Two high-throughput screening assays for aberrant RNA-protein interactions in myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG-repeat RNA. The resulting loss of MBNL1 activity causes misregulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high-throughput screens suitable assays using both homogenous time-resolved fluorescence energy transfer and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)(12) RNA. These assays are homogenous and successfully miniaturized to 1,536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z' factors.

Figure 1. RNA binding activity of MBNL1 constructs

Filter binding assay was used to compare (CUG)₁₀₉ binding affinity of full-length (MBNL1-FL) and truncated recombinant MBNL1 (MBNL1 105). Experimentally calculated K_D was 9.63 ± 1.02 for MBNL1 105 and 6.92 ± 0.78 for MBNL1-FL.

Figure 2. Schematic of assay principles

(a) In the HTRF assay, the MBNL1-His₆ and Biot-(CUG)₁₂ complex is captured by the HTRF reagents anti-His-Terbium and streptavidin-XL665. The formation of protein-RNA complex brings terbium cryptate within close proximity to XL665 to allow time-resolved fluorescence energy transfer. (b) In the AlphaScreen assay, streptavidin-conjugated donor beads are brought into close proximity to nickel-coated acceptor beads upon capture by the protein-RNA complex. Excitation with 680nm light induces release from donor bead of singlet oxygen that is capable of energy transfer with thioxene derivatives in the acceptor bead, culminating in 520-620nm photoemission by acceptor bead.

Figure 3. Assay optimization

(a) MBNL1-His₆ was titrated against 20 nM final concentration of Biot-(CUG)₁₂ in both HTRF and AlphaScreen assays performed in 384-well format. The optimal ratio of MBNL1 protein and (CUG)₁₂ ligand was 1:1 for both assays. (b) Detection reagents were titrated against 20 nM each of MBNL1-His₆ and Biot-(CUG)₁₂ for the HTRF and AlphaScreen assays. (c) The dose response curves for ssCAG-21, a competitive inhibitor of MBNL1-His₆/Biot-(CUG)₁₂ binding, was determined using the HTRF and AlphaScreen assays. RLU = relative luminescence unit; S/B = signal to background ratio.

Figure 4. Assay miniaturization and validation

(a) The assays were miniaturized to 4 ul final volume in 1536-well format. The dose response for inhibition by ssCAG-21 was similar to that of 384-well format. (b) Scatter plot representations of the DMSO plate test results for the HTRF and AlphaScreen assays. The treatments were used: column 1 was treated with titrations of ssCAG-21 (from 125 to 0.004 nM), column 2-3 and 5-48 with DMSO (total signal) and column 4 with DMSO and lacked MBNL1-His₆ (basal signal).

Figure 5. Confirmation of LOPAC¹²⁸⁰ hits

(a) Dose response of nor-Binaltorphimine dihydrochloride (nor-BNI) and NF449. The IC₅₀ values are 21.9 M in HTRF and 12.5 μM in AlphaScreen assays for nor-BNI, and 31.0 μM in HTRF and 31.5 M in AlphaScreen assays for NF449. (b) Nor-BNI has nonspecific RNA degradation activity. 100 pM of 5′-end labeled (CUG)₂₀ and (CAG)₂₀ was incubated for 30 min with varying concentrations of compounds in assay buffer at 37°C. Significant RNA degradation was observed for nor-BNI for both RNA sequences, while no degradation was observed for NF449 up to 800 μM compound concentration against either RNA species. (c) NF449 shows specificity to inhibit MBNL1 binding to CUG repeat hairpin. Filter binding assay results for two 5′-end labeled transcripts (CUG)₂₀ and (CAG)₂₀ incubated with different compound concentration and 200 nM MBNL1 protein. The IC₅₀ for (CUG)₂₀ is 29.7±4.2 M and for (CAG)₂₀, 98.7±9.6 μM.