A representation of the PTEN structure (PDB: 1DR5) was obtained by means of the PyMol program. PTEN domains corresponding to the phosphatase domain (PTPs) and the regulatory domain (C2) are displayed in this graphical representation. The peptide selected as target was contained on the PTP domain and was shown in blue color.

A. Protein pattern expression in E. coli of the recombinant proteins GST (E. coli) and GST-PTEN (+PTEN) in conjunction with the affinity column-purified proteins (GST and +PTEN). The identity was confirmed by binding of the goat antibody (Ab), resulting in similar patterns to the cloned aptamers derived from peptide (PTENz7 and PTENz14). B. Western blot using aptamers PTENz7 and PTENz14 (z7 and z14) and antibodies (Ab Rb) developed with peptide in the cerebellar homogenates. The synthetic aptamer strand of Anp32e was used as a negative control, as well as the omission of aptamers (–). The arrow indicates the most intense band corresponding to the 55-kDa PTEN isoform. C. Western blot detection made as in (B) using aptamers and antibodies developed against the GST- PTEN recombinant protein. The arrows indicate the band of 55 kDa and two bands of greater size, possibly due to ubiquitin-modified PTEN. D. PTEN aptamer conformations: Sequences from PTENz7 (left column) or PTENz14 (right column) were analyzed for internal interactions and the best three predicted structures (mFOLD, http://www.idtdna.com/Scitools/Applications/mFold) were presented in this panel. For the more stable conformation of PTENz7, a differential free energy of Gibbs of −19.04 kcal.mole-1 was observed and for PTENz14 this value was −17.3 kcal.mole-1.

The PTEN staining pattern using aptamers analyzed in cerebellar slices from mice. PTEN was detected with the peroxidase system within the cerebellar cortex of adult mice using the selected aptamers and a positive aptamer control developed from a peptide contained within the Anp32e protein sequence. The aptamer used is indicated in the column heading, while the designated treatment with recombinant proteins or antibodies is mentioned to the left of the rows. The first row shows the histological staining without any competition for the aptamers. The second row indicates aptamers pre-incubated with the recombinant protein before and during the histologic assay. The third row shows histologic samples where aptamers were pre-incubated before and during the histological assays with rabbit antiserum developed against the PTEN peptide. The cerebellar cortex layers are indicated in each micrograph as: Molecular layer (ML); Purkinje cell layer (PCL); Internal granular layer (IGL). Images were obtained at 100× magnification. The subcellular localizations are shown in the upper circle of each aptamer staining in the control row.

A. No crosslinking fixation. Histologic samples showing the peroxidase staining pattern of denatured proteins. 100× and 1000× magnifications are indicated at the top of each column. The denaturation was achieved through heating of the tissue, a technique commonly known as antigen retrieval (AR). The aptamer utilized for the staining is indicated on the left of each row. The cerebellar cortex layers are indicated in each micrograph as: molecular layer (ML); Purkinje cell layer (PcL); internal granular layer (IGL). The micrograph at 1000× magnification indicates Purkinje cells (Pc) and granule cells (Gc). B. Crosslinking fixation. Histologic samples fixed with paraformaldehyde (PFA) with and without antigen retrieval (+AR and -AR respectively) were stained simultaneously with PTENz7 (CY3-red) and PTENz14 (FITC-green). Colocalization was observed as yellow labeling at 400× magnification. C. Western blot subcellular fractions and homogenates. Homogenates and subcellular fractions were developed using a polyclonal antibody against the carboxy terminus of PTEN. Cytoplasmic fraction presented a large amount of PTEN, corresponding to the 55-kDa band. Another band of 120 kDa was also observed in this fraction (duplicates). The nuclear fraction presented a major band of 55 kDa with two minor bands of 80 kDa and 40 kDa. In homogenates, the major band of 55 kDa was observed (Ab), but required overexposure (Ab+) to detect the modified-mobility isoforms. The band of 55 kDa had a lower intensity than the antibody band. Both aptamers PTENz7 (z7) and PTENz14 (z14) detected the 80-kDa and 120-kDa bands. Controls were put in place using the negative chain corresponding to the lysozyme aptamer (Liso). D. Immunoprecipitation assays. Proteins from cerebellum homogenate were utilized to purify PTEN by means of the aptamers PTENz7 and PTENz14. The aptamer Anp32 was used as a negative control for the purification. The purified proteins were separated in denaturing 10% SDS-PAGE, transferred to a nitrocellulose membrane and detected using goat antibodies against GST-PTEN protein. Corresponding aptamers are indicated above each western blot lane, while the molecular weight is denoted to the left of the image. The PTEN protein was precipitated from the subcellular fractions and the presence of the protein was analyzed in the supernatants. Note that PTENz7 was able to diminish the PTEN staining from the nuclear fraction, whereas PTENz14 affects the cytoplasmic fraction.

A. In these assays, selected oligonucleotides were separated from the GST-PTEN target, indicated above the graph, and quantified by means of real-time PCR. The effect of amplification of the library on the quantity of oligonucleotides obtained is illustrated in this graph. The number of molecules, as indicated on the y axis, is obtained through extrapolation of the standard curve, while the number of cycles applied to the library used is indicated on the x axis. The three assays show a significant increase compared with the library without amplification, used as control. Libraries were amplified through PCR separately for each assay. Standard deviation is indicated using bars on each column. B. Results for assays as described above, using the synthetic peptide of PTEN as target, instead of GST-PTEN. Libraries were separated in aliquots from a larger pool that was amplified through PCR. C. Specificity of the oligonucleotides obtained. Aliquots of the selected oligonucleotides were amplified again through PCR, separated, and split into four dilutions. The filter binding assays were performed with different concentrations for four separate selections of aptamers, and each selection is indicated at the bottom of the graph. The gradient of concentration follows the slope of the triangle and the standard deviation is indicated using bars on each column. D. Affinity of the aptamers obtained. Autoradiography of radioactive aptamers bound to specific targets. The aptamer PTENz7 recognized the recombinant PTEN protein and the peptide without binding to the GST-Anp32e recombinant protein in filter binding assays. E. Selected oligonucleotides developed with 25 cycles of library amplification, using the synthetic peptide of PTEN as target, were cloned into vectors and sequenced. Plasmids from six different clones were labeled by PCR containing ATP-gamma-³⁵PO₄, incubated, and their affinity was measured in Scatchard binding assays. The example shown is the oligonucleotide from clone PTENz7, and the affinity constant measured corresponds to 75 nM, using GST-PTEN protein at a final concentration of 25 µM. At the bottom of the graph the autoradiographic results from the radioactive aptamers retained in the filter binding assays are shown. The concentration of the aptamers used corresponds to the measurements illustrated within the graph. On the x axis, the concentration of PTENz7 in nM is provided. On the y axis, the ratio between bound and free aptamers is shown.

Staining pattern in histologic samples of mouse cerebellum at two magnifications, 100× and 1000×, as indicated at the top of each column. To the left of each pair of panels the aptamer or antibody utilized for the histological staining assay of the PTEN protein was denoted. The layers of the cerebellum are indicated within each illustration as internal granular layer (IGL), Purkinje cell layer (PcL) and molecular layer (ML). The Purkinje cells are shown at the highest magnification, 1000×, where nuclear (N) or cytoplasmic (C) localization is distinguishable. The known aptamer Anp32e (Anp) was used as a positive control. The patterns belonging to clones 3, 4, 5, 6 and 9 corresponded to the aptamers developed against the recombinant protein GST-PTEN, while the patterns associated with z7 and z14 were the aptamers PTENz7 and PTENz14 developed against the peptides.

The results of precipitation are shown in terms of net amounts of molecules acquired through extrapolations from the standard curve of quantification. The backgrounds considered (PTENz7 and PTENz14) were attained through incubation and co-purification of the aptamers and antibodies in the absence of peptides. Quadruplicate samples showed the positive binding of PTENz14 to the peptide (PTENz14+Peptide). Findings also confirmed the blockade of PTENz7 recognition by the antibodies (PTENz7+Peptide). Extrapolations from the standard curve of quantification show 1.8×10¹¹ molecules of PTENz14 aptamer were bound to the peptide and co-precipitated with the antibodies attached to Sepharose – nearly a 20-fold increase with respect to the background (P<0.0001).

The figure shows the regions of interest: the amino domain of the PTEN protein surface and the backbone. The peptide surface exposed on the PTP domain is shown in the peptide sequence (detailed in the upper left hand corner). The ATP binding regions are shown in blue, and the target peptide is shown in red as shown in the upper sequence. Note that the ATP binding peptides correspond to the internal portion of the phosphatase structure. The graphic was developed using the PyMol program.