Dihua Shangguan

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.

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.

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.

Molecular Assembly of an Aptamer–Drug Conjugate for Targeted Drug Delivery to Tumor Cells

Special delivery! An aptamer‐directed anticancer drug was molecularly engineered to be delivered to target cells for efficient therapeutic application. The covalent conjugation of drug and aptamer creates alternative opportunities for targeted therapy, as multiple yet specific aptamers can be “generated” relatively easily by cell‐SELEX for any target cells; this demonstrates the full potential of cell‐SELEX as a molecular discovery tool for biomedical studies and drug development.

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.

Aptamer Directly Evolved from Live Cells Recognizes Membrane Bound Immunoglobin Heavy Mu Chain in Burkitt's Lymphoma Cells

The identification of tumor related cell membrane protein targets is important in understanding tumor progression, the development of new diagnostic tools, and potentially for identifying new therapeutic targets. Here we present a novel strategy for identifying proteins that are altered in their expression levels in a diseased cell using cell specific aptamers. Using an intact viable B-cell Burkitts lymphoma cell line (Ramos cells) as the target, we have selected aptamers that recognize cell membrane proteins with high affinity. Among the selected aptamers that showed different recognition patterns with different cell lines of leukemia, the aptamer TD05 showed binding with Ramos cells. By chemically modifying TD05 to covalently cross-link with its target on Ramos cells to capture and to enrich the target receptors using streptavidin coated magnetic beads followed by mass spectrometry, we were able to identify membrane bound immunoglobin heavy mu chain as the target for TD05 aptamer. Immunoglobin heavy mu chain is a major component of the B-cell antigen receptor, which is expressed in Burkitts lymphoma cells. This study demonstrates that this two step strategy, the development of high quality aptamer probes and then the identification of their target proteins, can be used to discover new disease related potential markers and thus enhance tumor diagnosis and therapy. The aptamer based strategy will enable effective molecular elucidation of disease related biomarkers and other interesting molecules.

Molecular Recognition of Small‐Cell Lung Cancer Cells Using Aptamers

Early diagnosis is the way to improve the rate of lung cancer survival, but is almost impossible today due to the lack of molecular probes that recognize lung cancer cells sensitively and selectively. We developed a new aptamer approach for the recognition of specific small‐cell lung cancer (SCLC) cell‐surface molecular markers. Our approach relies on cell‐based systematic evolution of ligands by exponential enrichment (cell‐SELEX) to evolve aptamers for whole live cells that express a variety of surface markers representing molecular differences among cancer cells. When applied to different lung cancer cells including those from patient samples, these aptamers bind to SCLC cells with high affinity and specificity in various assay formats. When conjugated with magnetic and fluorescent nanoparticles, the aptamer nanoconjugates could effectively extract SCLC cells from mixed cell media for isolation, enrichment, and sensitive detection. These studies demonstrate the potential of the aptamer approach for early lung cancer detection.

Molecular recognition of acute myeloid leukemia using aptamers

Cell surface proteins can play important roles in cancer pathogenesis. Comprehensive understanding of the surface protein expression patterns of tumor cells and, consequently, the pathogenesis of tumor cells depends on molecular probes against these proteins. To be used effectively for tumor diagnosis, classification and therapy, such probes would be capable of specific binding to targeted tumor cells. Molecular aptamers, designer DNA–RNA probes, can address this challenge by recognizing proteins, peptides and other small molecules with high affinity and specificity. Through a process known as cell-based SELEX, we used live acute myeloid leukemia (AML) cells to select a group of DNA aptamers, which can recognize AML cells with dissociation constants (Kds) in the nanomolar range. Interestingly, one aptamer (KH1C12) compared with two control cell lines (K562 and NB4) showed significant selectivity to the target AML cell line (HL60) and could recognize the target cells within a complex mixture of normal bone marrow aspirates. The other two aptamers KK1B10 and KK1D04 recognize targets associated with monocytic differentiation. Our studies show that the selected aptamers can be used as a molecular tool for further understanding surface protein expression patterns on tumor cells and thus providing a foundation for effective molecular analysis of leukemia and its subcategories.

Carbon Dots Based Dual-Emission Silica Nanoparticles as a Ratiometric Nanosensor for Cu2+

A simple and effective strategy for designing ratiometric fluorescent nanosensor has been described in this work. A carbon dots (CDs) based dual-emission nanosensor for Cu(2+) detection was prepared by coating CDs on the surface of Rhodamine B-doped silica nanoparticles. The fluorescent CDs were synthesized using N-(β-aminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS) as the main raw material, so that the residual ethylenediamine groups and methoxysilane groups on the surface of CDs can serve as the Cu(2+) recognition sites and the silylation reaction groups. The obtained nanosensor showed characteristic fluorescence emissions of Rhodamine B (red) and CDs (blue) under a single excitation wavelength. Upon binding to Cu(2+), only the fluorescence of CDs was quenched, resulting in the ratiometric fluorescence response of the dual-emission silica nanoparticles. This ratiometric nanosensor exhibited good selectivity to Cu(2+) over other substances, such as metal ions, amino acids, proteins, and vitamin C. The ratio of F467/F585 linearly decreased with the increasing of Cu(2+) concentration in the range of 0 to 3 × 10(-6) M, a detection limit as low as 35.2 nM was achieved. Additionally, this nanosensor was successfully applied for the ratiometric fluorescence imaging of Cu(2+) in cells and determination of Cu(2+) in real tap water.

Optimization and modifications of aptamers selected from live cancer cell lines.

Aptamers are single-stranded oligonucleotides derived from an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment). 2] The selected aptamers can recognize their target molecules with high affinity and specificity by folding into well-defined three-dimensional shapes. Recently, using a human T-cell acute lymphoblastic leukemia cell line as target, we generated a group of aptamers for the specific recognition of leukemia cells. The aptamers have an equilibrium dissociation constant (Kd) in the nm to pm range. They can specifically recognize target leukemia cells that have been mixed with normal human bone marrow aspirates, can identify cancer cells closely related to the target cell line in clinical specimens, and can also be used to enrich target cells spiked in blood samples. 6] These aptamers are very promising for molecular recognition in a variety of applications. Unlike antibodies, aptamers have low molecular weight, fast tissue penetration, and low toxicity, and can be reproducibly produced with a DNA synthesizer. They can be easily labeled with radioscopic, fluorescent, or other reporters. Moreover, aptamers remain stable during long-term storage and sustain reversible denaturation. These advantages make aptamers uniquely suitable as molecular probes for diagnosis or as drugs for targeted cancer therapy. Even though regular DNA molecules are sturdier than antibodies in vitro, in vivo stability is still a serious problem. To serve as effective therapeutic and diagnostic tools, aptamers must resist rapid degradation by exoand endonucleases. In this paper, we present the results of experiments for the design of aptamers with excellent biostability, affinity, and specificity for the study of diseases. Full-length aptamers generated by the SELEX process contain 70–100 nucleotides, which include two fixed primer sequences at each terminus for PCR amplification. Generally, not all nucleotides are necessary for direct interaction with the target or for folding into the structure that facilitates target binding. There is no doubt that longer sequences result in lower yield and higher cost in synthesis. The unnecessary nucleotides could also possess a higher probability of forming various secondary structures that destabilize the target-binding conformation of the aptamer. Additionally, reduced size has been reported to increase tissue penetration rates. Thus, in practical usage, it is always beneficial to obtain minimized sequences of aptamers that possess the same or better binding affinity to the target compared to the original full-length aptamer. For RNA aptamers, RNase footprinting or partial hydrolysis has been performed to determine the boundary and binding site of aptamers. For DNA aptamers, the minimal sequence has been determined by partially fragmenting a full-length aptamer, and then selecting the fragments that retained high affinity for the target. In these methods, radioactive labeling was needed to detect the aptamer fragments, and then the predicted potential minimal sequences had to be synthesized to confirm binding capacity. In this study, we utilized a relatively easy method to determine the secondary structures of aptamers as well as the critical sequences for target binding. At the end of the selection process, the enriched pool with high affinity to the target was sequenced. After alignment, the sequences were found to distribute into different families based on their sequence similarities, and many repeats were observed in each family. In the same family they sometimes showed very different affinities for the target even though sequences had the same motif and the difference was only a few nucleotides. The relationship ACHTUNGTRENNUNGbetween the sequences of the same family and their affinities can help us deduce the secondary structure of the aptamer that is needed for target binding. The potential secondary structures of DNA sequences can be predicted by a few algorithms. The free energy change (DG) of each structure was also calculated as an indication of structural stability. However, the lowest DG did not necessarily correspond with the actual target-binding structure, since target-induced refolding of the aptamer is common in aptamer–target interactions. On the other hand, sequences of the same family should usually bind to the same target with similar secondary structures but different Kd values. Thus, when searching for the target-binding structure, we gave higher priority to the calculated structures that were present for every sequence in the same family. Further analysis determined the target-binding structure which correlated well with the differences in the Kd values of the ACHTUNGTRENNUNGsequences. As an example, the two sequences Sga16 (Kd=5 nm) and Sgc8 (Kd=0.8 nm) in one of the sequence families, only differed by two nucleotides (G38, A69 in Sga16, and A38, T69 in Sgc8; Figure 1A). There are 11 potential structures of aptamer Sgc8 and thirteen of Sga16, as predicted by a program available on the internet. Among these structures, only two pairs of structures (Figure S1 in the Supporting Information) were commonly present. By further comparing the two structure pairs, we found that one structure pair could explain the different Kd values of Sga16 and Sgc8 (Figure 1A). In this structure pair, the differences in base sequence are in the stem section between loops 1 and 2 (indicated by arrows in Figure 1A). Namely, aptamer Sgc8 has a perfect-match stem while aptamer Sga16 has a one-base-mismatch stem. The mismatched base [a] Dr. D. Shangguan, Dr. Z. Tang, P. Mallikaratchy, Z. Xiao, Prof. Dr. W. Tan Department of Chemistry and Shands Cancer Center UF Genetics Institute and McKnight Brain Institute Center for Research at Bio/nano Interface, University of Florida Gainesville, FL 32611 (USA) Fax: (+1)352-846-2410 E-mail : tan@chem.ufl.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.