Liquid‒liquid phase separation induced by i-motif DNA under molecular crowding conditions

Abstract

Cytosine-rich sequences can fold into a four-stranded structure called the i-motif. These i-motif-forming DNA sequences are enriched in the promoter regions of cancer-related genes and telomeres, indicating their biological importance. Interestingly, an i-motif-forming DNA oligonucleotide derived from telomeres was reported to undergo liquid‒liquid phase separation (LLPS), suggesting that i-motifs regulate cellular processes involving gene expression via LLPS. However, it is still unclear whether i-motifs are able to undergo LLPS under physiological conditions because i-motif structures are thermodynamically stable only under acidic conditions. In this study, we systematically studied the thermodynamics of a series of i-motif-forming oligonucleotides and their ability to undergo LLPS under molecular crowding conditions that mimic the conditions inside cells at various pH values. The thermodynamic analyses revealed that crowding reagents with higher molecular weights stabilize the i-motif structure and increase its pKa. Moreover, we demonstrated that i-motif structures stabilized by molecular crowding undergo LLPS under neutral and even basic conditions. On the other hand, mutated oligonucleotides that do not form stable i-motif structures did not undergo LLPS. These results indicate that i-motif-forming DNAs can robustly and widely undergo LLPS depending on cellular environmental factors such as the solution pH, composition of crowding reagents and degree of molecular crowding in living cells. We showed that various i-motif-forming DNAs undergo LLPS with cationic peptide in acidic conditions. Considering cellular condition, we further investigated LLPS of DNAs in the molecular crowding conditions at physiological pH. LLPS is promoted by molecular crowding conditions at even in basic pH through stabilization of i-motif structure by molecular crowding condition.