reached the maximum value 30 minutes after adding the target, and the adenosine concentration-maximum fluorescence intensity function graph drawn from the results is shown in Figure 6C. In the presence of low concentrations of adenosine, there was a roughly linear relationship between target concentration and fluorescence intensity (Figure 6D). The slope of the curve of the Apt1a mutant was 3.8 times that of wild-type Apt2a, and both groups showed good target specificity (Figure 6E-F). The biosensor constructed based on mutant Apt1a has a detection limit of adenosine of 9.1 μM.

Figure 6 Biosensor performance characterization

In summary, this study used reasonable sequence design to remove each binding site of the adenosine aptamer individually, and verified that each group has similar binding affinity and specificity through isothermal titration calorimetry ITC, and discussed its biochemical significance. Additionally, the number of target binding sites can be increased, with up to four sites introduced in a single DNA sequence. Moreover, different aptamer sequences can also be used to assemble fluorescent biosensors. At lower adenosine concentrations, the sensitivity of the single-site aptamer increased 3.8-fold, with a detection limit of 9.1 μM adenosine. This work provides a solution for studying the relationship between the number of aptamer binding sites and detection sensitivity, and also has great reference significance for the field of biosensors.

Comments:

1. This article modified the classic adenosine aptamer, explored the independence and cooperation between different binding sites of the aptamer, and realized the optimization and tailoring of the aptamer, which is of great innovative significance;

2. Through reasonable sequence design, this paper uses the modified aptamer to construct a fluorescent biosensor. At a lower adenosine concentration, the sensitivity of the single-site aptamer is increased by 3.8 times, and the detection limit is 9.1 μM;

3. This article has innovative concepts, clear ideas, complete characterization, and combines thermodynamics with aptamer research to enrich related characterization methods.