Combining chemoselective ligation with polyhistidine-driven self-assembly for the modular display of biomolecules on quantum dots

Abstract

One of the principle hurdles to wider incorporation of semiconductor quantum dots (QDs) in biology is the lack of facile linkage chemistries to create different types of functional QD--bioconjugates. A two-step modular strategy for the presentation of biomolecules on CdSe/ZnS core/shell QDs is described here which utilizes a chemoselective, aniline-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto peptides and DNA. This specifically provides them the ability to ratiometrically self-assemble to hydrophilic QDs. The versatility of this labeling approach was highlighted by ligating proteolytic substrate peptides, an oligoarginine cell-penetrating peptide, or a DNA-probe to cognate hexahistidine peptidyl sequences. The modularity allowed subsequently self-assembled QD constructs to engage in different types of targeted bioassays. The self-assembly and photophysical properties of individual QD conjugates were first confirmed by gel electrophoresis and Forster resonance energy transfer analysis. QD-dye-labeled peptide conjugates were then used as biosensors to quantitatively monitor the proteolytic activity of caspase-3 or elastase enzymes from different species. These sensors allowed the determination of the corresponding kinetic parameters, including the Michaelis constant (K(M)) and the maximum proteolytic activity (V(max)). QDs decorated with cell-penetrating peptides were shown to be successfully internalized by HEK 293T/17 cells, while nanocrystals displaying peptide--DNA conjugates were utilized as fluorescent probes in hybridization microarray assays. This modular approach for displaying peptides or DNA on QDs may be extended to other more complex biomolecules such as proteins or utilized with different types of nanoparticle materials.

Figure 1

(A) Schematic of the aniline-catalyzed hydrazone ligation between aldehyde and hydrazine functionalities color-coded in blue and red, respectively. (B) Schematic of the two-step, modular design for the attachment of biomolecules to the surface of CdSe/ZnS core/shell QDs surface functionalized with charged DHLA or neutral DHLA-PEG ligands. Initially, a hexahistidine-appended, synthetic, starter peptide is ligated to the appropriately functionalized target biomolecule of interest using chemoselective ligation. Isolated conjugates are then ratiometrically self-assembled to QDs for use in targeted bioassays. (C) Structures of DHLA (deprotonated/negatively charged) and DHLA-PEG₆₀₀ QD surface modification ligands.

Figure 2

Chemical structures of starter and modified peptides (1a, 1b, 1d), DNA sequence (1c), and resulting chemoselective ligates (3–6) formed using aniline-catalyzed hydrazone coupling along with the corresponding assays they were assayed in. The location of the proteolytic sites present in 1a,b are highlighted in green and appropriate dye-labels are shown at the points of linkage to the peptides/DNA.

Figure 3

Representative FRET-data for the QD-conjugate systems. Absorption and emission spectra for 550 nm QDs and Texas Red (A), 520 nm QDs and AlexaFluor555 (B), and 550 nm QDs and TAMRA (C). Deconvoluted and direct-acceptor excitation corrected emission spectra for each QD donor-dye-labeled acceptor system for the relevant increasing number of self-assembled peptide conjugate 3 per 550 nm QD (D), peptide conjugate 4 per 520 nm QD (E), conjugate 5 per 550 nm QD (F). QD spectra were fit with a Gaussian profile. Normalized QD PL loss vs. n (acceptor valence) as indicated, FRET efficiency E, Poisson-corrected FRET E and acceptor reemission for each of the QD-conjugate systems are shown in corresponding panels G–I.

Figure 4

Representative proteolytic activity curves. (A) 520 nm QD DHLA-conjugate 4 assemblies were used to assay recombinant human caspase-3 activity under excess substrate conditions as described. Data from monitoring 580 nm QD DHLA-PEG₆₀₀-conjugate 3 assemblies digested with human neutrophil elastase (B) or porcine pancreatic elastase (C), both in excess enzyme formats. Insets show a close-up of the initial activities at low enzyme concentrations. Estimated K_M and V_max values derived for caspase-3 are shown along with the V_{max. app.} for the elastase assays.

Figure 5

Representative images of HEK 293T/17 cells incubated at 37°C for 1 hour with 50 nM of 550 nm QD DHLA-PEG₆₀₀ conjugated to CPP-ligate 6 (A) or starter peptide 2c (B) as a control. For each panel the corresponding differential interference contrast (DIC), QD fluorescence (~550 nm), AF647-Tfn fluorescence (~670 nm), DAPI fluorescence (~460 nm), and merged composite images are shown.

Figure 6

Images of microarrays hybridized with peptido-DNA ligate 5 self-assembled to 550 nm QDs surface functionalized with DHLA-PEG₆₀₀ (A) or DHLA (B). The slides were pre-spotted with varying concentrations of the ctxA sequence (0.08–250 pmol/μL, four replicate spots). Note, ctxB spots were used as a negative control.