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Screening and initial binding assessment of fumonisin b(1) aptamers.

McKeague M, Bradley CR, De Girolamo A, Visconti A, Miller JD, Derosa MC - Int J Mol Sci (2010)

Bottom Line: The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability.Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls.Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6; E-Mails: mmckeagu@connect.carleton.ca (M.M.); cbradle4@connect.carleton.ca (C.R.B.); david_miller@carleton.ca (J.D.M.).

ABSTRACT
Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

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Related in: MedlinePlus

Percent recovery of binding ssDNA to the FB1 immobilized magnetic beads after each selection round. With any observable significant increase or plateau in percent recovery, increasingly stringent selection conditions were implemented the following round (see Section 3 and Table 1).
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f2-ijms-11-04864: Percent recovery of binding ssDNA to the FB1 immobilized magnetic beads after each selection round. With any observable significant increase or plateau in percent recovery, increasingly stringent selection conditions were implemented the following round (see Section 3 and Table 1).

Mentions: To assess the enrichment of the library for target binding, as well as to determine when increased stringency could be applied to the selection, the percent binding of the DNA pool was monitored after each round using fluorescence and absorbance measurements (see Figure 2). Typical selection matrices include μmol amounts of target [36]. However, due to the cost and availability of the target FB1, as well as the possibility of using minute concentrations of target with the magnetic beads, our selection matrix contained a maximum of 5 nmol of target. In the later rounds, the amount of target available for binding could be as low as 20 pmol (1:5 ratio of FB1:DNA). This accounts for the seemingly low percent recovery of binding sequences with each round.


Screening and initial binding assessment of fumonisin b(1) aptamers.

McKeague M, Bradley CR, De Girolamo A, Visconti A, Miller JD, Derosa MC - Int J Mol Sci (2010)

Percent recovery of binding ssDNA to the FB1 immobilized magnetic beads after each selection round. With any observable significant increase or plateau in percent recovery, increasingly stringent selection conditions were implemented the following round (see Section 3 and Table 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100853&req=5

f2-ijms-11-04864: Percent recovery of binding ssDNA to the FB1 immobilized magnetic beads after each selection round. With any observable significant increase or plateau in percent recovery, increasingly stringent selection conditions were implemented the following round (see Section 3 and Table 1).
Mentions: To assess the enrichment of the library for target binding, as well as to determine when increased stringency could be applied to the selection, the percent binding of the DNA pool was monitored after each round using fluorescence and absorbance measurements (see Figure 2). Typical selection matrices include μmol amounts of target [36]. However, due to the cost and availability of the target FB1, as well as the possibility of using minute concentrations of target with the magnetic beads, our selection matrix contained a maximum of 5 nmol of target. In the later rounds, the amount of target available for binding could be as low as 20 pmol (1:5 ratio of FB1:DNA). This accounts for the seemingly low percent recovery of binding sequences with each round.

Bottom Line: The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability.Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls.Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6; E-Mails: mmckeagu@connect.carleton.ca (M.M.); cbradle4@connect.carleton.ca (C.R.B.); david_miller@carleton.ca (J.D.M.).

ABSTRACT
Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

Show MeSH
Related in: MedlinePlus