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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

Dot-blot analysis of the selected library after 15 rounds of systematic evolution of ligand by exponential enrichment (SELEX). (a) 1 µg of pure myelin basic protein (MBP) was spotted onto nitrocellulose membranes and incubated with equal nmol of different biotinylated aptamers. MBP C15 is the selected library against MBP; “unspecific” is another pool of aptamers selected against an unrelated target; “none” is without aptamers. Bound aptamers were detected with an alkaline phosphatase-conjugated anti-biotin antibody and developed with NBT/BCIP substrates. Relative intensities were measured with the ImageJ software. (b) Sequence alignment of the two most representative clones found in the enriched library selected for its affinity for MBP. After the 15th round of selection, the aptamer pool was cloned out and sequenced. MBPcl3 had the highest frequency among all nucleotide sequences followed by MBPcl9. Sequences corresponding to the constant primer regions are underlined. Letters in bold show differences between the two clones.
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fig1: Dot-blot analysis of the selected library after 15 rounds of systematic evolution of ligand by exponential enrichment (SELEX). (a) 1 µg of pure myelin basic protein (MBP) was spotted onto nitrocellulose membranes and incubated with equal nmol of different biotinylated aptamers. MBP C15 is the selected library against MBP; “unspecific” is another pool of aptamers selected against an unrelated target; “none” is without aptamers. Bound aptamers were detected with an alkaline phosphatase-conjugated anti-biotin antibody and developed with NBT/BCIP substrates. Relative intensities were measured with the ImageJ software. (b) Sequence alignment of the two most representative clones found in the enriched library selected for its affinity for MBP. After the 15th round of selection, the aptamer pool was cloned out and sequenced. MBPcl3 had the highest frequency among all nucleotide sequences followed by MBPcl9. Sequences corresponding to the constant primer regions are underlined. Letters in bold show differences between the two clones.

Mentions: Single-stranded DNA molecules exhibiting high affinity for mouse MBP were obtained by an in vitro aptamer selection procedure, named SELEX. Our initial ssDNA library was designed to contain a randomized sequence region of 35 nucleotides, and be flanked by fixed primer sites. High biases in composition of the nucleotide library may lead to an unsuccessful SELEX, such that equal representation of four nucleotides is preferred. To test for the nucleotide composition, our initial pool of aptamers was cloned out and 20 individual clones were sequenced, resulting in a composition of 27% A, 29.4% C, 19.7% G, and 23.4% T. Several variables were modified during the successive SELEX rounds in order to increase the stringency of the selection (Table 1). The SELEX was started with a library of ~3 × 1014 distinct ssDNA molecules. Aptamers obtained at the 15th cycle (MBP C15) were analyzed using a radio isotope-free dot-blot assay (Figure 1a), which has become a common technique for protein detection, and may be performed by most research laboratories due to its simplicity and speed. Figure 1a shows the MBP-binding activity of the selected pool of aptamers compared to the activity of another pool of aptamers that had been selected to an unrelated protein. Whereas the binding activity of the unspecific pool of aptamers is comparable to a control without aptamers, MBP C15 showed a higher binding activity suggesting that the pool of aptamers was enriched for molecules that have affinity for MBP. This aptamer pool was therefore cloned out and 15 individual clones were sequenced. Sequences were clustered into groups based on their nucleotide content, which revealed two prominent families with highly conserved sequences, represented by the clones MBPcl3 and MBPcl9 (Figure 1b). Interestingly, the length of the randomized region was shortened in both sequences. Indeed, while the original ssDNA library was designed to contain ssDNA molecules with a 35-nucleotide long randomized region, the aptamer obtained after 15 rounds of SELEX displayed a 26-nucleotide long randomized region. 5′ biotinylated versions of MBPcl9, MBPcl3, and of the unselected random library were generated by solid-phase synthesis for further studies in enzyme-linked immunosorbent assay (ELISA)-like assays. These assays followed the basic principles of an ELISA with the difference that the first antibody is replaced by an aptamer conjugated to a reporter for detection with a secondary horseradish peroxidase (HRP)-conjugated antibody. Our ELISA-like assay also differs from the enzyme-linked oligonucleotide assay,15 which only makes use of oligonucleotides for detection.


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

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

Dot-blot analysis of the selected library after 15 rounds of systematic evolution of ligand by exponential enrichment (SELEX). (a) 1 µg of pure myelin basic protein (MBP) was spotted onto nitrocellulose membranes and incubated with equal nmol of different biotinylated aptamers. MBP C15 is the selected library against MBP; “unspecific” is another pool of aptamers selected against an unrelated target; “none” is without aptamers. Bound aptamers were detected with an alkaline phosphatase-conjugated anti-biotin antibody and developed with NBT/BCIP substrates. Relative intensities were measured with the ImageJ software. (b) Sequence alignment of the two most representative clones found in the enriched library selected for its affinity for MBP. After the 15th round of selection, the aptamer pool was cloned out and sequenced. MBPcl3 had the highest frequency among all nucleotide sequences followed by MBPcl9. Sequences corresponding to the constant primer regions are underlined. Letters in bold show differences between the two clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Dot-blot analysis of the selected library after 15 rounds of systematic evolution of ligand by exponential enrichment (SELEX). (a) 1 µg of pure myelin basic protein (MBP) was spotted onto nitrocellulose membranes and incubated with equal nmol of different biotinylated aptamers. MBP C15 is the selected library against MBP; “unspecific” is another pool of aptamers selected against an unrelated target; “none” is without aptamers. Bound aptamers were detected with an alkaline phosphatase-conjugated anti-biotin antibody and developed with NBT/BCIP substrates. Relative intensities were measured with the ImageJ software. (b) Sequence alignment of the two most representative clones found in the enriched library selected for its affinity for MBP. After the 15th round of selection, the aptamer pool was cloned out and sequenced. MBPcl3 had the highest frequency among all nucleotide sequences followed by MBPcl9. Sequences corresponding to the constant primer regions are underlined. Letters in bold show differences between the two clones.
Mentions: Single-stranded DNA molecules exhibiting high affinity for mouse MBP were obtained by an in vitro aptamer selection procedure, named SELEX. Our initial ssDNA library was designed to contain a randomized sequence region of 35 nucleotides, and be flanked by fixed primer sites. High biases in composition of the nucleotide library may lead to an unsuccessful SELEX, such that equal representation of four nucleotides is preferred. To test for the nucleotide composition, our initial pool of aptamers was cloned out and 20 individual clones were sequenced, resulting in a composition of 27% A, 29.4% C, 19.7% G, and 23.4% T. Several variables were modified during the successive SELEX rounds in order to increase the stringency of the selection (Table 1). The SELEX was started with a library of ~3 × 1014 distinct ssDNA molecules. Aptamers obtained at the 15th cycle (MBP C15) were analyzed using a radio isotope-free dot-blot assay (Figure 1a), which has become a common technique for protein detection, and may be performed by most research laboratories due to its simplicity and speed. Figure 1a shows the MBP-binding activity of the selected pool of aptamers compared to the activity of another pool of aptamers that had been selected to an unrelated protein. Whereas the binding activity of the unspecific pool of aptamers is comparable to a control without aptamers, MBP C15 showed a higher binding activity suggesting that the pool of aptamers was enriched for molecules that have affinity for MBP. This aptamer pool was therefore cloned out and 15 individual clones were sequenced. Sequences were clustered into groups based on their nucleotide content, which revealed two prominent families with highly conserved sequences, represented by the clones MBPcl3 and MBPcl9 (Figure 1b). Interestingly, the length of the randomized region was shortened in both sequences. Indeed, while the original ssDNA library was designed to contain ssDNA molecules with a 35-nucleotide long randomized region, the aptamer obtained after 15 rounds of SELEX displayed a 26-nucleotide long randomized region. 5′ biotinylated versions of MBPcl9, MBPcl3, and of the unselected random library were generated by solid-phase synthesis for further studies in enzyme-linked immunosorbent assay (ELISA)-like assays. These assays followed the basic principles of an ELISA with the difference that the first antibody is replaced by an aptamer conjugated to a reporter for detection with a secondary horseradish peroxidase (HRP)-conjugated antibody. Our ELISA-like assay also differs from the enzyme-linked oligonucleotide assay,15 which only makes use of oligonucleotides for detection.

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