The research in our lab focuses on the development of novel imaging strategies and probes to monitor specific biological processes under physiological settings. Towards this goal, we are taking an interdisciplinary approach, combining synthetic and physical organic chemistry, radiochemistry, and molecular biology  with imaging techniques such as fluorescence microscopy, whole body fluorescence/bioluminescence imaging, and positron emission tomography (PET).

The research process varies depending on the nature of biological targets under investigation. A typical project normally begins from organic synthesis of designed probes, in vitro biochemical and biophysical evaluation, and imaging the biological process in living cells and/or in whole living animals (often in mice).  

Ongoing projects at CMIL:

I. Development of nanosensors for imaging and detection of tumor cells

Quantum dots are fluorescent semiconductor nanocrystals, and their superior optical properties make them attractive as fluorescent imaging probes. In order to develop quantum dot-based nanosensors and apply them to imaging and detection of tumor cells, we are exploring several approaches outlined below.

While quantum dots cannot serve as the acceptor for fluorescence resonance energy transfer (FRET), we have recently demonstrated that quantum dots can serve as an acceptor for bioluminescence energy resonance transfer (BRET), and by conjugating a BRET donor, a mutant of a bioluminescent protein Renilla luciferase, we prepared a new class of quantum dot conjugates that can glow in dark without excitation. This self-illuminating quantum dot conjugate offers much greater sensitivity for in vivo imaging. We are now applying it to cancer targeting and imaging.

Quantum dots are typically at a size of 10-20 nm in diameter depending on the coating materials on the surface, and thus are not cell-permeable. After transporting molecules are conjugated with quantum dots, the conjugates are then able to cross cell membranes. We have shown that the transducing activity of transporting molecules may be modulated by specific enzymes. Matrix metalloproteases (MMPs), a large family of extracellular proteases, are implicated in almost of all types of solid tumors. We are currently applying this approach to image MMPs activity in vivo.

Conjugation of large biomolecules to quantum dots is a critical step in preparing these nanosensors. Current conjugation methods  often do not permit site-specific conjugation and may result in the loss of the activity of conjugated proteins. We are developing new chemistries for site-specific conjugation of proteins to nanoparticles. Our recent example includes the HaloTag protein-mediated immobilization of a bioluminescent protein on quantum dots. This method may allow specific protein conjugation to quantum dots in vivo.

II. Visualizing RNA and RNA splicing activity in vivo

The Tetrahymena group I intron ribozymes can catalyze splicing reactions, and have been applied to repair mutant mRNA transcripts in mammalian cells. This ribozyme-mediated RNA repair is attractive in that the endogenous regulation of the targeted gene is maintained (as the repair targets endogenous mutant mRNA transcripts). But the use of this technique is limited by the poor ribozyme activity in vivo. In our group, we have developed a method to visualize and evaluate the splicing activity of these ribozymes in single living mammalian cells  by linking the ribozyme splicing activity to the activity of a reporter. Using this tool, we are performing directed in vivo evolution of group I intron ribozymes to identify mutants with improved splicing potential. Such new ribozymes may eventually lead to effective RNA repairs for gene therapy.

While trans-splicing ribozymes can be applied to correct mutant mRNA transcripts, they may also be employed to image target mRNAs in vivo. To image the low copy number of a specific mRNA per cell, a strategy employing a mechanism of signal amplification offers better possibility of success. Based on Tetrahymena ribozyme, we devise two strategies to detect and image target mRNA. We are evaluating both strategies for imaging p53 mRNA in vivo.

Our ribozyme-mediated imaging of mRNAs is an indirect approach of imaging mRNA. To develop a method that can directly image specific mRNAs, we are adopting an aptamer-based approach. We are designing small organic dye molecules that are not fluorescent but become fluorescent after binding to a target RNA aptamer. We will apply this method to image target mRNA in real time and space to study mRNA translocation and dynamics.

III. Multimodality imaging of gene expression

Recent advances in reporter gene assays allow real-time watching gene expression in living subjects. Those assays use a variety of reporter genes, from fluorescent proteins, bioluminescence proteins, to virus thymidine kinases, and tranferin with different imaging modalities such as fluorescence, bioluminescence, positron emission tomography (PET), and magnetic resonance imaging (MRI). None of these reporter genes can be imaged with more than one imaging modalities. We are interested in developing a single reporter gene that can be imaged with several imaging modalities and at both single living cells and whole living animals. Currently, our design is based on a bacterial ampcillin resistant gene (amp^r) encoding the enzyme beta-lactamase. We are designing substrate probes for imaging beta-lactamase expression in living mice with whole body fluorescence imaging and PET.