Two high-throughput screening assays for aberrant RNA–protein interactions in myotonic dystrophy type 1
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- Chen, C.Z., Sobczak, K., Hoskins, J. et al. Anal Bioanal Chem (2012) 402: 1889. doi:10.1007/s00216-011-5604-0
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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.
KeywordsMyotonic dystrophy type 1DM1Muscleblind-like 1MBNL1
Myotonic dystrophy type 1 (DM1), an autosomal dominant neuromuscular disorder affecting 1/8,000 individuals, is the most common form of muscular dystrophy in adults. The disease has a wide range of multi-systemic clinical presentations, which includes myotonia, cardiac conduction defects, progressive muscle wasting, endocrine imbalance and cataracts, causing progressive disability and premature death . The genetic lesion that causes DM1 is an expansion of CTG repeats in the 3′ untranslated region (UTR) of the DM protein kinase (DMPK) gene. The size of the triplet repeat expansion ranges from hundreds to thousands of repeats and correlates with disease severity [2, 3]. DM represents a novel disease paradigm where the mutant RNA exerts a dominant deleterious effect that is independent of the protein it encodes. The pathogenic expanded CUG-repeat (CUGexp) RNA accumulates in nuclear foci and sequesters proteins of the muscleblind-like (MBNL) family, of which MBNL1 is the most common family member in skeletal muscle [4, 5]. MBNL proteins regulate alternative splicing, and their sequestration alters the splicing patterns of a myriad of transcripts. Of the affected transcripts, cardiac troponin T (cTNT), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA1), insulin receptor (IR), and muscle-specific chloride channel (ClC-1) are the most well studied [6–9].
The binding interaction between MBNL proteins and CUG-repeat RNA is a key molecular step in the pathogenesis of DM1. Murine knockouts of MBNL1 recapitulate certain symptoms and splicing patterns of the disease , while over-expression of MBNL1 in a DM1 mouse model that expresses a (CUG)250 repeat transcript reverses myotonia and RNA missplicing . In addition, proof of principle that blocking the CUGexp/MBNL1 interaction in vivo could be beneficial for DM was demonstrated by the use of morpholino oligo to disrupt MBNL1-CUGexp binding, resulting in the correction of misregulated splicing .
Currently, there is no treatment to halt disease progression and therapy for DM1 is limited to supportive care. Recent drug development efforts have aimed at finding ligands that bind the expanded CUG RNA using combinatorial chemistry and peptoid synthesis, as well as a targeted small molecule screen. Several compounds that are able to inhibit the interaction between expanded CUG RNA and MBNL1 protein have been found [13–17]. Of these, only pentamidine has shown some in vivo activity but the therapeutic window is narrow due to its cytotoxicity . Continued development of small molecule inhibitors to disrupt the binding interaction between MBNL1 and CUG expansion remains an attractive approach for the treatment of DM1.
Protein–RNA interactions control many aspects of RNA splicing, translation and decay. Compounds that modulate these interactions could become useful research tools as well as potential therapeutics. Traditional methods for detection of protein–RNA binding include gel mobility shift, filter-binding and yeast-three-hybrid (Y3H) assays [15, 19–24]. While these assays are useful in low throughput applications, they lack adaptability to high-throughput screening (HTS) since they require either an electrophoresis apparatus or multiple wash steps. In addition, yeast-three-hybrid assay has added disadvantages due to the indirect nature of the assay. Two types of homogeneous assays, scintillation proximity and AlphaScreen technology, have been used to assay the bindings of HIV RNAs and host proteins in a high-throughput manner [24, 25]. A down side of scintillation proximity assays is the requirement of radio-labeled reagents that generate large amounts of radioactive waste. In addition, the advantages of a homogenous assay format are the simplification of assay workflow and reduction of assay noise due to plate wash steps. However, elimination of wash steps may lead to increased number of false positives. With sizes of many compound libraries currently expanding to over a million, there is increasing need of additional HTS-ready assays for orthogonal screening to eliminate detection related artifacts and allow fast hit triaging. Thus, the strategy of using one homogenous protein–RNA binding assay for HTS and the other as an orthogonal screen would facilitate hit prioritization efforts by rapidly eliminating detection-based false positives.
We have developed and compared two homogeneous assays to measure the binding of MBNL1 to CUG-repeat RNA. One uses the AlphaScreen technology and the other uses homogeneous time-resolved fluorescence energy transfer (HTRF). Since these two assays apply different detection methods, they can be used as an orthogonal pair to eliminate false positive compounds. Both assays were optimized, miniaturized to 1,536-well format and validated using a DMSO control plate. Our results indicate that while both assays are suitable for HTS, the HTRF assay is preferred for increased throughput and easier implementation, as no light shielding is needed. The HTRF assay was further validated in a pilot screen of 1280 pharmacologically active compounds (LOPAC1280, Sigma-Aldrich) and showed performance and hit rate that are acceptable for HTS. The hits were confirmed with the HTRF assay and assay artifacts were eliminated with the AlphaScreen assay as an orthogonal screen. The results indicate that the two assays can be paired for HTS and orthogonal screening use.
Materials and methods
5′-Biotinylated RNA oligo Biot-(CUG)12 with the sequence 5′-GCGCUGCUGCUGCUGCUGCUGCUGCUGCUGCUGCUGCUGCGC was purchased from IDT (Skokie, IL). (CUG)12 was designed to maintain a uniform hairpin structure after denaturation/renaturation, by adding a three nucleotides at both ends which form the GC clamp at the base of hairpin . The unlabeled mixed RNA/DNA oligo ssCAG-21 with the sequence 5′-rUrArGrCrArGrCrArGrCrArGrCrArGrCrArGrCTT (DNA sequence is underlined) was custom synthesized by IDT (Skokie, IL). The HTRF reagents, anti-His6-Terbium cryptate (anti-His-Tb) and streptavidin-conjugated XL665 (SA-XL665), were purchased from CisBio (Bedford, MA). The AlphaScreen reagents, nickel-coated acceptor beads and streptavidin-conjugated donor beads, were purchased from PerkinElmer (Waltham, MA). The library of pharmacologically active compounds (LOPAC1280) consists of a collection of small molecules with characterized biological activities and was purchased from Sigma-Aldrich (St. Louis, MO). More information on LOPAC1280 can be found at http://www.sigmaaldrich.com/chemistry/drug-discovery/validation-libraries/lopac1280-navigator.html.
Recombinant protein purification
BL21(DE3)pLysS E. coli (Invitrogen) were transformed with the pGEX-6P-MBNL1-Δ105-His plasmid, which expresses recombinant MBNL1-His6 protein with a 105 amino acid C-terminal deletion, an N-terminal GST tag and a C-terminal 6× His tag. 500 ml LB broth cultures with 200 μg/ml carbenicillin were grown at 37 °C to OD600 = 1.7–2.0, then induced with 250 μM IPTG overnight at 25 °C. Cells were pelleted (all centrifugation was performed at 4 °C), resuspended in 25 ml 1× His binding buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM imidazole and 0.1% IGEPAL CA-630) and incubated on ice for 30 min. Cell lysis was achieved with alternating rounds of sonication and icing. After clearing of cell debris via centrifugation, the supernatant, along with 1 mM PMSF, was added to 5 ml of Ni-agarose resin, prepared as 10 ml Ni-NTA Agarose slurry (Invitrogen) equilibrated with 1× His binding buffer. The lysate/bead mixture was incubated for 2 h at room temperature with gentle agitation. The supernatant was then discarded, and the resin was washed three times in 25 ml 1× His binding buffer with increasing imidazole concentrations of 5, 50, and 100 mM. The bound MBNL1-His6 protein was eluted in two rounds by vigorous shaking of the Ni-agarose resin in 3 ml 1× His binding buffer with 250 mM imidazole. The eluates were added to PD-10 Desalting Columns (GE Healthcare) that were equilibrated with 1× FBB buffer (50 mM Tris–HCl pH 8.0, 50 mM NaCl, 50 mM KCl, and 1 mM MgCl2). MBNL1 protein was eluted from the columns in two rounds with 2 ml 1× FBB buffer each. Glycerol was added to 50% in each sample, and total protein concentrations were determined with the BioRad Protein Assay. Each sample was resolved on a 10% SDS-PAGE Tris–glycine gel, stained by SYPRO Ruby (Invitrogen) and scanned with a GE Healthcare Typhoon 9400 Variable Mode Imager. MBNL1-His6 and contaminating protein bands were quantitated with ImageQuant software, and MBNL1-His6 purity was calculated as the ratio of the MBNL1 band versus the sum of all protein bands (typically ∼0.60–0.75). The MBNL1-His6 concentration for each sample was then calculated by multiplying this ratio by the total protein concentration.
Binding assay in 384-well plate format
Assay development and optimization was performed in white solid-bottom 384-well plates (Greiner Bio-One, Monroe, NC). For HTRF binding assays, reagents were sequentially added manually as 5 μl Biot-(CUG)12, 5 μl MBNL1-His6, 5 μl anti-His-Tb, and 5 μl SA-XL665. For AlphaScreen binding assays, reagents were sequentially added manually as 5 μl Biot-(CUG)12, 5 μl MBNL1-His6, 5 μl acceptor bead and 5 μl donor bead. All reagents were diluted in binding buffer composed of 25 mM HEPES pH 7.4, 110 mM KCL, 10 mM NaCl, 1 mM MgCl2, 15 μM ZnCl2, 0.02% Tween-20, 0.1% BSA and 5 mM freshly added DTT. After 60 min incubation at room temperature, the fluorescence (HTRF assay) or luminescence (AlphaScreen assay) was measured using an EnVision plate reader (PerkinElmer, Boston, MA). All results were generated from duplicate data points.
Binding assays in 1,536-well plate format
The binding assay was miniaturized to the 1,536-well plate format for HTS. Briefly, 2 μl/well of a premixed protein–RNA solution (20 nM Biot-(CUG)12 and 20 nM MBNL1-His6) was dispensed to a white solid-bottom 1,536-well plate (Greiner Bio-One, Monroe, NC), followed by the addition of 23 nl/well compound in DMSO solution. After a 15-min incubation period at room temperature, the detection reagents were simultaneously added as 1 μl/well each (0.22 ng/μl anti-His-Tb and 20 nM SA-XL665 for the HTRF assay or 25 ng/μl acceptor bead and 25 ng/μl donor beads for the AlphaScreen assay). The binding reactions were allowed to equilibrate for 60 min. The assay signal was then measured on an EnVision plate reader (PerkinElmer, Boston, MA). Dose–response of ssCAG-21 and confirmation of LOPAC1280 screen hits were tested in duplicates.
A BioRAPTR FRDTM Microfluidic Workstation (Beckman Coulter, Inc. Fullerton, CA) was used to dispense reagents into 1,536-well plates at volumes of 1–2 μl. For experiments using untagged oligos as binding inhibitors, the oligos were serially diluted in 384-well plates and then reformatted into 1,536-well plates as 7 μl/well using a CyBi-Well dispensing station with a 384-well head (Cybio Inc., Woburn, MA). For the DMSO plate tests and LOPAC1280 screen, 23 nl of liquids were transferred to 1,536-well assay plates using an automated pin-tool station (Kalypsys, San Diego, CA). An EnVision filter-based imaging plate reader (PerkinElmer, Boston, MA) was used for both the HTRF and AlphaScreen assays. For the two channel HTRF detection, an excitation of 340 nm, emission of 665 nm and 545 nm, count delay of 70 μs and count window of 400 μs was used. AlphaScreen assays were read using the AlphaScreen setting on the EnVision plate reader with either the 1,536- or 384-well aperture.
The RNAs used [(CUG)20 and (CAG)20] were chemically synthesized at Metabion International AG (Jena, Germany). Structures formed by these transcripts have been described earlier . All transcripts were 5′-end-labeled with T4 polynucleotide kinase and [γ32P]ATP (4500 Ci/mmol; ICN). The labeled RNAs were purified by electrophoresis in a denaturing 10% polyacrylamide gel, excised, eluted from the gel (0.3 M sodium acetate pH 5.2, 0.5 mM EDTA and 0.1% SDS) and precipitated. Prior to use, the 32P-labeled RNAs were subjected to a denaturation and renaturation procedure in 2× assay buffer containing 200 mM Tris–HCl (pH 8.0), 100 mM NaCl, 100 mM KCl, 2 mM MgCl2, by heating the sample at 85 °C for 1 min and then slowly cooling to 37 °C. RNA degradation assay was initiated by mixing 5 μl of the RNA sample (200 pM) with 5 μl of a compound solution. Incubation was performed at 37 °C for 30 min and stopped by adding an equal volume of stop solution (7.5 M urea and 20 mM EDTA with dyes). Samples were analyzed on a denaturing 12% polyacrylamide gel electrophoresis.
Filter-binding assay was performed as described earlier . Briefly, 15 μl of 5′-labeled RNA (200 pM) after denaturation–renaturation procedure (described above) was mixed with 15 μl of aqueous solution of different concentrations of compound and then 30 μl of 40 nM MBNL1 solution in 1 x assay buffer was added. After 30 min of incubation at 37 °C samples were loaded on filter sandwich (nitrocellulose [BioRad], Hybond N+ [Amersham], three sheets of 3 mm Whatman paper) mounted on slot-blot apparatus. To determine a kinetic of RNA/MBNL1 interaction similar procedure was used with exception of compound adding.
The quantitative data were calculated as mean ± standard error. The results for the assay optimization and inhibitor dose–response experiments were analyzed with Prism (Graphpad, San Diego, CA). For assay validation, signal-to-basal (S/B) ratios, Z′ factors and coefficient of variation (CV) values were calculated from 64 wells of DMSO only treatments (total signal) and 32 wells of binding reaction lacking MBNL-His6 (basal signal). For LOPAC1280 hit confirmation, percent binding is calculated from 48 wells of DMSO only treatments (normalized as 100% binding) and 24 wells of binding reaction without MBNL-His6 (normalized as 0% binding). The KD and IC50 values were calculated based on three independent experiments using the one-site binding model or dose–response inhibition curve, respectively, with use of GraphPad Prism 5.00 (GraphPad Software Inc.).
Results and discussion
Assay designs and principles
The second assay was developed with the same binding pair, MBNL1-His6 and Biot-(CUG)12, using the AlphaScreen assay format. This assay is a bead based assay using phthalocyanine (a photosensitizer) coated donor beads that convert ambient oxygen to an excited singlet oxygen upon excitation. The singlet oxygen has a 4-ms half-life, which allows a diffusion radius of approximately 200 nm in aqueous environment. Energy in the singlet oxygen form is transferred to an acceptor bead containing thioxene derivatives culminating in light production if the acceptor bead is within close proximity to the donor bead. Capture of the MBNL1-His6 and Biot-(CUG)12 complex by the nickel-charged acceptor beads and streptavidin-conjugated donor beads brings the AlphaScreen detection beads to close proximity that allows energy transfer in a homogeneous detection format (Fig. 2b). Both assays have the advantage of being homogenous and can be easily implemented in a “mix and read” format. The detection mechanisms in these two assays are different as the HTRF assay is based on the time-resolved fluorescence while AlphaScreen uses chemiluminance for detection. Thus, these two assays can be used as orthogonal screens to eliminate detection related false positive compounds.
The amount of detection reagents were titrated against a premixed solution containing 20 nM each of MBNL1-His6 and Biot-(CUG)12. For the HTRF assay, a titration of detection reagents carried out using the mixtures of anti-His-Tb (0.055 to 0.44 ng/μl) and the SA-XL665 (2.5 to 20 nM). The results showed that the optimal signal-to-basal ratio for the binding of 20 nM MBNL1-His6 to 20 nM Biot-(CUG)12 complex was achieved with 10 nM SA-XL665 to 0.11 or 0.22 ng/μl anti-His-Tb (Fig. 3b). Since 0.11 ng/μl and 0.22 ng/μl anti-His-Tb yielded almost identical S/B ratio, 0.11 ng/μl anti-His-Tb and 10 nM SA-XL665 was used for further experiments.
For the AlphaScreen assay, the manufacturers recommended using a 1:1 ratio of donor to acceptor beads. Therefore, the two detection reagents were titrated against premixed 20 nM MBNL1-His/Biot-(CUG)12 at a 1:1 ratio. Optimal S/B ratio was achieved at 12.5 ng/μl of each type of bead (Fig. 3b). This concentration was selected for further experiments.
Unlabeled oligonucleotide ssCAG-21 was used as a positive control to test for its ability to competitively inhibit binding of Biot-(CUG)12 to MBNL1 in both the HTRF and AlphaScreen assays (Fig. 3c). This oligonucleotide was shown previously as highly potent competitor of CUGexp/MBNL1 interaction using two different methodological platforms: the agarose gel electrophoretic mobility shift assay and β-galactosidase enzyme complementation assay [15, 17]. The dose-dependent inhibitory effects of ssCAG-21 on MBNL1-(CUG)12 binding in both HTS assays showed comparable and low IC50 values, indicating that both assays possessed similar detection sensitivity.
Assay miniaturization and validation
To further validate these two binding assays, we carried out a test with a plate containing DMSO, a solvent for the compound libraries. For the HTRF assay, the S/B ratio was 5.35-fold, CV was 2.4% and Z′ factor was 0.89 as determined from the DMSO plate test (Fig. 4b). Similarly, for the AlphaScreen assay, the S/B ratio was 55.5, CV was 6.7% and Z′ factor was 0.75 (Fig. 4b). These results demonstrate that both assays are robust and suitable for compound screens in the 1,536-well plate format.
Although both the AlphaScreen and HTRF assay gave statistical calculations that are suitable for HTS, the HTRF assay produced cleaner results (CV 2.4%) versus the AlphaScreen assay (CV 6.7%) in the DMSO plate test. The ratiometric calculation of the results from HTRF assay may contribute to the lower well-to-well variations. Additional advantages of the HTRF assay are that: (1) the HTRF assay lacks light sensitivity, which facilitates reagent additions and time course studies during assay development. (2) The detection speed of HTRF assay is much faster as it can be read on charge-coupled device (CCD) based microplate imager while AlphaScreen signal requires a laser-based excitation that is read by a photomultiplier tube. The ability of the CCD imager to collect signal from the entire plate at once allows for shorter read times and higher assay throughput for screens using the HTRF assay. For these reasons, the HTRF assay was chosen for validation screening against a small set of compounds.
LOPAC1280 library screening and hit confirmation
Summary of LOPAC1280 library screening hits
NF449 octasodium salt
SB 200646 HCl
We have developed two robust assays for detection of protein–RNA binding and validated their use for HTS and orthogonal screen. These assays apply AlphaScreen and HTRF technologies in a homogenous, easy to use format that is suitable for HTS. While the AlphaScreen system has previously been applied to a host protein–HIV RNA binding assay , the HTRF assay format has only been applied to protein–peptide, protein–protein and protein–DNA binding assays [30–32], but not protein–RNA binding. In addition to utilizing these assays for compound library screens to identify small molecule disruptors of the preformed RNA–protein complexes, these assays could also be utilized to screen for compounds that prevent complex formation by altering the order of addition of the target reagents and the compounds. Both the AlphaScreen and the HTRF assays were optimized in 1,536-well plate format for HTS. The HTRF assay was better suited for HTS based on assay throughput, while the AlphaScreen assay was useful as a homogenous orthogonal assay for hit confirmation and elimination of false positives that interfere with the HTRF assay. Therefore, pairing the HTRF and AlphaScreen assays in a primary and orthogonal secondary screen is an effective strategy for HTS to identify protein–RNA binding inhibitors.
This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research, National Institutes of Health, the Marigold Foundation research fellowship, the US grants MH087421, AR049077, and NS48843, and the Polish Ministry of Science and Higher Education grant N302 260938.