Weihong Tan

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 (Kd) 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.

Molecular Engineering of DNA: Molecular Beacons

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single-nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal-transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem-loop structure holds the fluorescence-donor and fluorescence-acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein-DNA interaction studies, and protein recognition.

Gold Nanoparticle-Based Colorimetric Assay for the Direct Detection of Cancerous Cells

Early and accurate detection of cancer often requires time-consuming techniques and expensive instrumentation. To address these limitations, we developed a colorimetric assay for the direct detection of diseased cells. The assay uses aptamer-conjugated gold nanoparticles to combine the selectivity and affinity of aptamers and the spectroscopic advantages of gold nanoparticles to allow for the sensitive detection of cancer cells. Samples with the target cells present exhibited a distinct color change while nontarget samples did not elicit any change in color. The assay also showed excellent sensitivity with both the naked eye and based on absorbance measurements. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamer used in the assay indicating the wide applicability of the assay for diseased cell detection. On the basis of these qualities, aptamer-conjugated gold nanoparticles could become a powerful tool for point of care diagnostics.

Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach.

Molecular medicine is an emerging field focused on understanding the molecular basis of diseases and translating this information into strategies for diagnosis and therapy. This approach could lead to personalized medical treatments. Currently, our ability to understand human diseases at the molecular level is limited by the lack of molecular tools to identify and characterize the distinct molecular features of the disease state, especially for diseases such as cancer. Among the new tools being developed by researchers including chemists, engineers, and other scientists is a new class of nucleic acid probes called aptamers, which are ssDNA/RNA molecules selected to target a wide range of molecules and even cells. In this Account, we will focus on the use of aptamers, generated from cell-based selections, as a novel molecular tool for cancer research. Cancers originate from mutations of human genes. These genetic alterations result in molecular changes to diseased cells, which, in turn, lead to changes in cell morphology and physiology. For decades, clinicians have diagnosed cancers primarily based on the morphology of tumor cells or tissues. However, this method does not always give an accurate diagnosis and does not allow clinicians to effectively assess the complex molecular alterations that are predictive of cancer progression. As genomics and proteomics do not yet allow a full access to this molecular knowledge, aptamer probes represent one effective and practical avenue toward this goal. One special feature of aptamers is that we can isolate them by selection against cancer cells without prior knowledge of the number and arrangement of proteins on the cellular surface. These probes can identify molecular differences between normal and tumor cells and can discriminate among tumor cells of different classifications, at different disease stages, or from different patients. This Account summarizes our recent efforts to develop aptamers through cell-SELEX for the study of cancer and apply those aptamers in cancer diagnosis and therapy. We first discuss how we select aptamers against live cancer cells. We then describe uses of these aptamers. Aptamers can serve as agents for molecular profiling of specific cancer types. They can also be used to modify therapeutic reagents to develop targeted cancer therapies. Aptamers are also aiding the discovery of new cancer biomarkers through the recognition of membrane protein targets. Importantly, we demonstrate how molecular assemblies can integrate the properties of aptamers and, for example, nanoparticles or microfluidic devices, to improve cancer cell enrichment, detection and therapy.

Pyrene‐Excimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids

China Scholarship Council (CSC); ACS; US NIH; China NSFC[20805038]; National Basic Research Program of China[2007CB935603, 2010CB732402]; China National Grand Program on Key Infectious Disease[2009ZX10004-312]; Key Project of Natural Science Foundation of China[90606003]; International Science & Technology Cooperation Program of China[2010DFB30300]; Hunan Provincial Natural Science Foundation of China[10JJ7002]

Development of DNA aptamers using Cell-SELEX

In the past two decades, high-affinity nucleic acid aptamers have been developed for a wide variety of pure molecules and complex systems such as live cells. Conceptually, aptamers are developed by an evolutionary process, whereby, as selection progresses, sequences with a certain conformation capable of binding to the target of interest emerge and dominate the pool. This protocol, cell-SELEX (systematic evolution of ligands by exponential enrichment), is a method that can generate DNA aptamers that can bind specifically to a cell type of interest. Commonly, a cancer cell line is used as the target to generate aptamers that can differentiate that cell type from other cancers or normal cells. A single-stranded DNA (ssDNA) library pool is incubated with the target cells. Nonbinding sequences are washed off and bound sequences are recovered from the cells by heating cell-DNA complexes at 95 °C, followed by centrifugation. The recovered pool is incubated with the control cell line to filter out the sequences that bind to common molecules on both the target and the control, leading to the enrichment of specific binders to the target. Binding sequences are amplified by PCR using fluorescein isothiocyanate–labeled sense and biotin-labeled antisense primers. This is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection. The enrichment of the selected pools is monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library. The procedure, from design of oligonucleotides to enrichment of the selected pools, takes ∼3 months.

Biochemically functionalized silica nanoparticles

In this report, we demonstrate the biochemical modification of silica based nanoparticles. Both pure and dye-doped silica nanoparticles were prepared, and their surfaces were modified with enzymes and biocompatible chemical reagents that allow them to function as biosensors and biomarkers. The nanoparticles produced in this work are uniform in size with a 1.6% relative standard deviation. They have a pure silica surface and can thus be modified easily with many biomolecules for added biochemical functionality. Specifically, we have modified the nanoparticle surfaces with enzyme molecules (glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH)) and a biocompatible reagent for cell membrane staining. Experimental results show that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization. The immobilized enzyme molecules on the nanoparticle surface have shown excellent enzymatic activity in their respective enzymatic reactions. The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications.

Dye-doped nanoparticles for bioanalysis

Understanding biological processes at the molecular level with accurate quantitation needs advanced bioanalysis. In this review, we describe dye-doped silica nanoparticles (NPs), their synthesis, bioconjugation, and applications in different bioanalysis formats. Silica-based nanomaterials have been developed with optical-encoding capabilities for the selective tagging of a wide range of biomedically important targets, such as bacteria, cancer cells, and individual biomolecules. We also briefly review other closely related nanomaterials, such as quantum dots, Au NPs, and magnetic NPs. We envisage that further development of these NPs will provide a variety of advanced tools for molecular biology, genomics, proteomics, drug discovery, and diagnosis and therapy of infectious disease and cancer.

Cell-Specific Aptamer Probes for Membrane Protein Elucidation in Cancer Cells

Disease biomarkers play critical roles in the management of various pathological conditions of diseases. This involves diagnosing diseases, predicting disease progression and monitoring the efficacy of treatment modalities. While efforts to identify specific disease biomarkers using a variety of technologies has increased the number of biomarkers or augmented information about them, the effective use of disease-specific biomarkers is still scarce. Here, we report that a high expression of protein tyrosine kinase 7 (PTK7), a transmembrane receptor protein tyrosine kinase-like molecule, was discovered in a series of leukemia cell lines using whole cell aptamer selection. With the implementation of a two-step strategy (aptamer selection and biomarker discovery), combined with mass spectrometry, PTK7 was ultimately identified as a potential biomarker for T-cell acute lymphoblastic leukemia (T-ALL). Specifically, the aptamers for T-ALL cells were selected using the cell-SELEX process, without any prior knowledge of the cell biomarker population, conjugated with magnetic beads and then used to capture and purify their binding targets on the leukemia cell surface. This demonstrates that a panel of molecular aptamers can be easily generated for a specific type of diseased cells. It further demonstrates that this two-step strategy, that is, first selecting cancer cell-specific aptamers and then identifying their binding target proteins, has major clinical implications in that the technique promises to substantially improve the overall effectiveness of biomarker discovery. Specifically, our strategy will enable efficient discovery of new malignancy-related biomarkers, facilitate the development of diagnostic tools and therapeutic approaches to cancer, and markedly improve our understanding of cancer biology.

Aptamers from Cell-Based Selection for Bioanalytical Applications

Weihong Tan,*,†,‡ Michael J. Donovan,†,‡ and Jianhui Jiang*,†,‡ †Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Biology and College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, People’s Republic of China ‡Center For Research at Bio/nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611, United States