Membranes were then washed three times with SB or TBS and incubated with 0

Membranes were then washed three times with SB or TBS and incubated with 0. 014 nmol of aptamers in SB or TBS made up of 0.1?mg/ml of yeast tRNA for 2 hours at 37 C. molecule as a therapeutic agent, diagnostic agent and research tool. Results Aptamer selection Single-stranded DNA molecules exhibiting high affinity for mouse MBP were obtained by an aptamer selection process, 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 equivalent 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 altered 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 Canrenone (MBP C15) were analyzed using a radio isotope-free dot-blot assay (Physique 1a), which has become a common technique for protein detection, and may be performed by most research laboratories due to its simplicity and velocity. Physique 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 this 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 (Physique 1b). Interestingly, the length of the randomized region was shortened in both sequences. Indeed, while the initial 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 this first antibody is usually 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 your enzyme-linked oligonucleotide assay,15 which only makes use of oligonucleotides for detection. Open in a separate window Physique 1 TMEM8 Dot-blot analysis of the Canrenone selected library after 15 rounds of systematic development of ligand by exponential enrichment (SELEX). (a) 1 g of real myelin basic protein (MBP) was spotted onto nitrocellulose membranes and incubated with equivalent nmol of different Canrenone biotinylated aptamers. MBP C15 is the selected library against MBP; unspecific is usually another pool of aptamers selected against an unrelated target; none is usually 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 experienced 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. Table 1 Protein and aptamer quantity, washing stringency and incubation time for each SELEX Open in a separate windows The MBP-binding activity of MBPcl3 and MBPcl9 was compared and no statistical difference was found (Physique 2a). Interestingly, the unselected aptamer pool also showed a strong conversation with immobilized MBP suggesting that this protein binds to nucleic acids irrespective of their sequence due to its highly positive net charge at physiological pH. However, it Canrenone is also possible that this binding activity resides in part within the fixed reverse primer-annealing region of the aptamer. Sequence analysis of both aptamer clones revealed a highly conserved nucleotide.