Difference between revisions of "Part:BBa K5321002"
 
(2 intermediate revisions by one other user not shown)
Line 13: Line 13:
 
</div>
 
</div>
 
</html><br>
 
</html><br>
−
'''Figure 1 | Binding affinity of thrombin_AYA1809004_40mer with thrombin.''' (a) Percent thrombin inhibition versus DNA concentration. Selection buffer containing human fibrinogen (2mg mi-1 final)and varying concentrations of DNA (~1 nM-150 nM) was incubated for 1 min at 37°C before adding thrombin. (b) Binding simulation predicted by Alphafold3. The thick yellow belt represents 15-mer aptamer. <br>
+
'''Figure 1 | Binding affinity of thrombin_TBA_15mer with thrombin.''' (a) Percent thrombin inhibition versus DNA concentration. Selection buffer containing human fibrinogen (2mg mi-1 final)and varying concentrations of DNA (~1 nM-150 nM) was incubated for 1 min at 37°C before adding thrombin.(Modified from [1]) (b) Binding simulation predicted by Alphafold3. The thick yellow belt represents 15-mer aptamer. <br>
−
 
+
−
+
  
 
===Characterization===
 
===Characterization===
Line 21: Line 19:
 
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.
 
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,
+
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''').
 +
 
 +
<html>
 +
<div align="center">
 +
        <img src="https://static.igem.wiki/teams/5321/result/emsa1.png" width="400" height="auto"/>
 +
</div>
 +
</html>
 +
 
 +
 
 +
'''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.
 +
 
 +
<html>
 +
<div align="center">
 +
        <img src="https://static.igem.wiki/teams/5321/result/emsa2.png" width="900" height="auto"/>
 +
</div>
 +
</html>
 +
 
 +
'''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.
 +
 
 +
<html>
 +
<div align="center">
 +
        <img src="https://static.igem.wiki/teams/5321/result/spr-result.png" width="800" height="auto"/>
 +
</div>
 +
</html>
 +
 
 +
'''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).
 +
 
 +
<html lang="zh"> 
 +
<head> 
 +
    <meta charset="UTF-8"> 
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0"> 
 +
    <title>居中表格示例</title> 
 +
    <style> 
 +
        /* 表格居中 */ 
 +
        table { 
 +
            margin: 0 auto; /* 左右居中 */ 
 +
            border-collapse: collapse; /* 边框合并为单线 */ 
 +
            width: 80%; /* 可根据需要调整宽度 */ 
 +
        } 
 +
        /* 表格单元格样式 */ 
 +
        th, td { 
 +
            border: 1px solid black; /* 所有单元格添加边框 */ 
 +
            padding: 8px; /* 内边距 */ 
 +
            text-align: center; /* 文字居中 */ 
 +
        } 
 +
        /* 表头样式 */ 
 +
        th { 
 +
            background-color: #f2f2f2; /* 表头背景色 */ 
 +
        } 
 +
    </style> 
 +
</head> 
 +
<body> 
 +
 
 +
<table id="af51de83-6f9e-47ac-80e5-50c78fbff04e" class="simple-table"> 
 +
    <tr id="5e2a59f0-2e9d-4074-85a9-f227fcd233a6"> 
 +
      <th id="CLGy" class="" style="width:98.57142857142857px"><strong>Aptamer</strong></th> 
 +
      <th id="ojEv" class="" style="width:98.57142857142857px"><strong>General Kinetics model</strong></th> 
 +
      <th id="mqTn" class="" style="width:98.57142857142857px"><strong>Quality Kinetics Chi² (RU²)</strong></th> 
 +
      <th id="ymfA" class="" style="width:98.57142857142857px"><strong>1:1 binding ka (1/Ms)</strong></th> 
 +
      <th id="uIQr" class="" style="width:98.57142857142857px"><strong>kd (1/s)</strong></th> 
 +
      <th id="X]?:" class="" style="width:98.57142857142857px"><strong>KD (M)</strong></th> 
 +
      <th id="zv:m" class="" style="width:98.57142857142857px"><strong>tc</strong></th> 
 +
    </tr> 
 +
    <tr id="012d2324-2411-4ed2-acd5-d1bc12f01b47"> 
 +
      <td id="CLGy" class="" style="width:98.57142857142857px">29</td> 
 +
      <td id="ojEv" class="" style="width:98.57142857142857px">1:1 binding</td> 
 +
      <td id="mqTn" class="" style="width:98.57142857142857px">1.03e0</td> 
 +
      <td id="ymfA" class="" style="width:98.57142857142857px">2.14e+5</td> 
 +
      <td id="uIQr" class="" style="width:98.57142857142857px">1.74e-4</td> 
 +
      <td id="X]?:" class="" style="width:98.57142857142857px">8.16e-10</td> 
 +
      <td id="zv:m" class="" style="width:98.57142857142857px">4.81e+7</td> 
 +
    </tr> 
 +
    <tr id="71feb723-4821-4129-aa3f-6a6973869eaf"> 
 +
      <td id="CLGy" class="" style="width:98.57142857142857px">40</td> 
 +
      <td id="ojEv" class="" style="width:98.57142857142857px">1:1 binding</td> 
 +
      <td id="mqTn" class="" style="width:98.57142857142857px">4.58e+1</td> 
 +
      <td id="ymfA" class="" style="width:98.57142857142857px">1.59e+5</td> 
 +
      <td id="uIQr" class="" style="width:98.57142857142857px">1.85e-1</td> 
 +
      <td id="X]?:" class="" style="width:98.57142857142857px">1.17e-6</td> 
 +
      <td id="zv:m" class="" style="width:98.57142857142857px">5.29e+7</td> 
 +
    </tr> 
 +
</table> 
 +
 
 +
</body> 
 +
</html>
 +
 
 +
'''Table 1 | Parameters fitted from 1:1 binding kinetic model.''' RU: resonance units; ka: association rate constant (M<sup>-1</sup>s<sup>-1</sup>); kd: dissociation rate constant (s<sup>-1</sup>); 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. Bock, Louis, et al. "Selection of single-stranded DNA molecules that bind and inhibit human thrombin." Nature 355, (1992): 564-566.<br>

Latest revision as of 10:48, 28 September 2024

thrombin_TBA_15mer

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_TBA_15mer is an aptamer originally characterized by Louis et al. in 1992. It specifically targets the heparin-binding site of thrombin.

Figure 1 shows the interaction of thrombin_TBA_15mer with human thrombin.


Figure 1 | Binding affinity of thrombin_TBA_15mer with thrombin. (a) Percent thrombin inhibition versus DNA concentration. Selection buffer containing human fibrinogen (2mg mi-1 final)and varying concentrations of DNA (~1 nM-150 nM) was incubated for 1 min at 37°C before adding thrombin.(Modified from [1]) (b) Binding simulation predicted by Alphafold3. The thick yellow belt represents 15-mer aptamer.

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).

居中表格示例

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. Bock, Louis, et al. "Selection of single-stranded DNA molecules that bind and inhibit human thrombin." Nature 355, (1992): 564-566.