Chaoyong James Yang

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.

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]

An Aptamer Cross-Linked Hydrogel as a Colorimetric Platform for Visual Detection†

US NIH ; China National Scientific Foundation of China [20805038, 20620130427]; National Basic Research Program of China [2007CB935603, 2010CB732402, 2009ZX10004-312]; ACS Division of Analytical Chemistry

High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets.

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.

Selection of DNA Aptamers against Epithelial Cell Adhesion Molecule for Cancer Cell Imaging and Circulating Tumor Cell Capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38 ± 9 and 67 ± 8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

Target-Responsive "Sweet" Hydrogel with Glucometer Readout for Portable and Quantitative Detection of Non-Glucose Targets

Portable devices with the advantages of rapid, on-site, user-friendly, and cost-effective assessment are widely applied in daily life. However, only a limited number of quantitative portable devices are commercially available, among which the personal glucose meter (PGM) is the most successful example and has been the most widely used. However, PGMs can detect only blood glucose as the unique target. Here we describe a novel design that combines a glucoamylase-trapped aptamer-cross-linked hydrogel with a PGM for portable and quantitative detection of non-glucose targets. Upon target introduction, the hydrogel collapses to release glucoamylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose for quantitative readout by the PGM. With the advantages of low cost, rapidity, portability, and ease of use, the method reported here has the potential to be used by the public for portable and quantitative detection of a wide range of non-glucose targets.

Twist2 contributes to breast cancer progression by promoting an epithelial-mesenchymal transition and cancer stem-like cell self-renewal

The epithelial to mesenchymal transition (EMT) is a highly conserved cellular programme that has an important role in normal embryogenesis and in cancer invasion and metastasis. We report here that Twist2, a tissue-specific basic helix-loop-helix transcription factor, is overexpressed in human breast cancers and lymph node metastases. In mammary epithelial cells and breast cancer cells, ectopic overexpression of Twist2 results in morphological transformation, downregulation of epithelial markers and upregulation of mesenchymal markers. Moreover, Twist2 enhances the cell migration and colony-forming abilities of mammary epithelial cells and breast cancer cells in vitro and promotes tumour growth in vivo. Ectopic expression of Twist2 in mammary epithelial cells and breast cancer cells increases the size and number of their CD44high/CD24low stem-like cell sub-populations, promotes the expression of stem cell markers and enhances the self-renewal capabilities of stem-like cells. In addition, exogenous expression of Twist2 leads to constitutive activation of STAT3 (signal transducer and activator of transcription 3) and downregulation of E-cadherin. Thus, the overexpression of Twist2 may contribute to breast cancer progression by activating the EMT programme and enhancing the self-renewal of cancer stem-like cells.

Label-Free Surface-Enhanced Raman Spectroscopy Detection of DNA with Single-Base Sensitivity

Direct, label-free detection of unmodified DNA is a great challenge for DNA analyses. Surface-enhanced Raman spectroscopy (SERS) is a promising tool for DNA analyses by providing intrinsic chemical information with a high sensitivity. To address the irreproducibility in SERS analysis that hampers reliable DNA detection, we used iodide-modified Ag nanoparticles to obtain highly reproducible SERS signals of single- and double-strand DNA in aqueous solutions close to physiological conditions. The phosphate backbone signal was used as an internal standard to calibrate the absolute signal of each base for a more reliable determination of the DNA structure, which has not been achieved before. Clear identification of DNA with single-base sensitivity and the observation of a hybridization event have been demonstrated.

A multifunctional nanomicelle for real-time targeted imaging and precise near-infrared cancer therapy

Simultaneous targeted cancer imaging, therapy and real-time therapeutic monitoring can prevent over- or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near-infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH-activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer-specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real-time imaging. Upon NIR irradiation, R16FP-mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.