Developing DNA origami based fluorescent protein assemblies using two-point fixation, as an accessible tool for quantum simulation

Despite recent advances in sensing and metrology for biomedicine, it is still challenging to experimentally assess delicate quantum phenomena in biological systems. Recently, the use of fluorescent proteins as a suitable model system to study these phenomena are increasingly explored due to its simple structures and prominent role in biology (Kim et al, Biophysical Journal, 2019, 116, 1918.). Here, we demonstrate a two-point fixation method to covalently attach two distinct single stranded oligonucleotides to monomeric enhanced green fluorescent protein (mEGFP) via the use of click chemistry and SMCC (Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) crosslinking. The resulting bifunctional mEGFP-DNA conjugates are anchored to a DNA origami square through sequence-specific annealing, enabling rigid two-point fixation. This dual anchoring reduces rotational flexibility and heterogeneity of fluorescent proteins onto DNA nanostructures. We aim for this construct to be used in applications requiring precise molecular positioning for investigating long-range coherent exciton coupling between fluorescent proteins.