Supporting Information. A general chemiluminescence strategy. for measuring aptamer-target binding and target concentration

Transcription

1 Supporting Information A general chemiluminescence strategy for measuring aptamer-target binding and target concentration Shiyuan Li, Duyu Chen, Qingtong Zhou, Wei Wang, Lingfeng Gao, Jie Jiang, Haojun Liang, Yangzhong Liu, Gaolin Liang and Hua Cui* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui , P. R. China. Corresponding author: Prof. H. Cui Tel: Fax: hcui@ustc.edu.cn S-1

2 Supporting information: S1. Optimization of CL detection conditions Several parameters influencing the CL intensity were investigated in order to establish optimal conditions for the detection of ATP using the proposed CL strategy. First, the effect of the concentration of biotin-f on the CL intensity was studied. As shown in Figure S1a, the CL intensity increased with the concentration of biotin-f. However, the increasing rate of the CL intensity started to slow down after the concentration of biotin-f reached 0.1 µm. Hence, 0.1 µm biotin-f was used in subsequent experiments. The effect of the ph of H 2 O 2 solution on the CL intensity was also examined. As shown in Figure S1b, when ph values range from 10.0 to 14.0 in H 2 O 2 solution, the CL intensity increased. However, ph 14.0 caused H 2 O 2 to strongly decompose, leading to poor reproducibility. Therefore, ph 13.0 was chosen as the optimum ph condition for the CL detection. The CL intensity of this system was also dependent on the concentration of H 2 O 2. As shown in Figure S1c, increased concentration of H 2 O 2 solution led to a great increase in CL intensity. However, the reproducibility of the CL signals became worse. In consideration of sensitivity, good stability and consumption of reagents, 0.15 M H 2 O 2 was chosen for the following experiments. Another factor affecting the sensitivity of the test is the incubation time in the presence of ABEI-Au colloid. As shown in Figure S1d, it was found that the signal rose sharply when incubation time increased from 0 to 10 min. The slope remained nearly the same after 20 min. As a result, 30 min was selected as incubation time for the following studies. S2. Binding fraction of 14-mer aptamer fragment for ATP The relationship between CL intensity and ATP concentration is shown in Figure S2. The CL intensity increased rapidly with lower ATP concentration. It increased slowly when ATP S-2

3 concentration was over 0.1 µm, but reached saturation when ATP concentration was over 1 µm. In order to estimate the binding fraction of 14-mer aptamer fragment for ATP, we assumed that the interaction between ATP and 14-mer aptamer was strong enough to capture most of the ATP added into the system. The amount of the forming complexes was approximately equal to the amount of the ATP added into the system. Based on the assumption, when the CL intensity reached saturation after 1 µm, all of the 14-mer aptamer fragments had bound with ATP (see Figure S2). Thus, the amount of the 14-mer aptamer fragments was approximately equal to 1 µm, and the plot of the fraction of 14-mer aptamer fragment and ATP binding as functions of ATP concentration is also shown in Figure S2 (inset). The estimate of the binding fraction of 14- mer aptamer fragment for ATP indicated that the binding fraction below 0.03 µm ATP was less than 3.2% and could thus be ignored. The curve from 0.1 to 1 µm was applied to measure binding constant. S3. Molecular Dynamic simulation of ATP-14mer aptamer fragment complex In order to investigate the binding mechanism of ATP and aptamer, Molecular Dynamic (MD) simulation was performed. Figure 5a-c represented the final binding conformation of ATP binding14-mer aptamer complex. Two regions of 14-mer fragment aptamer helped form the binding pocket that buried ATP: multi-g region consisting of consecutive G6-A10 residues and 5 -terminal residues A1. The center of the ATP-binding sites was characterized by ATP G9 recognition mismatch flanked by sheared A1 A10 and normal Watson-Crick C3 G8 pair. In order to investigate the conformational changes during the binding process, root meansquare deviation (RMSD) and radius of gyration (Rg) were calculated with respect to the initial structure to further analyze the simulation data (Figure S3). As suggested by the two platforms (10-30 ns and ns) of RMSD and Rg results, the binding process involved two S-3

4 conformational changes. The first one occurred when the ATP molecule approached the aptamer fragment, and the second one occurred when the aptamer fragment folded and formed a complex with the ATP molecule. To better understand the conformational changes during the binding process, a contact map, a binary two-dimensional matrix showing the distance between all possible residue-residue pairs, was also calculated from simulation results (Figure S4). By combining the contact maps averaged over two platform periods with snapshots collected at 15 ns and 68 ns, we found significant differences between the two conformations. During the first platform period, the 14- mer aptamer fragment was in its semi-expanded state, and the multi-g zone of the aptamer could form 2.75 hydrogen bonds with the ATP molecule, mainly through its phosphate group and adenosine. G9 residue was at the center of the binding zone and formed 0.46 hydrogen bonds with ATP. At the same time, the tail of the 14-mer aptamer fragment could also interact with the ATP molecule. During the second platform period, the 14-mer aptamer fragment folded and formed a binding pocket which consisted of the multi-g zone around G9 and tail residues such as A1 and C2. The ATP molecule was buried into this binding pocket with several G-A mismatches formed between ATP and G9, and three hydrogen bonds formed between the phosphate group of ATP and G6. By comparing our ATP-14-mer aptamer complex with the ATP-27-mer aptamer complex reported previously, we found an interesting peak shift of donor-acceptor distance distribution of hydrogen bonds between these two complexes (Figure S5). The average donoracceptor distance was 3.00 Å for the amino proton of G9 and imino proton of ATP pair (ATP N1-N2 G9) and 3.07 Å for the imino proton of G9 and amino proton of ATP pair (ATP N6-N3 G9) in the complex formed by ATP and 14-mer aptamer. In the case of 27-mer DNA aptamer, S-4

5 these two donor-acceptor pairs had distances of 3.06 Å and 2.82 Å, correspondingly. The peak shifts revealed that N1 of ATP retreated farther from N2 of G9, while N6 of ATP came closer to N3 of G9 in the complex formed by ATP and 14-mer aptamer. We also found that 1) the ATP G9 recognition mismatch was not as coplanar as that observed in the 27-mer DNA aptamer and 2) the adenosine of ATP was warped as a result of a less desirable binding platform and binding pocket. The specific hydrogen bonds formed between the aptamer and its target are responsible for the aptamer s high selectivity and affinity, including the 14-mer aptamer fragment. When the aptamer fragment folded and formed a complex with the ATP molecule, the average number of hydrogen bonds (averaged over time) formed between ATP and the 14-mer aptamer fragment had increased by 1.37, as shown in Figure S6. G7 contributed 1.83 out of the total 4.12 hydrogen bonds. This demonstrated the central role of the G9 in the ATP-binding process. In order to explore the driving force of the specific binding process, the time evolution of solvent accessible surface area (SASA) was studied, as shown in Figure S7. It was found that the hydrophilic SASA and the hydrophobic SASA stabilized during the first stage of the binding process, and then upon the folding of the 14-mer aptamer fragment, the areas of hydrophilic SASA and hydrophobic SASA slightly increased from to nm 2 and from 9.28 to 9.34 nm 2, respectively. The total SASA increased by only 4%, implying that the folding of the 14-mer aptamer fragment is not driven by hydrophobic force. According to the MM-PBSA method, [6] three energy terms constitute the total binding free energy: solvation contribution, entropy contribution and internal interaction energy of receptor and ligand. As the 14-mer aptamer fragment folds and binds the ATP molecule tightly, it is easy to see that the complex will lose the freedom of translation and rotation, leading to an unfavorable entropy contribution. Therefore, an S-5

6 assumption was made that the energy term driving the binding process should be the interaction energy term. Thus, focus was placed on the interaction between each aptamer residue and ATP molecule in order to understand the role of each individual residue in the selective binding process. Figure S8 showed the time evolution of interaction energy between ATP and the 14-mer aptamer fragment. The van der Waals energy between ATP and the 14-mer aptamer fragment was different during the two stages of the binding process. At the first stage, the Coulomb interaction fluctuated around kj/mol, while the van der Waals interaction remained stable at around kj/mol. Once the 14-mer aptamer fragment folded, we found that Coulomb interaction was lowered by kj/mol, while van der Waals interaction increased slightly by kj/mol. It can be clearly seen that the Coulomb interaction drives the binding process. The unfavorable change of van der Waals energy could be explained by the decrease of binding interface surface area and the exposure of more atoms to solvent as the binding pocket formed, resulting in the increase of SASA. Furthermore, the detailed interaction histograms incorporating the interactions between each individual residue and ATP during the two stages of the binding process were plotted (Figure S9). At the first stage, G7 had the most positive Coulomb interaction of kj/mol with ATP, while A10 had the most negative energy of kj/mol, which was the most favorable one. The other contributions came from G6 ( kj/mol), G8 ( kj/mol) and G9 ( kj/mol). The van der Waals energy showed a different distribution: multi-g region contributed most, including G6 ( kj/mol), G7 ( kj/mol), A10 ( kj/mol), G9 ( kj/mol), and G8 (-11.85kJ/mol). This may have resulted from the highly charged phosphate group of ATP, which prefers to interact with the backbones of G7 and G5, and the adenine base of ATP, which forms the ATP G9 mismatch with G9. S-6

7 The final stage of the binding process exhibited significantly different distributions. The tail base of A1 gained kj/mol van der Waals interaction energy with ATP. Meanwhile, the multi-g region folded around the ATP molecule, with van der Waals energy contribution from G8 ( kj/mol). The Coulomb energy contribution mainly came from G6 ( kj/mol) and G9 ( kj/mol), while the contribution from G7 decreased to kj/mol. Such a strong Coulomb interaction between G6 and ATP may have resulted from the highly charged phosphate group of ATP and guanine of G6. Furthermore, when the 14-mer aptamer fragment folded, A10 was far from the ATP molecule, which made its contribution negligible. Thus, in the final binding conformation, ATP mainly interacts with A1, G6, G7, G8 and G9. S-7

8 Supplementary Figures and Tables Figure S1 Effects of the reactant conditions on the CL measurement. (a), Effect of biothin- F concentration. (b), Effect of ph of H 2 O 2. (c), Effect of H 2 O 2 concentration. (d), Effect of incubation time. Reaction condition: 0.1 µm ATP. S-8

9 Figure S2 Relationship between CL intensity and ATP concentration. Inset: Plot of the fraction of 14-mer aptamer fragment and ATP binding as functions of ATP concentration. Reaction condition: 100 µl ph M H 2 O 2. S-9

10 Figure S3 RMSD and Rg analysis of MD simulation results. Both the radius of gyration (Rg) of 14-mer aptamer fragment (red) and root mean square derivations (RMSD) of complex (blue) were calculated with respect to the initial structure. S-10

11 Figure S4 Schematic representation of the binding conformation and contact maps analysis of ATP-14mer aptamer fragment complex. a, Conformation of the complex at 15 ns. b, Conformation of the complex at 68 ns. ATP molecule (Residue index 15) acquired more connection with 14-mer aptamer fragment, from the first platform period (10-20 ns) which contact by G6, G7, G8, G9, A10 to the second platform period (60-70 ns). During the first platform period the aptamer interacted with ATP through G6, G7, G8, G9, and A10, and during the second platform period, additional interaction was established between A1 and ATP, accompanied by folding of the 14-mer aptamer fragment. S-11

12 Figure S5 Distribution of donor-acceptor distances. Two hydrogen bonds that formed within the ATP G9 mismatch were used for analysis. The dashed lines show average distances of these two hydrogen bonds in the AMP-27-mer aptamer complex, respectively. S-12

13 Figure S6 Number of hydrogen bonds (H-Bonds) averaged over time. The numbers of H- Bonds between ATP and 14-mer aptamer fragment (blue curve) and between ATP and G9 of 14- mer aptamer fragment (red curve) were averaged every 20 ps. S-13

14 Figure S7 Time evolution of solvent accessible surface area (SASA). The area of 14-mer aptamer fragment exposed to solvent and the individual contributions of hydrophilic term and hydrophilic term are shown. Black curve, total; red curve, hydrophilic term; blue curve, hydrophobic term. S-14

15 Figure S8 Time evolution of the interaction energy. The short range coulomb interaction between ATP and 14-mer aptamer fragment is shown in red and the van der Waals interaction is shown in blue. S-15

16 Figure S9 Histogram of interaction energy between ATP and each residue of the 14-mer aptamer fragment. The van der Waals interaction is shown in black, and the short range Coulomb interaction is shown in red. Top: Data collected during the first binding stage (from 10 to 20 ns). Bottom: Data collected during the final binding stage (from 60 ns to 70 ns). S-16

17 Table S1. Sequences of aptamers used in this study Sequence biotin-f G9 G8 G7 G6 A1 C3 A10 S 14-mer fragment V-apt TBA 5 -biotin-acctgggggagtat-3 5 -biotin-acctggggtagtat-3 5 -biotin-acctgggtgagtat-3 5 -biotin-acctggtggagtat-3 5 -biotin-acctgtgggagtat-3 5 -biotin-ccctgggggagtat-3 5 -biotin-acttgggggagtat-3 5 -biotin-acctgggggcgtat-3 5 -biotin-tggcgtggacacgaagatcag-3 5 -ACCTGGGGGAGTAT-3 5 -biotin-tcacgtgcatgatagacggcgaagccgt CGAGTTGCTGTGTGCCGATGCACGTGA-3 5 -biotin-ggttggtgtggttgg-3 S-17

18 Table S2. The dissociation constants of original and mutated aptamers Dissociation constant This strategy Reference method biotin-f 0.65 µm - G9 - - G µm - G µm - G µm - A µm - C µm - A µm - AVP 0.90 µm 1.1 µm [7] (PCR-radio labeled assay) TB 66 nm nm [8] (PCR-chromogenic substrate assay) S-18

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