Screening and Initial Binding Assessment of Fumonisin B1 Aptamers
Abstract
:1. Introduction
2. Results and Discussion
2.1. Fumonisin B1 Immobilization
2.2. Selection of Aptamers
2.3. Binding Assays
3. Experimental Section
3.1. Reagents
3.2. Derivatization of Magnetic Beads
3.3. DNA Library and Primer Synthesis
3.4. SELEX Experiments
3.5. Cloning and Sequencing
3.6. Binding Assays
3.7. Determination of the Dissociation Constant
4. Conclusions
Acknowledgments
References
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Selection Round | Negative Selection Used | Number of FB1 derivatized beads used | Incubation time with FB1 derivatized beads(min) |
---|---|---|---|
1 | Unmodified | 5 × 108 | 60 |
2 | Unmodified | 5 × 108 | 60 |
3 | Unmodified | 5 × 108 | 60 |
4 | Unmodified | 5 × 108 | 60 |
5 | l-homocysteine | 1 × 108 | 60 |
6 | l-homocysteine | 1 × 108 | 60 |
7 | l-homocysteine | 1 × 108 | 45 |
8 | l-homocysteine | 1 × 108 | 45 |
9 | l-homocysteine | 1 × 108 | 45 |
10 | l-homocysteine | 5 × 107 | 45 |
11 | l-cysteine and l-methionine | 5 × 107 | 45 |
12 | l-cysteine and l-methionine | 5 × 107 | 30 |
13 | l-cysteine and l-methionine | 5 × 107 | 30 |
14 | l-cysteine and l-methionine | 5 × 107 | 30 |
15 | l-cysteine and l-methionine | 1 × 107 | 30 |
16 | l-glutamic acid | 1 × 107 | 30 |
17 | l-glutamic acid | 1 × 107 | 30 |
18 | l-glutamic acid | 1 × 107 | 20 |
Sequence | 5′-ATACCAGCTTATTCAATT-N60-AGATAGTAAGTGCAATCT-3′ | G Content (%) | Sequence Incidence (% of Total) |
---|---|---|---|
FB1 14 | CTATACGGAGTGGATATCGATCTGTAACGT GAGTGAGATAATGTGATGCATAGTCGTGG | 32 | 12.5 |
FB1 16 | CATCCAGTAACAAACACATAAGTAACGGC GATATGTCAAAGCGGTATCGGCTACAGATG | 22 | 12.5 |
FB1 23 | GCGGATGCGTAAATGACGATAAACATAGAT GGGGTATATCGCGATGCGACAGGGTGT | 35 | 12.5 |
FB1 31 | CGGGGACGTGTATACCAGCTTATTCAATTC ACAGTTATGTCCTATACCAGCTTATTCAATT | 17 | 25 |
FB1 32 | AATGTACGATGTGTGGGCAACATGAGTATG TCGTGTGATATCTAGATGAGGTAGCGGTGG | 37 | 12.5 |
FB1 39 | AATCGCATTACCTTATACCAGCTTATTCAAT TACGTCTGCACATACCAGCTTATTCAATT | 8 | 25 |
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McKeague, M.; Bradley, C.R.; Girolamo, A.D.; Visconti, A.; Miller, J.D.; DeRosa, M.C. Screening and Initial Binding Assessment of Fumonisin B1 Aptamers. Int. J. Mol. Sci. 2010, 11, 4864-4881. https://doi.org/10.3390/ijms11124864
McKeague M, Bradley CR, Girolamo AD, Visconti A, Miller JD, DeRosa MC. Screening and Initial Binding Assessment of Fumonisin B1 Aptamers. International Journal of Molecular Sciences. 2010; 11(12):4864-4881. https://doi.org/10.3390/ijms11124864
Chicago/Turabian StyleMcKeague, Maureen, Charlotte R. Bradley, Annalisa De Girolamo, Angelo Visconti, J. David Miller, and Maria C. DeRosa. 2010. "Screening and Initial Binding Assessment of Fumonisin B1 Aptamers" International Journal of Molecular Sciences 11, no. 12: 4864-4881. https://doi.org/10.3390/ijms11124864