QDS · New strategies for bioconjugation to quantum dots. Study of protein-nucleic acids and protein-protein interactions using fluorescence resonance energy transfer through quantum-dot-protein conjugates

Periodic Report Summary - QDS (New strategies for bioconjugation to quantum dots. Study of protein-nucleic acids and protein-protein interactions using fluorescence resonance energy transfer ...)

Semiconductor quantum dots (QDs) are a relatively novel class of nanomaterials with fascinating properties. More precisely, water solubilised QDs have a great potential in bioimaging and sensing applications due to their photophysical characteristics. However, the efficient functionalisation of QDs with complex biomolecules (proteins, nucleic acids) represents a significant challenge. We envision that the use of highly chemoselective reactions in aqueous solution will have a wide application for the conjugation of biomolecules to nanoparticles (NPs). We will apply straightforward arylhydrazone and Wittig reactions for the chemoselective covalent modification of QDs that are compatible with neutral pH and micro- / millimolar concentrations of the peptide targets. Furthermore, efficient cellular uptake and endosomal escape is a major challenge in nanoparticle delivery that we will address using a combination of polyprolines, positively charged amino acids and hydrophobic tails. Luminescent core-shell CdSe-ZnS QDs are typically stabilised by acidic-thiolated ligands or amphiphilic block copolymers in aqueous media. These ligands confer solubility and can be labeled in a nonselective manner to functional groups such as amines present on biomolecules. However, the efficient and controlled covalent conjugation of biomolecules to QDs and other NPs still requires further investigation. One of the reactions that have shown good chemoselectivity in aqueous buffer is hydrazone ligation. To facilitate rapid and quantitative conjugation of peptides to QDs, we selected the reaction between 4-formylbenzoyl group (4FB) and 2-hydrazinonicotinoyl (HYNIC). Benzaldehyde QDs were self-assembled from DHLA-PEG600 QDs with peptide 1 containing an N-terminal 4FB aldehyde (4FB-Ahx-Pro9Gly2His6, Ahx=Amino-hexanoyl) and ligated to protease substrate peptide 2 (HYNIC-GLYRGSGEGC-TAMRA). The utility of this new bioconjugation method for QDs was further demonstrated by carrying out enzymatic assays with either trypsin or chymotrypsin and the ligated peptide on the QD. The cleavage was monitored following QD donor photoluminiscence and Vmax and the KM were determined. The Wittig reaction takes place between a phosphorus ylide and an aldehyde/ketone resulting in the formation of a double bond. We found out that monosulfonated-water soluble phosphines react with N-terminal bromoacetyl-peptide derivatives 4 (BrAc-GERAFS) forming the desired Wittig-ylide type peptides at neutral pH and room temperature, achieving fast reaction times at millimolar concentrations. Furthermore, these ylides can ligate N-terminal glyoxilic-peptides 5 (CHOCO-GLYRAK) under the same smooth conditions. To evaluate the real scope of the Wittig ligation, we performed a variety of ligations on Myoglobin, a 17 KDa protein. N-terminal glycine was transaminated with pyridoxal phosphate and subsequently ligated to: a 5-mer peptide, a rhodamine labeled peptidic substrate, a Gadolinium complex used in MRI and a biotin derivatise d with a minipegylated linker. Delivery of QDs to intracellular medium has been a major field of investigation. We have recently discovered that assembling multiple copies of peptide 6 (Ac-WGDap(Palm)VKIKKP9G2H6, Dap = diaminopropionic, Palm = palmitate) to QDs leads to a fast internalisation and later escape of the QDs-6 conjugates, reaching a release peak around 48 h post-incubation. The results obtained so far have already been published in top journals: J. Am. Chem. Soc., ACS Nano, Bioconjugate Chemistry, Nature Materials. In addition, we have attended and presented the results in several symposiums. It is expected that the knowledge developed could be transferred as technology to biotech companies, specially important is the development of strategies that allow efficient internalisation of NPs in order that these can reach specific targets in altered cells and inactivate them, avoiding possible alterations and subsequent malfunctioning of the cellular machinery.

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