3.1 Analysis and shortening the aptamer
The length of aptamers screened by traditional SELEX technology was generally longer about 60 bases. some of these bases did not play a role in the binding to target but affected the affinity of the aptamer(Jia M et al,2020). In this study, when simulated with software MFold based on energy minimization principle, We have obtained a secondary structure that requires the least energy to form, that is, the easiest to form under target induction. The secondary structure of the SEQ.3 aptamer consisted of four parts: two central loops (A and B), a stem connecting the loops and a non-connecting stem (Figure.1A)(Yan Z et al,2019). It was obtained by software, base energy when binding to the target which from low to high was labelled as purple, blue, green, yellow, orange and red showed that loop A was the main binding domain according to the principle that the bases with higher binding energy were more likely to bind to the target(Kou Q et al, 2021). According to the reported cutting principle, the SEQ.3 aptamer was cut into two parts from the red dashed line in the stem, the part of the loop A with a length of 21 bases was named as SEQ.A, the other parts was combined at the cutting site and named as SEQ.B. Kd determination results (Figure.1B )showed that the Kd value of SEQ.A was only 132.26 nM, which was much lower than the initial 210.95 nM of SEQ.3, while the Kd of SEQ.B increased to 599.49 nM. This result was similar with that reported by Alsager et al, in which the affinity of 35-mer aptamer cut from an original length of the 75-mer aptamer based on structure simulation was increased by 2.5 times(Alsager.O. A, 2015). At the same time, this result showed that with the help of secondary structure simulation and base binding energy analysis, the key domains of aptamer recognition could be effectively predicted, which provided a basis for further mutation transformation to improve the recognition performance.
3.2 Aptamer structure mutation
Every base in the aptamer sequence played a certain role in the binding of the aptamer to the target. When the base pair changed, the affinity of the aptamer also changed due to the difference in chemical forces such as hydrogen bonds and van der Waals forces(Kaiser L et al,2018). We followed the principle of DNA mutation on the loop, and mutated in accordance with the principle of A-T base pair and G-C base pair replacement.The sequences of 7 new aptamers and their Kd values were listed in Table 1. Among these aptamers, except SEQ.A1 was mutated from the stem, the other 6 sequences were mutated in the loop (Figure.2A).
Table 1
The sequences and Kd of the aptamers obtained with base mutation
Number
|
Sequences (5’ to 3’)
|
Base mutation
|
Kd (nM)
|
SEQ.A
|
AGG GGG ACC CAT CAG GGG GCT
|
——
|
132.62
|
SEQ.A1
|
GGG GGG ACC CAT CAG GGG GCC
|
AT-GC
|
309.84
|
SEQ.A2
|
AAA GGG ACC CAT CAG GGG GCT
|
GG-AA
|
500.31
|
SEQ.A3
|
AGG GAA ACC CAT CAG GGG GCT
|
GG-AA
|
432.42
|
SEQ.A4
|
AGG GGG ATT CAT CAG GGG GCT
|
CC-TT
|
468.33
|
SEQ.A5
|
AGG GGG ACC CGC CAG GGG GCT
|
AT-GC
|
20.13
|
SEQ.A6
|
AGG GGG ACC CAT CGA GGG GCT
|
AG-GA
|
67.61
|
SEQ.A7
|
AGG GGG ACC CAT CAG GAA GCT
|
GG-AA
|
476.54
|
Something interesting was that when replaced CG with TA, including CC with TT (SEQ.A4) and GG with AA (SEQ.A2, SEQ.A3 and SEQ.A7) in the loop, the affinity of the new aptamer was reduced. But when replaced AT with GC bases (SEQ.A5), the affinity increased significantly (the Kd value decreased from 132.62 nM to 20.13 nM). Structure simulation showed that the secondary structure of SEQ.A5 hadn’t changed, but the base binding energy had changed obviously, the original low-energy A-T had been transformed into a high-energy G-C (Figure.2B). This indicated that the overall base binding energy of aptamer affected the affinity of the aptamer with the target(Liu X et al,2020). When the aptamer was combined with the target, a certain three-dimensional structure was formed by twisting and folding. In this process, the bases with high binding energy on the ring formed a bond-fixing structure, which overcame the flexibility of the aptamer and thus obtained better binding performance(Yang Q et al,2017).
At the same time, in order to ensure that other replacement methods didn’t produce better affinity and further determined the binding site, we had replaced a single base on the loop according to the replacement method of A-T base pair and G-C base pair replacement, thereby further verified our results. The Kd value was shown in Table 2 and the result was consistent with the double base substitution, when the base positions of SEQ.A5 and SEQ.A6 were substituted, the Kd value was the lowest.And, it provided a basis for us to determine the binding site later.
Table 2
The sequences and Kd of the aptamers obtained with single base mutation
Number
|
Sequences (5’ to 3’)
|
Base mutation
|
Kd (nM)
|
SEQ.A
|
AGG GGG ACC CAT CAG GGG GCT
|
——
|
132.62
|
1
|
AAG GGG ACC CAT CAG GGG GCT
|
G-A
|
654.30
|
2
|
AGA GGG ACC CAT CAG GGG GCT
|
G-A
|
633.29
|
3
|
AGG AGG ACC CAT CAG GGG GCT
|
G-A
|
588.31
|
4
|
AGG GAG ACC CAT CAG GGG GCT
|
G-A
|
571.69
|
5
|
AGG GGA ACC CAT CAG GGG GCT
|
G-A
|
537.24
|
6
|
AGG GGG GCC CAT CAG GGG GCT
|
A-G
|
503.93
|
7
|
AGG GGG ATC CAT CAG GGG GCT
|
C-T
|
498.32
|
8
|
AGG GGG ACT CAT CAG GGG GCT
|
C-T
|
501.55
|
9
|
AGG GGG ACC TAT CAG GGG GCT
|
C-T
|
422.58
|
10
|
AGG GGG ACC CGT CAG GGG GCT
|
A-G
|
87.22
|
11
|
AGG GGG ACC CAC CAG GGG GCT
|
T-C
|
64.03
|
12
|
AGG GGG ACC CAT TAG GGG GCT
|
C-T
|
102.13
|
13
|
AGG GGG ACC CAT CGG GGG GCT
|
A-G
|
98.42
|
14
|
AGG GGG ACC CAT CAA GGG GCT
|
G-A
|
111.58
|
15
|
AGG GGG ACC CAT CAG AGG GCT
|
G-A
|
506.33
|
16
|
AGG GGG ACC CAT CAG GAG GCT
|
G-A
|
532.66
|
17
|
AGG GGG ACC CAT CAG GGA GCT
|
G-A
|
511.34
|
18
|
AGG GGG ACC CAT CAG GGG ACT
|
G-A
|
601.09
|
19
|
AGG GGG ACC CAT CAG GGG GTT
|
C-T
|
611.78
|
3.3 Identification, Modification and Characterization of Aptamer
Autodock docking simulation has been used to determine the binding site of aptamer successfully(Zhou Y et al,2019), after site-directed mutation, the affinity of the aptamer was increased significantly, but the binding sites were not clear. When simulated with Autodock 4.2 software, it showed that the ENR was mainly combined with G11, C12 and G15 through the action of hydrogen bonds (Figure.3), so the five bases from G11 to G15 were identified as the binding sites. Among these bases, G11 and C12 were just the mutation sites in the site-directed mutation, which further proved the correctness of the site-directed mutation.
The five key bases (GCCAG) were cloned twice, three times and four times with TT base as the connecting arm, there new aptamers (SEQ.A52, SEQ.A52 and SEQ.A54) were obtained and their Kd values were determined.As showed in Table 3, the double cloned aptamer had the lowest Kd value, which was only11.07 nM. After triple cloning, the aptamer was 35-mer with the Kd value of 224.05 nM, which was similar to the that of the original SEQ.3. When cloned four times, the length of the poly-clonal was too long and the aptamers interfere with the formation of steric binding sites, resulting in a decrease in affinity. Structure simulation showed that the secondary structure of SEQ.A52 was different with the original SEQ.3, although it still had two central circles, but the stem length at the end of the SEQ.A52 circle was asymmetrical and the base difference between the two circles wasn’t as large as that of SEQ.3(Zhu Q et al,2017). It was speculated that after double cloning, the binding site changed from one to two, thereby binding more targets and improved the affinity.
Table 3
The sequences of the aptamers obtained with multiple clone
Number
|
Sequences (5’ to 3’)
|
Kd (nM)
|
SEQ.A5
|
AGG GGG ACC CGC CAG GGG GC
|
20.13
|
SEQ.A52
|
A GGG GGA CCC GCC AG TT GCC AGG GGGC
|
11.07
|
SEQ.A53
|
A GGG GGA CCC GCC AG TT GCC AGTT GCC AGG GGGC
|
224.05
|
SEQ.A54
|
A GGG GGA CCC GCC AG TT GCC AGTT GCC AGTT GCC AGG GGGCT
|
777.18
|
CD spectroscopy was used to determine the secondary structure changes of the aptamer during the combination with ENR(Okazawa A et al,2000). As showed in Fig. 5A, the original SEQ.3 presented the standard B-type aptamer absorption peaks formed of single stranded DNA, with the maximum and minimum absorption characteristic peaks at 280 nm and 245 nm respectively, which was consistent with the results reported by Nagatoishi et al(Nagatoishi, S et al,2007). After shortening, the characteristics of SEQ.A showed an obvious blue shift, the positive peak and negative peak absorption peaks were 270 nm and 240 nm, respectively. This might be due to the change in the length of the aptamer, resulting in the lack of the central circle of the B region in the secondary structure (Fig. 1A). As the site-directed mutant of SEQ.A, SEQ.A5 presented the similar characteristics to SEQ.A, but SEQ.A52 showed a significantly different spectrum. The negative peak couldn’t be found to shift to 235 nm and the positive peak at 270 nm was split into two peaks, a high peak at 260 nm and a low one at 290 nm, which belonged to the process intermediate from type B aptamer to type A aptamer(Zhang Y et al,2020). At the same time, the result was similar with the secondary structure simulation result, except that the secondary structure of SEQ.A52 was quite different from the secondary structure of SEQ.3, with two large and one small circles in the middle. It was precisely because of the difference in secondary structure that it had better binding properties.
In addition to the CD comparison of the four aptamers, ENR was also added to observe the changes in the secondary structure of the aptamers. after combination, the peak values of SEQ.3, SEQ.A and SEQ.A5 were decreased due to the interaction between the base stack and the spiral superstructure, the higher the affinity of aptamer, the more obvious the decrease of peak value, so SEQ.A5 had the most obvious effect because it had the highest affinity(Fig. 5D). To SEQ.A52, except the peak reduction, the positive peak at 260 nm also showed a split state, indicating that its binding to the target was different from other types(Ye B C et al,2008). This was also the reason for its high affinity with the target.
3.4 Sensitivity and specificity of the aptamer
The fluorescent detection method for ENR with aptamer was established based on the principle that the aptamers were adsorbed on the surface of AuNPs through electrostatic interaction and induced the quenching effect of AuNPs on fluorescence. When ENR wasn’t present, the aptamer modified with FAM was adsorbed on the surface of AuNPs and caused fluorescence quenching. When ENR was present, the aptamer competed from the AuNPs surface, the fluorescence value was restored and could be quantitative analyzed with the concentration of ENR(Song S H et al,2019)
The detection results with SEQ.3, SEQ.A, SEQ.A5 and SEQ.A52 were showed in Fig. 6, all the fluorescence values had a good linear correlation with the increase of ENR concentration. SEQ.A52 presented the highest sensitivity, in which the linear correlation was fitted as y = 0.2302x – 2.2942. The correlation coefficient was 0.9928 in low-concentration range of 50 pM to 1000 pM. The LOD was calculated as 41.35 pM by the concentration corresponding to the fluorescence value at three times standard deviation of 10 blank samples without ENR (Fig. 6A). This result was 100 times more sensitive than the fluorescence method established using SEQ.3 and the LOD was reduced by about 88 times (Fig. 6B). At the same time, the sensitivity has been greatly improved compared with the shortened aptamer (SEQ.A) and mutated aptamer(SEQ.A5) shown in Figure.6C and Figure.6D.
Specificity results (Figure.6E) showed that there were no significant changes between SEQ.A52 and SEQ.3 to the all the analogues except CIP and the cross-reaction rate was less than 15%. But to CIP, SEQ.A52 presented lower cross-reactivity than SEQ.3, the reason might be that the double cloning of the binding site to ENR in SEQ.A52 decreased the recognition to CIP. All the results indicated that with the optimization of the aptamer structure, reasonable shortening, rational modification through site-directed mutagenesis and the use of docking simulation to find the binding site while reasonably doubling could greatly improve the binding property of the aptamer(Tan D et al,2013).