Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates

Using a synthetic peptide modeled after the protein that the human immunodeficiency virus (HIV) uses to enter cells, a multi-institutional research team has created quantum dots that can penetrate the cell membrane and image internal structures in a cell.

This research team has also used peptide-coated quantum dots to monitor enzymatic activity within a cell.

In papers published in the journal Bioconjugate Chemistry and Nature Materials, researchers at the U.S. Naval Research Laboratory, Johns Hopkins University, and The Scripps Research Institute reported on their studies of developing a general method for creating self-assembled peptide-quantum dot conjugates for use in a variety of biomedical research applications. Their method involves two key steps. The first step is to create quantum dots and then coat them with the molecule dihydrolipoic acid (DHLA), a biocompatible, water-soluble molecule. The second is to attach a short peptide composed entirely of the amino acid histidine to one end of a larger peptide with a specific application, such as targeting. Poly(histidine) binds tightly to DHLA, so that when a poly(histidine)-modified peptide and DHLA-coated quantum dots are mixed the two components self-assemble into a sturdy nanosized particle.

To demonstrate the utility of this method, the researchers developed two different quantum-dot based research tools. In one application, the researchers created multiple colors of cadmium selenide quantum dots coated with TAT, the peptide that HIV uses to enter human cells. They then used these peptide-quantum dot conjugates to study how quantum dots move inside cells as a model for gaining a better understanding of nanoparticle trafficking within cells. The researchers note that this proof-of-principle experiment suggests that it should be possible to use this self-assembly process to create multifunctional quantum dots coated with more than one peptide, or to use uniquely colored quantum dots, each coated with a different peptide, to image multiple functions within a single cell.

In a second demonstration, the investigators used several different peptides to detect and quantify the activity of four different proteases using fluorescence energy resonance transfer (FRET). Proteases are enzymes that degrade specific proteins and measurements of protease activity are used to identify some types of malignant cells.

To create these FRET sensors, the investigators attached peptides containing cleavage sites for the various enzymes and a dye molecule that quenches the light emission of the quantum dot by absorbing the fluorescent energy released by a stimulated quantum dot. When a particular protease is present in an assay mixture, it cleaves the peptide, releasing the dye molecule. When the dye is no longer in close proximity to the quantum dot, the quantum dot fluoresces brightly. By using quantum dots of four different colors, each linked to a peptide substrate for one of the four proteases, the researchers were able to create an assay capable of simultaneously measuring the activity of each of the four enzymes present in a single assay solution. The investigators also showed that they could use these peptide-quantum dot conjugates in drug discovery assays aimed at identifying protease inhibitors.

The work on intracellular delivery is detailed in a paper titled, “Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery.” This paper was published online in advance of print publication. An abstract is available at the journal’s website. View abstract.

The work on monitoring protease activity is detailed in a paper titled, “Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates.” This paper was published online in advance of print publication. An abstract is available through PubMed. View abstract.