Protein-DNA condensates mediate various processes, including transcription. Relative to proteins, much less is known about how DNA can regulate condensate material properties. We previously found that mixtures of protamine, an arginine-rich disordered protein, with a dsDNA formed amorphous aggregates, whereas those with a ssDNA formed liquid droplets. Unexpectedly, mixing protamine with additional ssDNA produced aggregates. These ssDNA sequences contain self-complementary segments and can self-dimerize. To systematically study the roles of DNA secondary structure, we designed three classes of DNA sequences: (A) self-dimerizing ssDNA, comprising a 10- or 20-nt self-complementary segment and an extension, which become one strand of a duplex and an overhang, respectively, upon self-dimerization; (B) hairpins, with a 10-bp stem and a 6- or 16-nt loop; and (C) a 20-bp dsDNA with a 12-nt guanine-rich segment on one strand primed for G-quadruplex formation. The expected secondary structures were confirmed by UV melting and circular dichroism. In particular, the CD spectrum of the guanine-rich dsDNA implicated both G-quadruplex (by the 12-nt guanine-rich segment) and duplex (∼8 bp). At 0.15 M KCl, the material state transitioned from aggregates to droplets for the self-dimerizing ssDNA as the overhang length increased beyond 5 nt, was aggregates for the hairpins, and droplets for the guanine-rich dsDNA. Overall, duplex favors aggregate formation, whereas ssDNA free of secondary structures favors droplet formation. As overhang or loop length increased, condensates became more resistant to dissolution by high salt, implicating contributions of cation- π ; interactions between arginine sidechains and nucleobases. Optical tweezers-directed measurements showed that the fusion speed of protamine-DNA droplets decreased with increasing lengths of both the duplex segment and the overhang or loop. By changing secondary structure, DNA sequence can tune the material states and material properties of protein-DNA condensates to suit their specific functions.
Mudiyanselage et al. (Sun,) studied this question.