Fluorescent Functionalization across Quaternary Structure in a Virus-like Particle

Abstract

Proteinaceous nanomaterials and, in particular, virus-like particles (VLPs) have emerged as robust and uniform platforms that are seeing wider use in biomedical research. However, there are a limited number of bioconjugation reactions for functionalizing the capsids, and very few of those involve functionalization across the supramolecular quaternary structure of protein assemblies. In this work, we exploit the recently described dibromomaleimide moiety as part of a bioconjugation strategy on VLP Qβ to break and rebridge the exposed and structurally important disulfides in good yields. Not only was the stability of the quaternary structure retained after the reaction, but the newly functionalized particles also became brightly fluorescent and could be tracked in vitro using a commercially available filter set. Consequently, we show that this highly efficient bioconjugation reaction not only introduces a new functional handle "between" the disulfides of VLPs without compromising their thermal stability but also can be used to create a fluorescent probe.

One-pot thiol–amine bioconjugation to maleimides: simultaneous stabilisation and dual functionalisation

“…To overcome these limitations we, and others, have reported extensively on the use of dibromomaleimides (DBMs), [16][17][18][19][20][21][22][23] and related substituted maleimides known collectively as next generation maleimides (NGMs). 20,24 These reagents retain the favourable kinetics of maleimides 25,26 whilst avoiding the formation of stereoisomers, and through the addition of two thiols allow the construction of dual conjugates (Fig. 1B).…”

Site Selective Nucleation and Size Control of Gold Nanoparticle Photothermal Antennae on the Pore Structures of a Virus and Confined Drug Delivery

“…In our initial strategy, we assumed that reduction of the disulfides was occurring with sodium borohydride, but we were able to rule this out as we detected no such reduction by gel electrophoresis (Figure S8) nor was there any appreciable difference in the Ellman's assay before and after addition of borohydride (Figure S10). It is important to point out that, during the synthesis, even spectroscopically pure virus particles are not fully oxidized-that is to say, it appears from non-reducing SDS-PAGE that monomeric proteins exist when there should be none [55][56] (Figure S9). Curiously, reducing the disulfides to free sulfurs also fails to yield AuNP formation on the surface of the virus.…”

PhotoPhage: A Virus-based Photothermal Therapeutic Agent

“…The formulation is an effective photo-immunotherapy system in a triple negative breast cancer tumor model in BALB/c mice with complicating lung metastasis. Specifically, we show VLP Qβ (Figure 1A), a 30 nm icosahedral nanoparticle that can be expressed in high yields, possesses exceptional thermostability, and has multiple functional handles for bioconjugation, [49][50][51][52][53] serves as a good PhotoPhage following functionalization with hundreds of croconium dyes. Not only can Pho-toPhage sustain significant bulk photothermal heating in vitro and in vivo without denaturing, the nanoparticle formulations show a significant enhancement in photothermal performance over free dye and considerable improvements in solubility.…”