Constrained peptides mimic a viral suppressor of RNA silencing

Abstract

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.

Figure 1.

(A) Crystal structure (PDB ID: 2ZI0) showing two TAV2b peptides (orange) bound to pal–RNA (gray). Close-up highlights the wt33 sequence (K21–R53, orange, cartoon representation) in complex with ds pal-RNA (gray and white, surface representation). Selected wt33 side chains are shown in ball-and-stick representation. (B) Representative ITC of unlabeled wt33 with the pal–RNA duplex (for full data see Supplementary Table S3 and Figure S5). (C) CD spectra of wt33 (c = 4 μM), pal-RNA (c(duplex) = 2 μM), spectra of pal-RNA (c(duplex) = 2 μM) with wt33 (c = 4 μM) in solution (pal-RNA + wt33), and the sum of the two individual spectra ([pal-RNA +wt33]). Buffer: 10 mM sodium phosphate, pH 7.4, 100 mM NaCl. (D) Sequences of TAV2b-derived peptides highlighting macrocyclization points. (E) EMSA of ds pal-RNA incubated with fluorescein-labeled TAV2b-derived peptides (A1–A3, B–B3, C–C3). Experiments employed 10% native PAGE (c(RNA) = 3 μM, c(peptide) = 6 μM). Running buffer: 25 mM Tris, pH 8.0, 200 mM glycine, 20 mM NaCl. For full gels including fluorescein readout, see Supplementary Figure S6 (ds: double-stranded, ss: single-stranded).

Figure 2.

(A) ITC-derived thermodynamic properties of pal-RNA binding to unlabeled wt33, B1, B3 and C1, including K_d-values (measurements were performed in triplicate, errors account for 1σ; for full data see Supplementary Table S3 and Figure S11–S13). (B) Percentage of α-helical secondary structure content for each fluorescein-labeled peptide. Buffer: 10 mM sodium phosphate, pH 7.4 (for complete secondary structure distribution, see Supplementary Table S4). (C) Overlaid CD spectra of fluorescein-labeled wt33, B1, B3 and C1 (c(peptide) = 75 μM). Buffer: 10 mM sodium phosphate, pH 7.4.

Figure 3.

(A) Half-life (t_1/2) values for fluorescein-labeled peptides (c = 50 μM) in the presence of proteinase K (c = 10 μg·ml⁻¹). Buffer: 10 mM Tris, pH 8.0, 150 mM NaCl. (B) Percentage of intact fluorescein-labeled peptide remaining over time in the presence of proteinase K. (C) mFI-Values of K562 human leukemia cells treated with fluorescein-labeled peptides (c = 1 μM, t = 90 min, triplicate of measurements, errors account for 1σ). (D) Fluorescence intensity distribution of K562 cells exposed to fluorescein-labeled wt33, B1, B3 and C1.

Figure 4.

(A) Sequence of the pre-miR-21 hairpin (black/gray) with the miR-21 duplex shown in black. Locations of Dicer cleavage sites are indicated (red). (B) Melting profiles of miR-21 in the absence and presence of double-stapled peptides B3, C1 and D1 (λ = 267 nm, c(miR-21) = 2 μM, c(peptide) = 4 μM. Buffer: 10 mM sodium phosphate pH 7.4, 100 mM NaCl). (C, D) Enrichment of miR–21 and pre–miR–21, respectively, after treatment of K562 cells with the biotinylated peptides B3, C1 and D1 and subsequent streptavidin-mediated selection of peptide-bound RNA relative to total RNA after cell lysis (triplicate of measurements, errors account for 1σ). (E) ITC-derived thermodynamic properties of B3 binding to miR–21 and pre–miR–21 (measurements were performed in triplicate; for full data see Supplementary Table S3 and Figures S22 and S23). (F) SimRNP model of a complex involving three wt33 peptide ligands (white, cartoon representation) and pre-miR–21 (black/gray, cartoon representation). Dicer cleavage sites are indicated (red). Coordinates of the model can be found as supplementary data. (G) Relative miR-21 abundance after Dicer processing of pre-miR-21 in the presence and absence of B3 or D1 using qRT-PCR (relative to absence of peptide control; c(pre-miR-21) = 40 nM, c(Dicer) = 4 U·ml⁻¹; triplicate of measurements, errors account for 1σ).