Preparation and Biological Properties of Oligonucleotide-Functionalized Virus-like Particles

Oligonucleotides are powerful molecules for programming function and assembly. When arrayed on nanoparticle scaffolds in high density, the resulting molecules, spherical nucleic acids (SNAs), become imbued with unique properties. We used the copper-catalyzed azide–alkyne cycloaddition to graft oligonucleotides on Qβ virus-like particles to see if such structures also gain SNA-like behavior. Copper-binding ligands were shown to promote the click reaction without degrading oligonucleotide substrates. Reactions were first optimized with a small-molecule fluorogenic reporter and were then applied to the more challenging synthesis of polyvalent protein nanoparticle–oligonucleotide conjugates. The resulting particles exhibited the enhanced cellular uptake and protection from nuclease-mediated oligonucleotide cleavage characteristic of SNAs, had similar residence time in the liver relative to unmodified particles, and were somewhat shielded from immune recognition, resulting in nearly 10-fold lower antibody titers relative to unmodified particles. Oligonucleotide-functionalized virus-like particles thus provide an interesting option for protein nanoparticle-mediated delivery of functional molecules.


Qb-(AF647)30(azide)550
To a solution of Qβ (1 mL from 8 mg/mL stock solution in 1x PBS, pH 7.4; 0.57 µmol in coat protein (CP) subunit), AFDye 647 NHS ester (16 µL from 25 mM stock solution in DMSO; approx. 0.7 equivalents relative to CP subunit concentration) was slowly added in a microcentrifuge tube. The reaction mixture was wrapped in foil to protect from light and placed on a slowly rotating rotisserie shaker at room temperature (r.t.). After 2 h, azido-PEG4-NHS ester (160 µL from 250 mM stock in DMSO; approx. 70 equivalents relative to CP subunits) was added to the reaction mixture without purification. The reaction was let proceed for another 2 h at room temperature, and the resulting VLPs were purified using PD-10 Sephadex G-25 columns. Eluted particles were concentrated using Amicon Ultra-4 10 kDa centrifugal filters and passed through 0.2 µm PTFE syringe filters. Protein recovery was quantified using the Coomassie Plus Bradford Assay Kit (Pierce). The density of particle-displayed azide linkers was determined by ESI-TOF HRMS (550 ± 50 azides per particle, 3.1 ± 0.3 per subunit), and the density of particle-displayed AF647 dyes was determined by UV-vis spectroscopy. Particle integrity was assessed through DLS and FPLC.

Representative procedure for Qb-DNA conjugation using THPTA
To a solution of Qβ-(AF647)30(azide)500 (0.1 mL from 6 mg/mL stock solution in 1x PBS; 38 nmol CP subunit, 106 nmol reactive azides) was added oligonucleotide-alkyne (17 µL from a 10 mM solution in nuclease-free water; 1.6 equivalents relative to particle-azide), a premixed solution of 5:1/THPTA:Cu (8.5 µL from a solution of 450 mM THPTA and 90 mM Cu(II)SO4 in water; 7.2 equivalents copper relative to particle-azide), aminoguanidine (8.5 µL from a 1 M stock in water; approx. 80 equivalents to azide), and sodium ascorbate (8.5 µL from a freshly prepared 1 M stock in water; approx. 80 equivalents to azide). Particles were mixed by gentle pipetting, spun down, and incubated at 50 °C for 3 h in a thermocycler. VLP-oligonucleotide conjugates were purified by using PD-10 Sephadex G-25 column, followed by repeated cycles of centrifugation against Amicon Ultra-4 100 kDa centrifugal filters. Protein recovery was quantified by Bradford assay, as described above. V-SNAs were characterized by UV-Vis spectroscopy and by microchip electrophoresis using the Protein 80 kit on a 2100 BioAnalyzer (Agilent) to determine the loading density of oligonucleotides on particles, and by DLS and FPLC to determine particle integrity.

Representative procedure for Qb-(18mer)270 conjugation using BimC4A
To a solution of Qβ-(AF647)30(azide)500 (5 µL from 29 mg/mL stock; 9.2 nmol CP subunit, 25.6 nmol reactive azides) was added oligonucleotide-alkyne (8.6 µL from 6 mM stock; 2.0 equivalents relative to particle-azide), BimC4A (8.4 µL from 4.2 mM stock; 35.3 nmol), CuSO4 (8.4 µL from 8.4 mM stock; 70.6 nmol, 2 equivs relative to ligand), aminoguanidine (2 µL from a 1 M stock in water; approx. 80 equivalents to azide), and sodium ascorbate (2 µL from a freshly prepared 1 M stock in water; approx. 80 equivalents to azide). Particles were mixed by gentle pipetting, spun down, and incubated at 50 °C for 3 h in a thermocycler. Mixtures of the BimC4A ligand and copper in water form precipitates that can be resuspended by dilution to 1-2 mM in water.         Figure S9. Z-stack image series for C166 treated with 5 nM Qb-VLPs for 180 minutes (data also shown in Figure 4a). The '0 µm' position was selected based on VLP-binding to the bottom of the plate. The contrast in the 'Qβ' image series was adjusted independently.

In vitro microscopy
Figure S10. Images taken from z-stack analysis of HeLa cells treated with 5 nM Qb-VLPs for 180 minutes, as described above for Supplemental Movies S4-S6. In the right-hand image (unmodified Qb-VLPs), arrows point to examples of staining of the outer cell surface by the particles, a pattern that is not observed with DNA-decorated VLPs; the contrast in the right-hand image was adjusted independently.
from Supplemental Movie S6 from Supplemental Movie S5 from Supplemental Movie S4