Molecular beacons for protein-DNA interaction studies.

Real-time monitoring of DNA-protein interactions involving molecular beacon (MB) and molecular beacon aptamer (MBA) was discussed in this chapter. MBs are single-stranded oligonucleotide probes with a hairpin structure. MBs have been designed for oligonucleotide recognition and protein-DNA interaction studies. Real-time monitoring of enzymatic reactions, such as cleavage, ligation, and phosphorylation of single-stranded DNA by specific enzyme, has been studied using MBs. Meanwhile, a new generation of molecular probes, MBA, was designed by combining the excellent signal transduction properties of MBs with the specificity of aptamers for protein recognition. Two different aptamers, the one for thrombin and that for platelet-derived growth factor, have been successfully used to construct MBA probes. The interaction between the proteins and the MBA probes was investigated by fluorescence resonance energy transfer, fluorescence anisotropy, and time-resolved fluorescence. This chapter has reviewed our recent progress in this area.

Identification of liver cancer-specific aptamers using whole live cells.

Liver cancer is the third most deadly cancers in the world. Unfortunately, there is no effective treatment. One of the major problems is that most cancers are diagnosed in the later stage, when surgical resection is not feasible. Thus, accurate early diagnosis would significantly improve the clinical outcome of liver cancer. Currently, there are no effective molecular probes to recognize biomarkers that are specific for liver cancer. The objective of our current study is to identify liver cancer cell-specific molecular probes that could be used for liver cancer recognition and diagnosis. We applied a newly developed cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) method for the generation of molecular probes for specific recognition of liver cancer cells. The cell-SELEX uses whole live cells as targets to select aptamers (designed DNA/RNA) for cell recognition. In generating aptamers for liver cancer recognition, two liver cell lines were used: a liver cancer cell line BNL 1ME A.7R.1 (MEAR) and a noncancer cell line, BNL CL.2 (BNL). Both cell lines were originally derived from Balb/cJ mice. Through multiple rounds of selection using BNL as a control, we have identified a panel of aptamers that specifically recognize the cancer cell line MEAR with Kd in the nanomolar range. We have also demonstrated that some of the selective aptamers could specifically bind liver cancer cells in a mouse model. There are two major new results (compared with our reported cell-SELEX methodology) in addition to the generation of aptamers specifically for liver cancer. The first one is that our current study demonstrates that cell-based aptamer selection can select specific aptamers for multiple cell lines, even for two cell lines with minor differences (MEAR cell is derived from BNL by chemical inducement); and the second result is that cell-SELEX can be used for adhesive cells and thus open the door for solid tumor selection and investigation. The newly generated cancer-specific aptamers hold great promise as molecular probes for cancer early diagnosis and basic mechanism studies.

Aptamers evolved from cultured cancer cells reveal molecular differences of cancer cells in patient samples.

BACKGROUND: Molecular-level differentiation of neoplastic cells is essential for accurate and early diagnosis, but effective molecular probes for molecular analysis and profiling of neoplastic cells are not yet available. We recently developed a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy to generate aptamers (designer DNA/RNA probes) as molecular probes to recognize neoplastic cells. METHODS: We tested 6 cell-SELEX-generated aptamers with equilibrium dissociation constants in the nanomolar to subnanomolar range: sgd5, selected from Toledo cells, a human diffuse large-cell lymphoma cell line (B-cell), and sgc8, sgc3, sgc4, sgd2, and sgd3 from CCRF-CEM cells, a human precursor T cell acute lymphoblastic leukemia (T-ALL) cell line. Aptamers were labeled with fluorescein isothiocyanate fluorophores and then used to recognize, by flow cytometric analysis, neoplastic cells in cultured hematopoietic cell lines and clinical samples. RESULTS: Aptamer sgd5 recognized only its target cells. Aptamers sgc3, sgd2, sgd3, sgc4, and sgc8, selected from a T-cell leukemia cell line, identified all of the cultured T-cell leukemia cell lines with relatively high fluorescence intensity. Aptamers sgc8, sgc3, and sgd3 showed good selectivity toward T-ALL cells and almost no binding to normal hematopoietic cells or lymphoma and myeloma cells. Selected aptamers also detected targets on the cell membranes of neoplastic cells in patient samples. CONCLUSIONS: Aptamers selected against cultured neoplastic cells can effectively be used as molecular probes for recognition of neoplastic cells in patient samples. Cell-based aptamer selection can be used to generate aptamer probes to obtain molecular signatures of neoplastic cells in patient samples.

Aptamers evolved from live cells as effective molecular probes for cancer study.

Using cell-based aptamer selection, we have developed a strategy to use the differences at the molecular level between any two types of cells for the identification of molecular signatures on the surface of targeted cells. A group of aptamers have been generated for the specific recognition of leukemia cells. The selected aptamers can bind to target cells with an equilibrium dissociation constant (K(d)) in the nanomolar-to-picomolar range. The cell-based selection process is simple, fast, straightforward, and reproducible, and, most importantly, can be done without prior knowledge of target molecules. The selected aptamers can specifically recognize target leukemia cells mixed with normal human bone marrow aspirates and can also identify cancer cells closely related to the target cell line in real clinical specimens. The cell-based aptamer selection holds a great promise in developing specific molecular probes for cancer diagnosis and cancer biomarker discovery.