Min-kyung So

Self-illuminating quantum dot conjugates for in vivo imaging.

Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 103 for 5 pmol of conjugate.

Multiplex Detection of Protease Activity with Quantum Dot Nanosensors Prepared by Intein-Mediated Specific Bioconjugation

We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few nanograms per milliliter and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown.

Creating self-illuminating quantum dot conjugates

Semiconductor quantum dots are inorganic fluorescent nanocrystals that, because of their unique optical properties compared with those of organic fluorophores, have become popular as fluorescent imaging probes. Although external light excitation is typically required for imaging with quantum dots, a new type of quantum dot conjugate has been reported that can luminesce with no need for external excitation. These self-illuminating quantum dot conjugates can be prepared by coupling of commercially available carboxylate-presenting quantum dots to the light-emitting protein Renilla luciferase. When the conjugates are exposed to the luciferases substrate coelenterazine, the energy released by substrate catabolism is transferred to the quantum dots through bioluminescence resonance energy transfer, leading to quantum dot light emission. This protocol describes step-by-step procedures for the preparation and characterization of these self-illuminating quantum dot conjugates. The preparation process is relatively simple and can be done in less than 2 hours. The availability of self-illuminating quantum dot conjugates will provide many new possibilities for in vivo imaging and detection, such as monitoring of in vivo cell trafficking, multiplex bioluminescence imaging and new quantum dot-based biosensors.

HaloTag protein-mediated specific labeling of living cells with quantum dots

Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging.

Improved QD-BRET conjugates for detection and imaging

Self-illuminating quantum dots, also known as QD-BRET conjugates, are a new class of quantum dot bioconjugates which do not need external light for excitation. Instead, light emission relies on the bioluminescence resonance energy transfer from the attached Renilla luciferase enzyme, which emits light upon the oxidation of its substrate. QD-BRET combines the advantages of the QDs (such as superior brightness and photostability, tunable emission, multiplexing) as well as the high sensitivity of bioluminescence imaging, thus holding the promise for improved deep tissue in vivo imaging. Although studies have demonstrated the superior sensitivity and deep tissue imaging potential, the stability of the QD-BRET conjugates in biological environment needs to be improved for long-term imaging studies such as in vivo cell tracking. In this study, we seek to improve the stability of QD-BRET probes through polymeric encapsulation with a polyacrylamide gel. Results show that encapsulation caused some activity loss, but significantly improved both the in vitro serum stability and in vivo stability when subcutaneously injected into the animal. Stable QD-BRET probes should further facilitate their applications for both in vitro testing as well as in vivo cell tracking studies.

Imaging Target mRNA and siRNA-Mediated Gene Silencing In Vivo with Ribozyme-Based Reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans‐splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme‐mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180‐fold, and by ten‐fold for the stably expressed target. siRNA‐mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme‐based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.