Colin D. Medley

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.

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.

Aptamer-Conjugated Nanoparticles for Cancer Cell Detection

Aptamer-conjugated nanoparticles (ACNPs) have been used for a variety of applications, particularly dual nanoparticles for magnetic extraction and fluorescent labeling. In this type of assay, silica-coated magnetic and fluorophore-doped silica nanoparticles are conjugated to highly selective aptamers to detect and extract targeted cells in a variety of matrixes. However, considerable improvements are required in order to increase the selectivity and sensitivity of this two-particle assay to be useful in a clinical setting. To accomplish this, several parameters were investigated, including nanoparticle size, conjugation chemistry, use of multiple aptamer sequences on the nanoparticles, and use of multiple nanoparticles with different aptamer sequences. After identifying the best-performing elements, the improvements made to this assays conditional parameters were combined to illustrate the overall enhanced sensitivity and selectivity of the two-particle assay using an innovative multiple aptamer approach, signifying a critical feature in the advancement of this technique.

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.

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.

Monitoring Nucleic Acids Using Molecular Beacons

The ability to observe the dynamic of RNA in single living cells offers many exciting opportunities in biology and medicine. In the last few years, molecular beacons (MBs) have shown great potential in monitoring RNA synthesis, transportation, and localization with good sensitivity and selectivity. A hairpin structure probe, MB is a dual-labeled single stranded oligonucleotide that only fluoresces in the presence of target sequences. In this paper, the basic principle and design of MB will be described. The application of MB for RNA imaging in living cells will be reviewed. The limitations of MB for in vivo application will be identified. In the last section of the article, the efforts on designing better MBs for highly sensitive and selective RNA imaging will be discussed.

Stochasticity of Manganese Superoxide Dismutase mRNA Expression in Breast Carcinoma Cells by Molecular Beacon Imaging

Visual and quantitative monitoring of cell‐to‐cell variation in the expression of manganese superoxide dismutase (MnSOD) mRNA by using novel ratiometric imaging with molecular beacons (MB) reveals a distinct change in patterns following induction of human breast‐carcinoma cells with lipopolysaccharide. Interestingly, the pattern of cell‐to‐cell variation in a cell line stably transfected with a plasmid bearing a cDNA clone of MnSOD and overproducing the enzyme is significantly different from the pattern associated with MnSOD induction. The levels and the patterns of cell‐population heterogeneity for β‐actin mRNA expression do not show distinct changes either following induction or in stably transfected cells. These results are significant in light of the reported relationship between this enzyme and malignant phenotype of breast‐carcinoma cells. Use of MBs in ratiometric image analyses for cytoplasmic mRNAs represents a novel means of directly examining the stochasticity of transcription of MnSOD and other genes implicated in cellular phenotype regulation.

Antibody-functionalized nano test tubes target breast cancer cells

AIM To develop nano test tubes that will deliver a biomedical payload to a specific cell type. METHODS The template-synthesis method was used to prepare silica nano test tubes. An antibody that is specific for breast cancer cells was attached to the outer tube surfaces. A fluorophore was attached to the inner surfaces of the nano test tubes. The tubes were incubated with the breast cancer cells and the extent of attachment to the cell surfaces was investigated by fluorescence microscopy. RESULTS Tubes modified on their outer surfaces with the target antibody showed enhanced attachment to breast-cancer cells, relative to tubes modified on their outer surfaces with a species and isotype-matched control antibody. CONCLUSIONS This work is a first step toward demonstrating that nano test tubes can be used as cell-specific delivery vehicles.

A DNA-conjugated magnetic nanoparticle assay for assessing genotoxicity

The genotoxicity of a molecule refers to its ability to interact with DNA in a way that inhibits normal DNA replication and transcription possibly leading to mutagenesis or carcinogenesis. Assessing the genotoxicity of a compound is critical in the development of pharmaceuticals and other products designed for human consumption or use. Typically genotoxicity is established using expensive and time consuming methods using animals or bacteria like the Ames test, mouse lymphoma assay, or mouse and rat carcinogenicity tests. We have developed a magnetic nanoparticle-based assay that uses conjugated double-stranded DNA to serve as a substrate for interaction with genotoxic molecules. After application of a magnetic field, the genotoxic molecules are extracted with the DNA-conjugated magnetic nanoparticles. The genotoxic molecules can then be released and detected. To evaluate the potential of this assay, we have screened several genotoxic and non-genotoxic compounds and have demonstrated the ability to extract a genotoxic compound in the presence of a non-genotoxic molecule. The assay demonstrates suitable analytical performance and the ability to differentiate between genotoxic and non-genotoxic molecules providing a rapid and inexpensive alternative to more traditional methods of evaluating genotoxicity.