Smart Nucleic Acid Chaperones: Phase-Separating Intrinsically Disordered Proteins for Accelerating DNA Hybridization...

Nucleic acid (NA) hybridization reactions are central to molecular biology and NA nanotechnologies, which exploit base-pairing to generate intricate structures and dynamic reaction systems. NA chaperones are molecules (typically proteins) that can catalyze the formation of thermodynamically favorable NA structures through inter- and intramolecular hybridization. This work develops smart NA chaperones (SNACs) which combine the catalytic properties of NA chaperones with the dynamic behavior of intrinsically disordered proteins (IDPs) engineered to undergo programmable, triggered, and reversible liquid-liquid phase separation. We present model SNACs that are fusions of NA chaperones and IDPs, or are IDPs that have been designed to have NA chaperoning function. We demonstrate the ability of SNACs to enhance the kinetics of nucleic acid strand annealing (SA) and toehold-mediated strand displacement (TMSD) reactions. In the case of SA, SNACs, including engineered elastin-like proteins fused with NA-binding domains, overcome kinetic barriers in the hybridization of structured ssDNA oligonucleotides and form protein-DNA coacervates that accelerate hybridization reactions. Similarly, SNACs significantly enhance TMSD kinetics, both in their soluble state and within phase-separated, SNAC coacervates. This work establishes SNACs as tools for controlling NA hybridization, leveraging phase-separation to enhance reaction kinetics, and highlighting their potential as nanoassemblers for complex NA systems.