Research in the Rao lab uses physical, chemical and biological tools to develop novel imaging strategies. We develop new molecular probes to monitor specific biological targets under physiological settings, and in general our projects fit into one or more of the following three interconnected lines.
1. Enzyme activity-based in vivo imaging
Many receptors and enzymes play crucial roles in cellular and physiological functions. While receptors generally function by binding ligands, enzymes catalyze biochemical transformations. Employing a common strategy for imaging receptors, enzymes may also be imaged by binding labeled and irreversible inhibitors. Such an approach however does not take advantage of an enzyme's biological function, catalysis. Enzyme activity-based approaches rely upon and benefit from enzymatic function, creating signal amplification and producing high sensitivity with little perturbation to the biological functions of targetted enzymes. We have long-standing interests in imaging real-time enzyme activity in vivo. Three examples are outlined below.
Beta-lactamases are a class of bacterial enzymes responsible for bacterial resistance to beta-lactam containing drugs, notably penicillins. We have developed small, activatable fluorogenic and bioluminogenic substrates designed to image and detect beta-lactamase activity in living cells and mice. (Angew Chem 46:7031) (JACS 127:4158)
We currently collaborate with microbiologists at Texas A&M to develop next generation beta-lactamase probes that can detect Mycobacterium tuberculosis (TB) in small volumes of biological samples, such as sputum samples, with high sensitivity and specificity. We are also working to directly observe real-time TB viability and localization during infection, and using preclinical models to evaluate the efficacy of therapeutics.(PNAS 27:107) We will next non-invasively image TB in patients to monitor treatment efficacy and to develop new diagnositic tests.
Proteolytic processing of biomolecules is one of the most common kinds of posttranslational modifications, often functionally activating biological molecules. Such processing is therefore important in many cellular and physiological contexts. Aberrant protease activity is often observed in diseases such as cancers and arthritis. For example, matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases crucial for regulated degradation and processing of extracellular matrices, as well as many other biological processes. Over-expression of MMPs has been observed in nearly every type of human cancer, and found to correlate with advanced tumor stage, increased cancer invasion and metastasis, and shortened patient survival times.
We develop novel sensors and probes for in vitro detection and in vivo imaging of MMP activity. Centered upon fluorescent semiconductor quantum dots (QDs) and bioluminescence resonance energy transfer (BRET), we developed a QD-BRET detection system for highly sensitive detection of MMPs in vitro. (Anal Chem 80:8649) Currently we design new strategies for multimodality imaging of MMPs and other proteases. (Nano Lett 6:1988)
Tetrahymena group I intron ribozymes are RNA molecules that catalyze splicing reactions, and that were applied to repair mutant mRNA transcripts in mammalian cells. Ribozyme-mediated RNA repair is a specific repair process in the sense that it preserves the endogenous regulation of genes by targetting only mutanted mRNA transcripts. We have developed a genetically encoded reporter to visualize and evaluate the splicing activity of these ribozymes in single, living mammalian cells and in living whole animals by linking the ribozyme splicing activity to the activity of a reporter enzyme, luciferase or beta-lactamase. (Chembiochem 7:925) (PNAS 100:14892) (JACS 126:7158) This novel reporter system was used to screen for ribozyme mutants with improved splicing activitiy from a ribozyme library. Such new ribozymes may allow development of effective RNA repair applications for gene therapy, as well as in vivo imaging of targetted mRNAs and studies of mRNA inhibition by small interference RNAs (siRNAs).
2. In vivo protein and RNA labeling
A second major area of interest in the lab is developing general strategies to label proteins and RNAs in living cells for in vivo imaging. Our approach is to design small organic dye molecules that are not fluorescent initially but that become fluorescent after binding to a receptor or tag fused to either a protein or RNA molecule of interest. While the idea seems straightforward, designing novel probes that bind new receptors or tags with high affinity and selectivity remains a critical challenge throughout the research world. We are combining rational design and library selection methods, such as SELEX and phage display, to develop new in vivo labeling systems. In collaboration with W.E. Morener’s group in the Chemistry department, we are evaluating new labeling strategies for super high resolution single-molecule imaging in living cells. (BBRC 374:419) (Chembiochem 9:2682)
3. New sensing and imaging technologies
Our third research focus is to develop novel sensing and imaging technologies. We mimicked the naturally occurring bioluminescence resonance energy transfer (BRET) system in the sea pansy, Renilla reniformis. This species expresses a bioluminescent enzyme that non-radiatively transfers its biochemical energy generated by substrate oxidation to a fluorescent acceptor molecule, green fluorescent protein (GFP). We employed fluorescent nanocrystals, quantum dots, as BRET acceptors, and developed a QD-BRET technology widely applicable for in vitro biosensing and in vivo imaging.(Nature Biotech 24:339) (Nature Protocols 1:1160) (Curr Opin Biotechnol 18:17)
Our QD-BRET technology eliminates the need for external illumination or excitation when using QDs for in vivo imaging, thus avoiding the problems of high background autofluorescence during whole-body fluorescence imaging. We are exploring this technology as a general nanoplatform to examine a variety of biological processes in vitro and in vivo.