Programmed Aptamer Screening, Characterization, and Rapid Detection for α-Conotoxin MI
Abstract
:1. Introduction
2. Results and Discussion
2.1. The Isolation of Aptamers for CTX-MI
2.2. The Selection of Candidate Aptamers
2.3. The Determination of Affinity and Specificity
2.4. The Interaction Mechanism between MBMI-01c and CTX-MI
2.5. The Development of a BLI-Based Aptasensor for CTX-MI Detection
3. Conclusions
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Aptamers Selection In Vitro
4.2.1. Preparation of MBs
4.2.2. Random ssDNA Library and Primers
4.2.3. Aptamer Selection by MB-SELEX
4.3. The Preparation of HTS Samples
4.4. The Determination of Affinity by BLI Assay
4.5. Computer Simulation
4.5.1. Three-Dimensional Structure Prediction of MBMI-01c
4.5.2. Molecular Docking
4.5.3. MD Simulation
4.6. Performance Evaluation of the BLI Aptasensor
4.7. Treatment of Real Samples
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
Abbreviation | Full Name |
BLI | biolayer interferometry |
CTX-GI | α-conotoxin GI |
CTX-GI-MBs | CTX-GI coated MBs |
CTX-MI | α-conotoxin MI |
CTX-MI-MBs | CTX-MI coated MBs |
CTXs | conotoxins |
CV | coefficient of variation |
DA | domoic acid |
GQ | G-quadruplet |
HPLC | high-performance liquid chromatography |
HTS | high-throughput sequencing |
LOD | limit of detection |
LOQ | limit of quantification |
MALDI | matrix-assisted laser desorption/ionization |
MB | magnetic beads |
MD | molecular dynamics |
MS | mass spectrometry |
N-J tree | neighbor-joining tree |
OA | okadaic acid |
PME | Particle-Mesh-Ewald |
QGRS prediction | G-rich sequences prediction |
RMSD | root mean square deviation |
SB | selection buffer |
SELEX | systematic evolution of ligands by exponential enrichment |
SSA | super streptavidin |
STX | saxitoxin |
TdMD | temperature-dependent molecular dynamics simulation |
TTX | tetrodotoxin |
3D | three-dimensional |
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Ligand | Etotal (kcal/mol) | Eshape (kcal/mol) | Eforce (kcal/mol) | RMS |
---|---|---|---|---|
CTX-MI | −531.44 | −531.44 | 0 | −1.00 |
CTX-GI | −439.61 | −439.61 | 0 | −1.00 |
OA | −264.49 | −264.49 | 0 | −1.00 |
STX | −388.01 | −388.01 | 0 | −1.00 |
CTX-MI (μM) | Detection Value (μM) | CV (%) |
---|---|---|
5 | 5.48 | 3.25 |
10 | 10.21 | 2.45 |
20 | 20.14 | 2.00 |
CTX-MI (μM) | Detection Value (μM) | CV (%) | Recovery (%) |
---|---|---|---|
10 | 10.47 | 4.31 | 102.11 |
25 | 24.71 | 4.90 | 98.83 |
50 | 50.53 | 2.21 | 101.06 |
ID | Sequence (5′ to 3′) |
---|---|
Lib V1 | ATTGGCACTCCACGCATAGG-N40-CCTATGCGTGCTACCGTGAA |
F1 | ATTGGCACTCCACGCATAGG |
F2 | AAAGCAATTGGCACTCCACGCATAGG |
F3 | AACGCCATTGGCACTCCACGCATAGG |
F4 | AAGGCGATTGGCACTCCACGCATAGG |
F5 | ACAGGAATTGGCACTCCACGCATAGG |
F6 | ACCGGCATTGGCACTCCACGCATAGG |
R1 | A20-Spacer18-TTCACGGTAGCACGCATAGG |
R2 | TTCACGGTAGCACGCATAGG |
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Guo, H.; Deng, B.; Zhao, L.; Gao, Y.; Zhang, X.; Yang, C.; Zou, B.; Chen, H.; Sun, M.; Wang, L.; et al. Programmed Aptamer Screening, Characterization, and Rapid Detection for α-Conotoxin MI. Toxins 2022, 14, 706. https://doi.org/10.3390/toxins14100706
Guo H, Deng B, Zhao L, Gao Y, Zhang X, Yang C, Zou B, Chen H, Sun M, Wang L, et al. Programmed Aptamer Screening, Characterization, and Rapid Detection for α-Conotoxin MI. Toxins. 2022; 14(10):706. https://doi.org/10.3390/toxins14100706
Chicago/Turabian StyleGuo, Han, Bowen Deng, Luming Zhao, Yun Gao, Xiaojuan Zhang, Chengfang Yang, Bin Zou, Han Chen, Mingjuan Sun, Lianghua Wang, and et al. 2022. "Programmed Aptamer Screening, Characterization, and Rapid Detection for α-Conotoxin MI" Toxins 14, no. 10: 706. https://doi.org/10.3390/toxins14100706
APA StyleGuo, H., Deng, B., Zhao, L., Gao, Y., Zhang, X., Yang, C., Zou, B., Chen, H., Sun, M., Wang, L., & Jiao, B. (2022). Programmed Aptamer Screening, Characterization, and Rapid Detection for α-Conotoxin MI. Toxins, 14(10), 706. https://doi.org/10.3390/toxins14100706