RNA selectively modulates activity of virulent amyloid PSMα3 and host-defense LL-37 via phase separation and aggregation dynamics.

In vitro biophysical and biochemical study examining the effects of RNA and EGCG on peptide assembly and activity

“Here, RNA acts as a concentration-dependent regulator of two sequence-related α-helical peptides with fundamentally different assembly behaviors: the cross-α amyloid-forming Staphylococcus aureus virulence factor PSMα3 and the non-amyloidogenic human host-defense peptide LL-37.”

Staphylococcus aureus virulence factor PSMα3, human host-defense peptide LL-37, Escherichia coli, and human HeLa cells

“The antimicrobial activity of PSMα3 against Escherichia coli was evaluated under varying RNA concentrations and incubation times using the PrestoBlue Cell Viability Assay (Figure 4A).”

Experiments were performed in technical triplicates across three independent biological experiments

“The experiments were performed in technical triplicate across three independent biological experiments (n=3) to ensure result reliability.”

Incubation times ranged from 30 minutes to 24 hours, with specific heat shock treatments of 15 minutes

“In contrast, after 24 hr of incubation, PSMα3’s antibacterial activity was significantly reduced (Figure 4A), suggesting a decrease in its effective concentration or changes in its morphology.”

RNA drives PSMα3 through distinct assembly states, from liquid-like condensates to fibrillar polymorphs, while preserving cytotoxic and antimicrobial activity over time

“RNA drives PSMα3 through distinct assembly states, from liquid-like condensates to fibrillar polymorphs, while preserving cytotoxic and antimicrobial activity over time.”

RNA attenuates LL-37 cytotoxicity toward host cells while maintaining antibacterial activity

“In contrast, RNA attenuates LL-37 cytotoxicity toward host cells while maintaining antibacterial activity, consistent with a host-protective immunomodulatory effect.”

Epigallocatechin gallate (EGCG) redirects both peptides into amorphous assemblies, abolishing their bioactivity

“Together with the opposing effects of epigallocatechin gallate, which redirects both peptides into amorphous assemblies, these findings support a mechanistic model in which biological activity is governed by supramolecular architecture, assembly trajectory, and dynamics rather than by monomer abundance or mature fibrils alone.”

The experimental timescales examined do not allow reliable determination of whether diffusion-driven coalescence kinetics would support classical droplet ripening dynamics

“The experimental timescales examined do not allow us to reliably determine whether diffusion-driven coalescence kinetics would support classical droplet ripening dynamics (Amico et al., 2024; Ray and Buell, 2024).”