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Myelin Basic Protein and a Multiple Sclerosis-related MBP-peptide Bind to Oligonucleotides.

Rozenblum GT, Kaufman T, Vitullo AD - Mol Ther Nucleic Acids (2014)

Bottom Line: Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting.Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length.Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Investigaciones Biomédicas y Biotecnológicas, Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico, CEBBAD-Universidad Maimónides, Buenos Aires, Argentina.

ABSTRACT
Aptamer ligands for myelin basic protein (MBP) were obtained using the systematic evolution of ligand by exponential enrichment (SELEX) method. Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting. Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length. Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody. In addition, we showed the ability of the aptamer to detect myelin-rich regions in paraffin-embedded mouse brain tissue. Therefore, the MBP-binding activity of the selected oligonucleotide may prove useful as a tool for life science and medical research for myelin detection and might be a good lead for testing it in autoimmune diseases such as multiple sclerosis.

No MeSH data available.


Related in: MedlinePlus

Myelin basic protein (MBP)-binding activity of the MBPcl3 aptamer in a complex mixture of proteins. A cleared lysate of Hela cells was immobilized onto a microtiter plate containing 1 µg, 0.5 µg, or nothing of pure MBP. Wells were then coated with 3% BSA such that wells with lesser amount of MBP contained a higher amount of either the Hela cell lysate or the BSA protein. Therefore, the observed difference between the two treatments may only be explained by the difference in the amount of MBP. Experiments were performed in triplicate. The asterisk indicates a significant difference when compared to MBPcl3 1 µg (P < 0.05, Student's t-test). Data are shown as mean ± SD.
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fig4: Myelin basic protein (MBP)-binding activity of the MBPcl3 aptamer in a complex mixture of proteins. A cleared lysate of Hela cells was immobilized onto a microtiter plate containing 1 µg, 0.5 µg, or nothing of pure MBP. Wells were then coated with 3% BSA such that wells with lesser amount of MBP contained a higher amount of either the Hela cell lysate or the BSA protein. Therefore, the observed difference between the two treatments may only be explained by the difference in the amount of MBP. Experiments were performed in triplicate. The asterisk indicates a significant difference when compared to MBPcl3 1 µg (P < 0.05, Student's t-test). Data are shown as mean ± SD.

Mentions: In order to analyze whether the MBPcl3 aptamer is able to detect MBP in a complex mixture of proteins, 1 or 0.5 µg of MBP was immobilized onto wells of a microtiter plate and a cleared Hela cells lysate was added to the wells. Wells were then coated with bovine serum albumin (BSA) to block the remaining free surface. In consequence, if there was a high unspecific binding activity of MBPcl3, a higher absorbance should be expected in wells containing 0.5 µg of MBP due to a higher surface availability for the binding of soluble Hela cell proteins. The absorbance measured in wells containing 0.5 µg of MBP was much lower than in wells originally coated with 1 µg of MBP, reflecting a reduced protein–aptamer interaction in the former, thereby indicating that MBPcl3 specifically detected MBP in a mixture of proteins (Figure 4).


Myelin Basic Protein and a Multiple Sclerosis-related MBP-peptide Bind to Oligonucleotides.

Rozenblum GT, Kaufman T, Vitullo AD - Mol Ther Nucleic Acids (2014)

Myelin basic protein (MBP)-binding activity of the MBPcl3 aptamer in a complex mixture of proteins. A cleared lysate of Hela cells was immobilized onto a microtiter plate containing 1 µg, 0.5 µg, or nothing of pure MBP. Wells were then coated with 3% BSA such that wells with lesser amount of MBP contained a higher amount of either the Hela cell lysate or the BSA protein. Therefore, the observed difference between the two treatments may only be explained by the difference in the amount of MBP. Experiments were performed in triplicate. The asterisk indicates a significant difference when compared to MBPcl3 1 µg (P < 0.05, Student's t-test). Data are shown as mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222649&req=5

fig4: Myelin basic protein (MBP)-binding activity of the MBPcl3 aptamer in a complex mixture of proteins. A cleared lysate of Hela cells was immobilized onto a microtiter plate containing 1 µg, 0.5 µg, or nothing of pure MBP. Wells were then coated with 3% BSA such that wells with lesser amount of MBP contained a higher amount of either the Hela cell lysate or the BSA protein. Therefore, the observed difference between the two treatments may only be explained by the difference in the amount of MBP. Experiments were performed in triplicate. The asterisk indicates a significant difference when compared to MBPcl3 1 µg (P < 0.05, Student's t-test). Data are shown as mean ± SD.
Mentions: In order to analyze whether the MBPcl3 aptamer is able to detect MBP in a complex mixture of proteins, 1 or 0.5 µg of MBP was immobilized onto wells of a microtiter plate and a cleared Hela cells lysate was added to the wells. Wells were then coated with bovine serum albumin (BSA) to block the remaining free surface. In consequence, if there was a high unspecific binding activity of MBPcl3, a higher absorbance should be expected in wells containing 0.5 µg of MBP due to a higher surface availability for the binding of soluble Hela cell proteins. The absorbance measured in wells containing 0.5 µg of MBP was much lower than in wells originally coated with 1 µg of MBP, reflecting a reduced protein–aptamer interaction in the former, thereby indicating that MBPcl3 specifically detected MBP in a mixture of proteins (Figure 4).

Bottom Line: Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting.Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length.Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Investigaciones Biomédicas y Biotecnológicas, Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico, CEBBAD-Universidad Maimónides, Buenos Aires, Argentina.

ABSTRACT
Aptamer ligands for myelin basic protein (MBP) were obtained using the systematic evolution of ligand by exponential enrichment (SELEX) method. Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting. Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length. Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody. In addition, we showed the ability of the aptamer to detect myelin-rich regions in paraffin-embedded mouse brain tissue. Therefore, the MBP-binding activity of the selected oligonucleotide may prove useful as a tool for life science and medical research for myelin detection and might be a good lead for testing it in autoimmune diseases such as multiple sclerosis.

No MeSH data available.


Related in: MedlinePlus