Using the selected aptamers to recognize FFPE normal or glioma tissue sections.

<p>FFPE tissue sections were incubated with cy5-labeled aptamers. A =  Normal brain tissue with GBM128; B =  Glioblastoma tissue with GBM128; C =  Anaplastic oligodendroglima with GBM128; D =  Oligoastrocytoma with GBM128; E =  Pilocytic astrocytoma with GBM128; F =  Normal brain tissue with GBM131; G =  Glioblastoma tissue with GBM131; H =  Anaplastic oligodendroglima with GBM131; I =  Oligoastrocytoma with GBM131; J =  Pilocytic astrocytoma with GBM131. The final concentration of Cy5-labeled aptamers was 250 nM.</p

Enrichment of DNA sequences that bind to AGR2-GST-beads.

<p>a) Flow cytometry assay to monitor the binding of a selected pool with AGR2 (target protein) and GST (control protein). The red curve represents the background binding of unselected library. For the target protein AGR2, there was an increase in binding capacity of the pool as the selection progressed, while there was no observable change for the control protein GST. The final concentration of the selected pool in binding buffer was 100 nM. b) Determination of the dissociation constant of the enriched library and unselected library to AGR2. c) Fluorescence measurements to determine the dissociation constant of selected aptamer C14B. Two negative controls, GST-beads and GSH-beads, were carried out. The data were the average of triplicate experiment results.</p

Schematics of systematic evolution of DNA aptamers against AGR2.

<p>In sepharose-beads-based SELEX process for protein AGR2, the GST-beads were incubated with the ssDNA library for counter-selection to remove nonspecific sequences. The unbound DNAs were then incubated with AGR2-GST-beads for target-selection. After harshly washing, the AGR2-specific DNA sequences were subsequently amplified by PCR for the next round of selection, or for cloning and sequencing to identify individual aptamers after flow cytometry analysis.</p

Confocal images of cultured U118-MG and SVGp12 cells staining with FAM-labeled aptamers.

<p>Cells were incubated with FAM labeled initial library and aptamers. The initial library showed the background binding and aptamers showed significant binding over the background. For U118-MG cells: A =  initial library; B =  GBM128; C =  GBM131. For SVGp12 cells; D =  initial library; E =  GBM128; F =  GBM131. The final concentration of FAM-labeled aptamers was 250 nM.</p

Evolution of DNA Aptamers through in Vitro Metastatic-Cell-Based Systematic Evolution of Ligands by Exponential Enrichment for Metastatic Cancer Recognition and Imaging

Metastasis, the capability of tumor cells to spread and grow at distant sites, is the primary factor in cancer mortality. Because metastasis in sentinel lymph nodes suggests the original spread of tumors from a primary site, the detection of lymph node involvement with cancer serves as an important prognostic and treatment parameter. Here we have developed a panel of DNA aptamers specifically binding to colon cancer cell SW620 derived from metastatic site lymph node, with high affinity after 14 rounds of selection by the cell-SELEX (systematic evolution of ligands by exponential enrichment) method. The binding affinities of selected aptamers were evaluated by flow cytometry. Aptamer XL-33 with the best binding affinity (0.7 nM) and its truncated sequence XL-33-1 with 45 nt showed excellent selectivity for recognizing target cell SW620. The binding entity of the selected aptamer has been preliminarily determined as a membrane protein on the cell surface. Tissue imaging results showed that XL-33-1 was highly specific to the metastatic tumor tissue or lymph node tissue with corresponding cancer metastasis and displayed an 81.7% detection rate against colon cancer tissue with metastasis in regional lymph nodes. These results suggest that XL-33-1 has great potential to become a molecular imaging agent for early detection of lymph node tissue with colon cancer metastasis. More importantly, this study clearly demonstrates that DNA ligands selectively recognizing metastatic cancer cells can be readily generated by metastatic-cell-based SELEX for potential applications in metastatic cancer diagnosis and treatment

The binding affinity assay of the enriched pools with U118 MG and SVGp12 cells.

<p>(A) The fluorescence intensity of U118-MG cells binding with selected pools increased gradually as the selection progressed, indicating that the enhanced binding affinity of enriched pools. (B)The selected pools also showed increased affinity with SVGp12 cells, but the response was apparently weaker than with U118-MG cells. Final concentration of FAM-labeled ssDNA pools was 250 nM in binding buffer.</p

Working principle of allosteric molecular beacon probe for sensitive and selective detection of AGR2 based on fluorescent flow cytometry analysis.

<p>Working principle of allosteric molecular beacon probe for sensitive and selective detection of AGR2 based on fluorescent flow cytometry analysis.</p

Characterization of the aptamer C14B1.

<p>a) CD spectra of the aptamer C14B1 in the presence of different cations. This aptamer has a positive band near 260 nm that can be assigned to a parallel quadruplex structures. b)The structural stability of the aptamer relies heavily on [K⁺]. c) Fluorescent signal migration of C14B1 binding to AGR2-beads at different concentration of K⁺ was investigated. d) The binding constants of C14B1 in the buffer w/ and w/o K⁺. The data were the average of triplicate experiment results.</p

Schematics of systematic evolution of DNA aptamers against U118-MG cells.

<p>Schematics of systematic evolution of DNA aptamers against U118-MG cells.</p

Specificity of aptamer C14B1 against AGR2 and three control proteins, BSA, Trypsin and Thrombin.

<p>The signal-to-background ratio (SBR) is defined as the fluorescence signal ratio of FAM-labeled aptamer to FAM-labeled random sequence when treating with protein modified beads. The data were the average of triplicate experiment results.</p