Yanrong Wu

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.

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]

DNA aptamer–micelle as an efficient detection/delivery vehicle toward cancer cells

We report the design of a self-assembled aptamer–micelle nanostructure that achieves selective and strong binding of otherwise low-affinity aptamers at physiological conditions. Specific recognition ability is directly built into the nanostructures. The attachment of a lipid tail onto the end of nucleic acid aptamers provides these unique nanostructures with an internalization pathway. Other merits include: extremely low off rate once bound with target cells, rapid recognition ability with enhanced sensitivity, low critical micelle concentration values, and dual-drug delivery pathways. To prove the potential detection/delivery application of this aptamer–micelle in biological living systems, we mimicked a tumor site in the blood stream by immobilizing tumor cells onto the surface of a flow channel device. Flushing the aptamer–micelles through the channel demonstrated their selective recognition ability under flow circulation in human whole-blood sample. The aptamer–micelles show great dynamic specificity in flow channel systems that mimic drug delivery in the blood system. Therefore, our DNA aptamer–micelle assembly has shown high potential for cancer cell recognition and for in vivo drug delivery applications.

Carbon Nanotubes Protect DNA Strands During Cellular Delivery

To protect against nuclease digestion, or single-strand binding protein interactions, oligonucleotides for targeted delivery into intracellular systems must be stable. To accomplish this, we have developed single-walled carbon nanotubes as a carrier for single-stranded DNA probe delivery. This has resulted in superior biostability for intracellular application and, hence, has achieved the desired protective attributes, which are particularly important when DNA probes are used for intracellular measurements. Specifically, when bound to single-walled carbon nanotubes, DNA probes are protected from enzymatic cleavage and interference from nucleic acid binding proteins. Moreover, and equally important, our study shows that a single-walled carbon nanotube-modified DNA probe, which targets a specific mRNA inside living cells, has increased self-delivery capability and intracellular biostability when compared to free DNA probes. Therefore, this new conjugate provides significant advantages for basic genomic studies in which DNA probes are used to monitor intracellular levels of molecules.

DNA‐Based Micelles: Synthesis, Micellar Properties and Size‐Dependent Cell Permeability

Functional nanomaterials based on molecular self-assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure, and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape, and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self-assemble into monodispersed, three-dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine-tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while retaining their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into the cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size-dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity are all excellent features that make our DNA-micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems.

Single-walled carbon nanotube as an effective quencher

Over the past few years, single-walled carbon nanotubes (SWNTs) have been the focus of intense research motivated by their unique physical and chemical properties. This review specifically summarizes recent progress in the development of fluorescence biosensors that integrate the quenching property of SWNTs and the recognition property of functional nucleic acids. SWNTs are substantially different from organic quenchers, showing superior quenching efficiency for a variety of fluorophores, with low background and high signal-to-noise ratio, as well as other advantages derived from the nanomaterial itself. As the second key component of biosensors, functional nucleic acids can bind to either their complementary DNA or a target molecule with the ability to recognize a broad range of targets from metal ions to organic molecules, proteins, and even live cells. By taking advantage of the strengths and properties of both SWNTs and nucleic acid based aptamers, a series of fluorescence biosensors have been designed and fabricated for the detection of a broad range of analytes with high selectivity and sensitivity.

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.

Nucleic Acid Beacons for Long-Term Real-Time Intracellular Monitoring

Real time intracellular monitoring of biological molecules inside living cells is important in many biomedical studies and reveals valuable information unobtainable by conventional molecular biology techniques. A variety of methods and molecular probes have been developed, but long term (from a few hours to days) intracellular monitoring with high sensitivity and selectivity is impossible and has not been accomplished. We have used locked nucleic acids (LNA) to engineer novel molecular beacons (MBs) for long-term intracellular monitoring. The LNA-MBs were made of a mixed LNA and DNA bases to have extremely high biostability. The new beacons were tested with MDA-MB-231 cancer cells and used effectively to monitor mRNA expression levels in real-time for 5-24 h. After 24 h inside living cells, the LNA-MBs were still functional, demonstrating a greatly enhanced stability enabling the measurement of intracellular gene expression over an extended period of time.

Locked nucleic acid based beacons for surface interaction studies and biosensor development.

DNA sensors and microarrays permit fast, simple, and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective, and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacons poor stability because of the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics, and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity, and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization.