Part:BBa_K5321000

Thrombin_HD22_29mer

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In order to perform our proof of concept of our project, we choose thrombin as a model to mimic disease biomarkers, and its aptamers reported previously. Thrombin_HD22_29mer (DNA ligand 60-18[29]) is an aptamer originally characterized by Tasset et al. in 1997. It specifically targets the heparin-binding site of thrombin through hydrophobic interactions of the duplex.

Figure 1 shows the interaction of Thrombin_HD22_29mer with human thrombin.

Figure 1 | The interaction of the Thrombin_HD22_29mer with human thrombin. (a) the site of crosslinking between thrombin_HD22_29mer and thrombin. The crosslinked tryptic peptide of thrombin is shown in boldface letters, and the arrow denotes the crosslink between T12 within the G-quadruplex of the DNA and Phe245 of thrombin. (b) the ribbon diagram of human thrombin, shown in purple, interacted with Thrombin_HD22_29mer in blue.

Characterization

Electrophoretic mobility shift assay (EMSA)

An electrophoretic mobility shift assay (EMSA) is a common affinity electrophoresis technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence. In the present study, EMSA was employed for affinity test of the aptamers.

After thrombin and aptamers were diluted with proper buffer, reaction systems were built with a gradient of aptamers. 15-mer, 29-mer and 40-mer aptamers were tested, and a gradient of concentration of thrombin were applied to reflect the binding affinity. After the aptamers were co-incubated with thrombin for 60 min, an 12% non-denaturing polyacrylamide gel electrophoresis was performed. The gel was then stained by fluorescent dye. GelRed was used as the DNA dye. Random extension was added to aptamer, in order to enhance GelRed incorporation (Figure 2).

Figure 2 | The design of aptamer-linker-probe complex. This structure enlarged the DNA molecule while its binding activity was not weakened.

The electrophoresis showed that the aptamers showed rather strong binding affinity (Figure 3). The shift bands became more clear as the concentration of thrombin increased. 15-mer and 29-mer aptamers had a clear shift band and a clear non-shift band. 40-mer aptamer was suspected to form multimers, causing a strong band at the sampling hole and unclear bands at the target sites.

Figure 3 | Native-PAGE results of EMSA. In the two figures, lane 1, marker; lane 2, control group with 15-mer, 29-mer and 40-mer aptamers and NO thrombin. All aptamers were at a concentration of 10 pM. A: lane 3-5, 0.45 pM thrombin incubated with respectively 29-mer+40-mer, 15-mer+40-mer, 15-mer+29-mer; lane 6-8, 15-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM. B: lane 3-5, 29-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.; lane 6-8, 40-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.

Surface Plasmon Resonance (SPR)

EMSA provided a relatively rudimentary validation of the binding interactions. To achieve a more quantitative and precise characterization of the interactions between the 29-mer/40-mer aptamers and thrombin, Surface Plasmon Resonance (SPR) was employed for testing. Briefly, streptavidin (SA) was amino-conjugated to capture biotinylated aptamers and seal the chip with bovine serum albumin (BSA) to prevent non-specific binding of thrombin. Then gradient diluted thrombin was loaded to obtain the corresponding curves within SPR buffer. Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD). Detailed operational procedures can be found on the Experiments-Surface Plasmon Resonance (SPR) page.

Figure 4 | Surface Plasmon Resonance (SPR) results of aptamer-thrombin binding. The sensorgrams illustrate the binding interactions between the 29-mer/40-mer aptamers and thrombin, showing real-time changes in refractive index. Curves represent the association and dissociation phases, providing insights into the binding kinetics and affinity of the aptamers for thrombin. A & C: Thrombin was subjected to binding and dissociation tests by flowing gradient-diluted samples over the chip. B & D: Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD).

Aptamer	General Kinetics model	Quality Kinetics Chi² (RU²)	1:1 binding ka (1/Ms)	kd (1/s)	KD (M)	tc
29	1:1 binding	1.03e0	2.14e+5	1.74e-4	8.16e-10	4.81e+7
40	1:1 binding	4.58e+1	1.59e+5	1.85e-1	1.17e-6	5.29e+7

Table 1 | Parameters fitted from 1:1 binding kinetic model. RU: resonance units; ka: association rate constant (M^-1s^-1); kd: dissociation rate constant (s^-1); KD: equilibrium dissociation constant (M); tc: flow rate-independent component of the mass transfer constant.

As shown above, aptamer 29 demonstrated a strong affinity for thrombin, with a dissociation constant (KD) of 0.816 nM, while aptamer 40 had a much lower affinity, with a KD of 1170 nM. Due to the high affinity between aptamer 29 and thrombin, the dissociation was incomplete when thrombin concentrations were high, this could be improved by increasing the flow rate during the experiment. A 1:1 binding kinetic model was used based on the assumption that each aptamer binds to a single site on thrombin. Surface Plasmon Resonance (SPR) experiments meticulously verified the binding interactions between aptamers and thrombin. By accurately determining the association and dissociation constants, we have significantly bolstered our confidence in the results obtained from our Electrophoretic Mobility Shift Assays (EMSA), laying a solid groundwork for the foundational concepts necessary for our subsequent system development.

References

1. Tasset, D. M., Kubik, M. F., & Steiner, W. (1997). Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. Journal of molecular biology, 272(5), 688–698. https://doi.org/10.1006/jmbi.1997.1275