Part:BBa_K5321003

MAGA_A thrombin aptamer

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

Accurate and timely detection of disease biomarkers is crucial for effective diagnosis and treatment across various medical conditions. Traditional methods, such as SELEX, used for screening Aptamers that bind to specific biomarkers, are often slow, costly, and lack the precision needed for diverse applications. To overcome these limitations, we introduce MAGA: Make Aptamer Generally Applied—a universal machine learning-based platform designed to predict Aptamer sequences that can target a wide range of disease biomarkers with high specificity and affinity. MAGA_A thrombin aptamer is derived by inputting the thrombin sequence into the MAGA model.

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. (Modified from [1])

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. MAGA_A and MAGA_C 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.

The electrophoresis showed that the aptamers showed strong binding affinity (Figure 2). 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.

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