Extended Data Figure 2. Base-pairing interactions impart solid-like properties to DNA-spermine complexes.

Electrostatic interactions between polymeric anions (such as nucleic acids) and multivalent cations can lead to phase separation via formation of polyelectrolyte complexes, a phenomenon known as complex coacervation. We find that spermine, a tetravalent cation at pH 7, can induce phase separation of single-stranded DNA oligonucleotides. (a) Mixing 10 mM spermine pH 7 (left tube) with 10 μM T-90 DNA (90-mer polyT DNA oligonucleotide) (right tube) immediately results in a turbid solution (center tube). (b) Examination by brighfield microscopy revealed numerous spherical droplets. Using a fluorescently-labeled T-90, we confirmed that the droplet phase is enriched in DNA. Representative bright field (left), fluorescence (center) and overlay (right) images for the DNA-spermine complexes. (c–f) We investigated the effect of base-pairing interactions on DNA-spermine complexes. The T-90 DNA-spermine complexes are liquid-like, as evidenced by their spherical geometry (c) and a rapid fluorescence recovery after photobleaching (f, 99 ± 1% recovery, τ_FRAP-T90 = 5 ± 2 s, mean ± s.d., n = 3 droplets). Next, we designed a 90 bases long DNA with five 8-bp long palindromic hybridization sites separated by poly-dT spacers (sequence S1, sequences in Supplementary Table 2). S1 DNA also phase separated and formed spherical liquid-like droplets in the presence of spermine (d). However, the S1-spermine droplets exhibited reduced fluidity, as evidenced by a slower recovery upon photobleaching (f, 90 ± 4% recovery, τ_FRAP-S1 = 335 ± 41 s, mean ± s.d., n = 5). (e) We performed similar spermine-mediated coacervation experiments with a (dAdT)₄₅ oligonucleotide (AT-45) that may form multivalent A:T base-pairing interactions. AT-45 DNA formed interconnected network-like structures spanning hundreds of microns or gels (e). These AT-45 DNA gels are in a solid-like state as evidenced by lack of fluorescence recovery after photobleaching (f, 14 ± 5% recovery, mean ± s.d., n = 4 clusters). Scale bars represent 5 μm.