Co-incubation of Short Amphiphilic Peptides with Dicer Substrate RNAs Results in β-Sheet Fibrils for Enhanced Gene Silencing in Cancer Cells

Abstract

RNA can interact with positively charged, amphiphilic peptides to cooperatively assemble into fibrils that enable RNA transport across cancer cellular membranes. RNA decreases the folding energy barrier imposed by the electrostatic repulsion between these charged peptides, thus partaking in RNA-peptide self-assembly along particular pathways in the energy landscape. Specific amphiphilic peptides capable of protecting and transporting RNA across a membrane have Type II' β-turn hairpin forming motifs in their structures, which aids self-assembly into β-sheet fibrils. We employed a set of such cationic, amphiphilic peptides that have random coiled structures in the absence of folding stimuli, to characterize the (peptides):(RNA) assembly. We subjected these complexes to extensive biophysical characterization in vitro and in cell culture. We show that short RNAs (such as Dicer substrate RNAs) can lead these peptides to self-assemble into β-sheet fibrils that have RNA transport capabilities and can act as non-viral delivery vectors for RNA. Modulation in the peptide sequence implicitly alters the way they bind RNA and influence the peptides' ability to transport nucleic acids across membranes.

Keywords: Dicer substrate RNA; RNA interference; RNA-peptide co-evolution; amphiphilic peptides; cancer; peptide folding energy landscape; β-sheet fibrils.

Fig. 1.

Schematic representation of peptide-assisted transmembrane delivery of nucleic acids.

Fig. 2.

(Peptides):(DsiRNA) complexes. (A) Schematic representation of (peptide):(RNA) complexes. m and n are the number of molecules of peptides and nucleic acids. Various m:n ratios were tested. (B-C) CD studies of peptides at room temperature. (B) Peptides in water without RNA. (C) Peptides co-incubated with DsiRNA were assembled in assembly buffer, pH=8.3 and then diluted in water to final pH of 7.2–7.4 (shown here). All peptides except MAX8V16E form β-sheets in the presence of DsiRNAs.

Fig. 3.

Characterization of (peptide):(DsiRNA) complexes in vitro. (A-C) TEM images and data analyses. (A) Preformed fibers of HPL24 peptides alone; (B) Adding DsiRNAs to preformed HPL24 fibers displays slightly thicker fibril formation upon complexation; (C) HPL24 peptides co-incubated with DsiRNA shows big structural changes with significant thickening of the fibrils; Scale bar corresponds to 200 nm. (D) Proposed energy landscape for β-sheet forming peptides (see Discussion for details).

Fig. 4.

(A) Relative binding affinities assessed with FA experiments. All the peptides show various degrees of binding with Alexa488 labeled DNA duplexes, MAX8V16E shows significantly less. (B) Principle of nuclease degradation. (C). Nuclease degradation assay of fluorescently quenched DNA duplexes alone or complexed with peptides. MAX8V16E showed the least protection.

Fig. 5.

Cellular studies of amphiphilic peptides complexed with DNA/RNA hybrids or DsiRNAs in the breast cancer cell line MDA-MB-231. (A) Relative cellular uptake of fluorescently labeled hybrids complexed with the studied peptides. (B) Visualization of cellular uptake of (HPL24):(hybrid labeled with Alexa488) complexes. (C) (Peptide):(DsiRNA) complexes’ cytotoxicities (D) Percentage of eGFP silencing by DsiRNA mediated by amphiphilic peptides. The eGFP expression was analyzed with flow cytometry and normalized to untreated cells. (E) Visualization of eGFP silencing in MDA-MB-231/eGFP by (HPL24):(DsiRNA) complexes.

Fig. 6.

Characterization of (peptide):(nucleic acid) complexes entry mechanisms (A) Tb³⁺:DPA leakage is induced by HPL24 peptide alone but not by the co-incubated (peptides):(DsiRNA) complexes. (B) eGFP silencing in MDA-MB-231/eGFP with DsiRNAs co-incubated with HPL24 or sequentially incubated with HPL24 preformed fibrils. (C-D) Temperature dependent uptake at 0°C and 37°C in MDA-MB-231 cells of hybrids labeled with Alexa488 mediated by HPL24 (C) and MAXR6Q2 (D).