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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
ACS Chem Biol. Author manuscript; available in PMC 2013 December 21.
Published in final edited form as:
PMCID: PMC3528830

Rational Design of Bioactive, Modularly Assembled Aminoglycosides Targeting the RNA that Causes Myotonic Dystrophy Type 1


Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)exp) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6′-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)exp. However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a D-Arg9 molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r(CUG)exp; pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA.

Potential RNA drug targets are plentiful in the transcriptome; however, only the bacterial rRNA, and hence the ribosome, are tried and true targets for small molecules.(1) Ideally, both coding and non-coding RNAs that have important biological functions could be targeted with small molecules.(2) There are significant challenges for the development of small molecules that modulate RNA function, either by screening or rational design. These issues are mainly centered on the identification of selective small molecule ligands that target specific RNAs and parallel efforts to identify the RNA motifs that selectively bind small molecule ligands.(3)

The current state of the art in developing compounds that target RNA is the use of antisense nucleic acids or interfering RNA.(46) Although both of these strategies are powerful, oligonucleotide-based therapeutics can have undesirable properties such as non-specific stimulation of the immune system and off-target effects.(7, 8) In addition, the compounds have poor cellular permeability and are more expensive to manufacture than small molecules. The advantage of oligonucleotides is their unparalleled simplicity of design based on base-pairing rules.

In an effort to develop methods to target RNA with small molecules, our group has developed a program to define a database of RNA motif-ligand interactions by using multidimensional combinatorial screening.(912) In this approach, a library of small molecules is probed for binding to a library of discrete RNA motifs that are commonly found in the repertoire human RNA structures (hairpins or internal loops, for example). By selecting RNA motif-ligand binding partners, the optimal RNA motifs that bind small molecules are defined and deposited into the database. This database can be mined against transcriptomic data and secondary structure predictions to determine if a particular RNA has ligand-targetable motifs. The small molecules that bind to these motifs serve as lead compounds to target the RNA of interest.(1316)

In previous investigations, it was determined that 6′-N-5-hexynoate kanamycin A (K), binds a 2×2 nucleotide pyrimidine-rich internal loop that is present in the RNA that causes myotonic dystrophy type 2 (DM2).(9, 12, 13) DM2 is caused by an expanded r(CCUG) repeat in intron 1 of the zinc finger 9 protein (ZNF9). The expanded repeat folds into a hairpin with an array of 5′CCUG/3′GUCC motifs. These loops serve as a high affinity-binding site for Muscleblind-like 1 (MBNL1) protein, a regulator of pre-mRNA splicing.(17) DM2 is associated with the inactivation of MBNL1, which leads to a variety of pre-mRNA splicing defects.(18, 19) By using the information that K binds to RNA motifs like those present multiple times in r(CCUG)exp, a potent in vitro inhibitor of the r(CCUG)exp-MBNL1 interaction was designed. Specifically, the optimal multivalent compound displays the K module with the same periodicity as the array of 5′CCUG/3′GUCC motifs present in r(CCUG)exp.(13)

During the course of studies to understand the RNA targets of 6′-N-5-hexynoate kanamycin A, we determined that a suboptimal motif for ligand binding is 5′CUG/3′GUC, the motif that is highly reiterated in the expanded r(CUG) repeat (r(CUG)exp) that causes myotonic dystrophy type 1 (DM1). DM1 and DM2 share a similar molecular basis of disease as both expanded repeats bind and inactivate MBNL1. The r(CUG) expansion is also located in a non-coding sequence, the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA.(20, 21)

It was hypothesized that the optimal distance between K modules would be shorter for the DM1 RNA than the DM2 RNA due to the smaller size of the internal loop (Figure 1). Indeed, by decreasing the distance between K modules, a modularly assembled compound that was selective for r(CUG) repeats and potently inhibitory for the r(CUG)exp-MBNL1 interaction in vitro was identified.(15) These studies established that both the nature of the RNA-binding module and the spacing between modules are independent determinants of RNA-binding properties of modularly assembled ligands.

Figure 1
The disease mechanism for DM1 and DM2 and the strategy employed to design modularly assembled small molecules that target the RNAs that cause disease. A, the secondary structures of r(CCUG)exp and r(CUG)exp, the causative agents of DM2 and DM1, respectively. ...

In this report, we disclose that second generation modularly assembled compounds that target r(CUG)exp are effective in cell culture and animal models of DM1. These compounds were engineered for enhanced cellular permeability and nuclear localization via conjugation to a D-Arg9 (DR9) molecular transporter.(2225) Specifically, the designer compounds improve pre-mRNA splicing defects in cell culture and animal models, improve translational defects in a cell-based model system, and disrupt the formation of nuclear foci.


We previously reported that modularly assembled small molecules displaying 6′-N-5-hexynoate kanamycin A (K) inhibit the formation of the r(CUG)exp-MBNL1 complex in vitro.(13, 15, 16) The optimal compounds consist of a peptoid backbone in which the K ligand modules are separated by two propylamine spacers. The nomenclature for these structures is nK-2, where n is the number of RNA-binding modules displayed on a single chain (or valency), K indicates the RNA-binding module (a conjugated version of 6′-N-5-hexynoate kanamycin A), and the number after the dash indicates the number of propylamine spacers between K modules. The structures of these and related control compounds are shown in Figure 1.

The Bioactivity of nK-2 Compounds in DM1 Cell-Based Model Systems

The presence of r(CUG)exp causes various defects in vivo, including (i) dysregulation of pre-mRNA splicing controlled by MBNL1;(19, 26) (ii) nuclear retention and hence decreased translation of r(CUG)exp-containing transcripts;(27, 28) and, (iii) formation of nuclear foci, which consist of r(CUG)exp-protein aggregates.(29, 30)

Two cell-based models were used to determine if the optimal compound from in vitro studies, 4K-2, could improve DM1-associated defects. These assays were completed as described previously.(31) First, the effect of 4K-2 on pre-mRNA splicing was assayed in HeLa cells.(32) Briefly, cells were co-transfected with a DM mini-gene that expresses 960 interrupted r(CUG) repeats and a cardiac troponin T (cTNT) pre-mRNA mini-gene.(31, 32) After transfection, the cells were treated with compound in growth medium. cTNT alternative splicing (Figure 3) was then analyzed by RT-PCR and denaturing gel electrophoresis as previously described.(31)

Figure 3
Assessing the bioactivity of modularly assembled small molecules targeting r(CUG)exp in a pre-mRNA splicing defect assay. A, top, a schematic of the cTNT mini-gene that was used in this assay. Middle, representative gel autoradiogram assaying improvement ...

The second model system mimics the DMPK translation defect (Figure 4). The C2C12 cell line was stably transfected with the firefly luciferase gene in which r(CTG)800 was placed in the 3′UTR.(31) Expression of luciferase is low in this cell line due to the binding of r(CUG)800 to MBNL1 and other proteins, resulting in nuclear retention of the luciferase mRNA. If a compound is efficacious, then an increase in luciferase activity in cell lysates is observed.

Figure 4
Assessing the bioactivity of modularly assembled small molecules targeting r(CUG)exp in a translational assay. Top, a schematic of the translational assay used to assess the effect of compounds on the DM1 translation defect. Bottom, 2K-2-DR9 and 4K-2-DR ...

In both model systems, 4K-2 was not active, or only very slightly active at 10 μM (Figure 3). Previous studies of the cellular permeability of 2K-2 and 4K-2 showed that, although the compounds are cell permeable, they localize mainly to the perinuclear region.(13, 15, 16, 33) We hypothesized that if the cellular permeability and nuclear localization of the compounds could be improved, then the compounds might be efficacious.

Cellular Permeability of nK-2-DR9 Compounds

To develop compounds with increased cellular permeability and nuclear localization, the molecular transporter D-R9 (DR9) (23, 24) was conjugated onto 4K-2 to yield 4K-2-DR9 (Figure 2). Previous studies have shown that multiple guanidinium units facilitate cellular uptake of cargo ranging from small molecules to peptides and proteins.(23, 34) Furthermore, mechanistic studies have shown that poly arginines enter mammalian cells through a variety of pathways that include binding to cell surface heparin sulfate and endocytotic uptake.(35) Since many cell and tissue types present heparin sulfate,(36) we envisioned that DR9 conjugation could engender compounds with the ability to more efficiently enter a variety of cell lines and mouse tissues.

Figure 2
The synthetic scheme used to provide modularly assembled small molecules that target r(CUG)exp. The modularly assembled small molecules were synthesized to contain a D-Arg9 tag to facilitate cellular permeability. Bioactive peptoids contain K modules ...

To study if the nK-2-DR9 compounds have enhanced cellular uptake relative to the parent molecules, flow cytometry experiments were complete using the HeLa cell line since it was also used to assay pre-mRNA splicing defects. Compounds were added in growth medium to the cells and incubated for 1.5 h. The cells were trypsinized from the surface and stained with propidium iodide (detects dead or damaged cells with compromised cell membranes). Since the compounds are labeled with fluorescein, it was used to quantify cellular permeability. Compound 4K-2 was only taken up by ca. 1% of the cells in these conditions, while 4K-2-DR9 was taken up by 13-fold higher number of cells. Two related compounds were also studied, 4N-2-DR9 and 4Az-2-DR9 where N indicates the conjugation of 6′-N-5-hexyonate neamine to the peptoid backbone and Az indicates the unconjugated (azide-displaying backbone). 4N-2-DR9 and 4K-2-DR9 have similar cellular permeabilities while 4Az-2-DR9 is taken up by 75-fold more cells than 4K-2. It is likely that the decreased cellular permeability of 4N-2-DR9 and 4K-2-DR9 relative to 4Az-2-DR9 is due to the highly cationic aminoglycoside cargo. Confocal microscopy images confirm that 4K-2-DR9 is permeable to almost all cells after longer incubation times (16 h, Figure 6). In all cases there is no change in the number of cells that are stained by propidium iodide, which indicates cell death relative to cells that are not treated with compound (Table S-3). Thus, addition of a DR9 tag enhances cell uptake by greater than 10-fold while not at the expense of cell toxicity. Furthermore, addition of cargo (K or N modules) onto a peptoid with DR9 decreases uptake.

Figure 6
4K-2-DR9 improves pre-mRNA splicing defects in the muscle-specific chloride ion channel (Clcn1) and sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1) pre-mRNAs in a DM1 mouse model. The DM1 mouse model expresses the human skeletal actin (HSA) ...

In Vitro Potency and Affinity of nK-2-DR9 Compounds

The potency of the second-generation compounds for disruption of the r(CUG)10-MBNL1 complex are summarized in Table 1 (and Supporting Information). 2K-2-DR9 and 4K-2-DR9 disrupt the r(CUG)10-MBNL1 complex in vitro with IC50’s of 1430 ± 160 nM and 240 ± 5 nM while the corresponding monomer, FITC-K has an IC50 >250 μM. Once normalized for the number of K units, the multivalent effect (37) for 4K-2-DR9 is >250-fold. Control peptoids in which the backbone in unconjugated (4Az-2-DR9) or conjugated to a neamine derivative (4N-2-DR9) have IC50’s of 5400 ± 510 and 1030 ± 90 nM, respectively. Thus display of the appropriate module, K, imparts improved potency (by at least 5-fold) for the disruption of the pre-formed r(CUG)10-MBNL1 complex. The observation that both 4Az-2-DR9 and 4N-2-DR9 inhibit the r(CUG)10-MBNL1 complex suggests that addition of the DR9 tag causes some level of non-specific binding of the compounds to RNA, which is not unexpected. This is further verified by the IC50 for 4K-2, which is 16300 μM in this assay. The large difference in IC50 between 4K-2 and 4K-2-DR9 is likely because the DR9-conjugate occupies a larger amount of the RNA’s surface area. A larger difference in potency was previously observed for 4K-2 and 4N-2 (>33-fold) than for 4K-2-DR9 and 4N-2-DR9, although these experiments were completed using a different assay.(15)

Table 1
The binding affinity and stoichiometry for RNA and the potency for inhibition of the r(CUG)-MBNL1 complex.

We previously reported that the distance between K modules also affects potency and affinity.(15) As shown in Figure 1, the optimal distance for r(CUG)exp is afforded by two propylamine spacing modules while the optimal distance for r(CCUG)exp is four propylamines. In order to determine if conjugation of DR9 affects the optimal distance between K modules for r(CUG)exp, the potencies of 2K-4-DR9, 3K-4-DR9, and 2N-4-DR9 were determined (Table 1). As expected, 2K-4-DR9 is a 38-fold weaker inhibitor of the r(CUG)-MBNL1 complex (IC50 = 55 μM) than 2K-2-DR9. Increasing the valency to 3K-4-DR9 improves potency by ~2-fold (26 μM) but it is still a less potent inhibitor by ~18-fold than 2K-2-DR9 and ~100-fold weaker inhibitor than 4K-2-DR9. Interestingly, 2N-4-DR9 is a better inhibitor than 2K-4-DR9 (IC50 = 9 μM; ~6-fold worse than 2K-2-DR9), suggesting that the optimal distance between RNA-binding modules is ligand-dependent.

To further understand the nature of inhibition of the complex and the effect of affinity of the RNA-ligand complex, binding measurements were completed with 4K-2-DR9 and the control compounds (Table 1). The RNA used in these studies contains 12 5′CUG/3′GUC motifs or 24 r(CUG) repeats (r(CUG)12×2) embedded in a hairpin cassette (15). This construct was used so that comparisons could be made to binding affinities reported previously.(15) The data are summarized in Table 1.

The RNA-binding module, FITC-K, a fluorescently labeled derivative of 6′-N-5-hexynoate kanamycin A has a previously reported Kd of 1 μM.(13) The affinities of the modularly assembled compounds, however, are much higher. For example, 4K-2 has a binding affinity of 4 nM and 4K-2-DR9 has a Kd of 3.5 nM. 4K-2-DR9 binds to r(CUG)12×2 with a stoichiometry of 3.7±1.2. Since the RNA target contains 12 copies of the 5′CUG/3′GUC motif, the stoichiometry indicates that the designed ligand is approximately interacting with each 5′CUG/3′GUC motif. This was expected based on previous experiments with 4K-2 and other related compounds.(13)

Additionally, 4K-2-DR9 was tested for binding to potential cellular bystander RNA, using bulk yeast. The compound interacts with tRNAs very weakly with a Kd of greater than 2 μM. The control compounds, 4Az-2-DR9 and 4N-2-DR9, bind tRNA and r(CUG)12×2 very weakly; binding curves indicate that the Kd’s are greater than 2 μM. The addition of the uptake tag does induce some non-specific RNA binding, as expected and as evidenced by the protein displacement data (Table 1).

Biological Efficacy of nK-2-DR9 Compounds in Cell-Based Model Systems of DM1

Next, the compounds and their appropriate controls were studied for modulating the toxicity of r(CUG)exp in cell-based models of DM1. Three models were used that probe (i) r(CUG)exp toxicity derived from pre-mRNA splicing defects due to sequestration of MBNL1;(19, 26) (ii) r(CUG)exp toxicity derived from nuclear retention, and thus reduced translation, of the DMPK mRNA;(27, 28) and, (iii) formation of nuclear foci due to r(CUG)exp-protein complexes.(29, 30)

Improvement of Pre-mRNA Splicing Defects

Pre-mRNA alternative splicing was assayed in HeLa cells as described above.(32) Briefly, cells were transfected with a DM1 mini-gene that expresses 960 interrupted r(CUG) repeats and a pre-mRNA splicing reporter mini-gene of interest.(31, 32) We first investigated the effect of the compounds on the alternative splicing of the cTNT mini-gene,(21) the parent gene of which is mis-spliced in DM patients.(21, 38, 39) In healthy cells, MBNL1 binds upstream of exon 5 in the cTNT pre-mRNA and represses its inclusion.(38, 40) In the DM1 model system, approximately 65% of exon 5 is included in cTNT mRNA in the absence of r(CUG)exp while approximately 90% of exon 5 is included in the presence of r(CUG)exp (Figure 3).

As shown in Figure 3, 2K-2-DR9 and 4K-2-DR9 improve the pre-mRNA splicing defect observed in the cTNT mini-gene towards healthy/wild type levels (no r(CUG)exp expression) at micromolar concentrations. For 2K-2-DR9, pre-mRNA splicing defects improve ~50% when cells are treated with 2 and 20 μM (two-tailed p value = 0.0418) while no effect is observed at lower concentrations. For 4K-2-DR9, pre-mRNA splicing defects are only modestly affected at 1 and 0.1 μM; however, pre-mRNA splicing is restored to levels observed in the absence of r(CUG)exp when cells are treated with 10 μM compound (two-tailed p value = 0.0309). Thus, designed compounds improve pre-mRNA alternative splicing towards a non-DM1-like state to varying extents, with 4K-2-DR9 being more efficacious in vitro and in vivo.

A series of control experiments were also completed. First, 4Az-2-DR9 and 4N-2-DR9 were also studied for affecting pre-mRNA splicing. The control compounds were chosen to investigate the role of the RNA-binding module. The compounds are weak in vitro inhibitors (Table 1). As shown in Figure 3, neither compound improves cTNT pre-mRNA splicing. Additional control experiments demonstrated that neither 2K-2-DR9 nor 4K-2-DR9 affect (i) the alternative splicing of the cTNT mini-gene in the absence of r(CUG)exp (Figure 3B); (ii) the alternative splicing of a PLEKHH2 mini-gene, the alternative splicing of which is not regulated by MBNL1 (Figure 3B); and (iii) the alternative splicing of endogenous genes (CAMKK2 and TTC8) which are also not regulated by MBNL1 (data not shown).

Improvement of Translational Defects

In order to determine if 2K-2-DR9 or 4K-2-DR9 can improve DM1-associated translational defects, a stably transfected cell line in which r(CUG)800 was placed in the 3′ UTR of firefly luciferase mRNA was used (Figure 4).(31) As mentioned above, expression of luciferase is low due to the binding of r(CUG)800 to MBNL1 and other proteins, resulting in nuclear retention of the luciferase mRNA. In good agreement with the results of the pre-mRNA splicing assays described above, 2K-2-DR9 and 4K-2-DR9 increase the nuclear export and translation of the luciferase mRNA as determined by an increase in luciferase activity. For example, 0.3 and 1.2 μM of each compound stimulates luciferase production by at best 20%. However, both compounds stimulate luciferase production by over 50% and by as much as 90% when cells are dosed with 5 or 20 μM compound. In contrast, no effect on luciferase activity was observed when the cells were treated with as much as 20 μM of the two control compounds, 4Az-2-DR9 and 4N-2-DR9.

Control assays were completed in which 2K-2-DR9 and 4K-2-DR9 were tested for non-specific production of luciferase by using a luciferase mRNA without r(CUG)exp in the 3′ UTR. No change in luciferase production was observed when the cells were treated with as much as 20 μM 2K-2-DR9 or 4K-2-DR9.

Disruption of Nuclear Foci

Another hallmark of DM1-affected cells is the presence of nuclear foci that consist of r(CUG)exp-protein complexes (26). Therefore, a fluorescence in situ hybridization assay (FISH) was used to probe if 4K-2-DR9 can decrease the occurrence of nuclear foci. HeLa cells were transfected with the DM1 mini-gene and treated with 4K-2-DR9. The cells were then probed with a 2′-O-methyl oligonucleotide labeled with Cy3 that is complementary to r(CUG)exp. The cells were then imaged via confocal microscopy (Figure 5). In the absence of 4K-2-DR9, multiple nuclear foci are observed in each cell, which correspond to r(CUG)exp-protein complexes (Figure 5a). Upon addition of 4K-2-DR9, however, there is a marked reduction in the number of the nuclear foci and the small number of foci that remain are much smaller in size compared to those observed in untreated cells. Since 4K-2-DR9 is labeled with fluorescein, cellular permeability and localization can also be imaged. Fluorescence from the compound is observed in almost every cell and is highly abundant in the cytoplasm with some nuclear localization.

Figure 5
4K-2-DR9 disrupts the formation of nuclear foci in a DM1 model system as assayed by fluorescence in situ hybridization. A, confocal microscopy images of untreated cells that express r(CUG)exp. From left to right: DAPI fluorescence (nuclear stain), Cy3 ...

The microscopy data support the results obtained from the luciferase reporter system used to assay the DM1 translational defect. For example, if 4K-2-DR9 was completely localized to the nucleus, then it could cause a further decrease in the production of luciferase by increasing the transcript’s nuclear retention. The observation that 4K-2-DR9, however, enhances luciferase production and is mainly cytoplasmic with some nuclear localization lends some support to a mechanism in which 4K-2-DR9 binding to r(CUG)exp, displaces MBNL1 and enables cytoplasmic transport.

4K-2-DR9 Improves pre-mRNA Splicing Defects in a Mouse Model of DM1

A mouse model of DM1 has been reported in which expanded r(CUG) repeat are expressed using a skeletal actin promoter (HSALR).(20) The presence of the repeats causes the mis-splicing of the muscle-specific chloride ion channel (Clcn1) and the sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1) pre-mRNAs.(4144) Normal adult mice have a Clcn1 exon 7a exclusion rate of 96%; DM1 mice have an exclusion rate of 61% (Figure 6). When DM1 mice are dosed with 80 mg/kg of 4K-2-DR9, the exclusion rate is partially rescued to 71% (Figure 6). These improvements in splicing are statistically significant as determined by a t test (p = 0.0022). Atp2a1 mis-splicing is also partially rescued. In normal adult mice, the inclusion rate for exon 22 is 100% while the inclusion rate in the HSALR line is only 10% (Figure 6). When mice are dosed with 80 mg/kg of 4K-2-DR9, splicing is partially rescued with an inclusion rate of 26% (Figure 6). Again, the improvement in splicing is statistically significant (p = 0.0491).

Comparison to other studies

Previous studies have reported three other compounds that improve DM1-associated defects in cell culture. They include pentamidine,(32) a bis-benzimidazole (H1),(45) and modularly assembled compounds displaying a derivative of Hoechst 33258 as the RNA-binding module (2H-4, 3H-4, and 4H-4).(31) The concentrations required to afford bioactivity is much greater with the lower molecular weight and thus more “drug-like” small molecules (pentamidine and H1) than with the modularly assembled structures. For example, the IC50’s of H1 and pentamidine that improve pre-mRNA splicing defects are 500 and 50 μM, respectively.(32, 45) The modularly assembled structure 2H-4, 3H-4, and 4H-4 restore splicing patterns to levels that are observed in the absence of r(CUG)exp at low micromolar concentrations (10, 50, or 50 μM, respectively).(31) Thus, 2K-2-DR9 and 4K-2-DR9 are as effective in these cell-based assays as other modularly assembled compounds targeting r(CUG)exp but are much more effective than bioactive monomeric ligands.

Improvement of the translational defect was also probed with the modularly assembled Hoechst 33258 compounds.(31) 2H-4, 3H-4, and 4H-4 increased translation by 100% at 6, 3, and 3 μM, respectively. 2K-2-DR9 and 4K-2-DR9 also stimulate translation; dosing of 20 μM of either compound increases translation by 80–90%. Thus, 2K-2-DR9 and 4K-2-DR9 are slightly less effective than the nH-4 compounds that were previously described.

Future Directions and Conclusions

In this report, we determined that multivalent small molecules that display an aminoglycoside derivative can mitigate the toxicity of r(CUG)exp RNA that causes DM1. By conjugating a cellular uptake tag onto first-generation compounds, the compound has improved cellular permeability and improves DM1-assoicated defects in cell and animal models. Traditionally, most RNA drug targets have been difficult to exploit as targets for small molecules. This and other studies, however, have shown that small molecules that target RNA and modulate its function in both cellular and animal models of disease can be designed.

Further developments in this area will focus on the study of modularly assembled small molecules targeting the r(CCUG)exp that causes DM2;(13, 16) it is likely that these compounds will be bioactive upon conjugation to a DR9 tag. Previous studies have shown that alterations in the spacing submonomer, propylamine in 4K-2, can affect cell uptake and localization into a variety of cell lines.(33) Thus, there is the possibility that the DR9 tag may no longer be required for bioactivity. Development in these areas will be reported in due course.


Quantitative Time-Resolved Fluorescence Resonance Energy Transfer (qTR-FRET) Assay

The qTR-FRET assay used to identify lead inhibitors of the r(CUG)10-MBNL1 complex is based on previously published report.(46) Briefly, 5′-biotinylated r(CUG)10 was folded in 1X Folding Buffer (20 mM HEPES, pH 7.5, 110 mM KCl, and 10 mM NaCl) by heating at 60 °C followed by slowly cooling to room temperature on the bench top. The buffer for r(CUG)10 was adjusted to 1X Assay Buffer (20 mM HEPES, pH 7.5, 110 mM KCl, 10 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM DTT, 0.1% BSA, and 0.5% Tween-20) and MBNL1-His6 was added. The final concentrations of RNA and MBNL1 were 80 nM and 60 nM, respectively. The sample was allowed to equilibrate at room temperature for 5 min, and then the compound of interest was added. After 15 min, strepatividin-XL665 (cisbio Bioassays) and anti-His6-Tb (cisbio Bioassays) were added to final concentrations of 40 nM and 0.44 ng μL−1, respectively, in a total volume of 10 μL. The samples were incubated for 1 h at room temperature and then transferred to a well of a white 384-well plate.

Time-resolved fluorescence was measured on a Molecular Devices SpectraMax M5 plate reader. Fluorescence was first measured using an excitation wavelength of 345 nm and an emission wavelength of 545 nm (fluorescence due to Tb). TR-FRET was then measured by using an excitation wavelength of 345 nm, an emission wavelength of 665 nm, a 200 μs evolution time, and a 1500 μs integration time.

The ratio of fluorescence intensity of 545 nm and 665 nm as compared to the ratios in the absence of ligand and in the absence of RNA were used to determine IC50’s. The percentage of MBNL1 binding that was inhibited was plotted versus ligand concentration and the resulting curve was fit to SigmaPlot’s 4-parameter logistic function in order to determine the IC50 (Equation 1):

Equation 1

where y is the percentage of MBNL1 bound, D is the minimum response plateau, A is the maximum response plateau, and x is the concentration of ligand. A and D are typically 100% and 0%, respectively. In cases of weak inhibition, IC50’s were determined by fitting the curves to a straight line.

RNA Binding Assays

The affinities of RNA-ligand complexes were determined as described using a fluorescence emission-based assay. Briefly, RNA was annealed in 1X MBNL Buffer (50 mM Tris HCl, pH 8.0, 50 mM NaCl, 50 mM KCl, 1 mM MgCl2) without MgCl2 by incubating at 60 °C for 5 min followed by slowly cooling to room temperature. Then, MgCl2, BSA, and ligand of interest were added to final concentrations of 1 mM, 40 μg mL−1, and 100 nM, respectively. The RNA was serially diluted in 1X MBNL buffer containing 40 μg mL−1 BSA and 100 nM ligand and incubated for 1 h at room temperature. Fluorescence intensity was determined using a BioTek FLX-800 plate reader. Scatchard analyses were completed to determine stoichiometry and dissociation constants, accounting for statistical effects by using a functional form of the Scatchard equation for large ligands binding to a lattice (Equation 2) (47, 48):

Equation 2

where v is the moles of ligand per moles of RNA lattice, [L] is the concentration of ligand, N is the number of repeating units on the RNA, l is the number of consecutive lattice units occupied by the ligand, and k is the microscopic dissociation constant. This equation simplifies to the commonly used form of the Scatchard equation for simple systems.(47, 48) Experiments were completed in triplicate and the reported errors are the standard deviations in those measurements.

Improvement of Splicing Defects in a Cell Culture Model Using RT-PCR

In order to determine if the compounds improve splicing defects in vivo, a previously reported method was employed.(32) Briefly, HeLa cells were grown as monolayers in 96-well plates in growth medium (1X DMEM, 10% FBS, and 1X GlutaMax (Invitrogen)). After the cells reached 90–95% confluency, they were transfected with 200 ng of total plasmid using Lipofectamine 2000 reagent (Invitrogen) per the manufacturer’s standard protocol. Equal amounts of a plasmid expressing a DM1 mini-gene with 960 CTG repeats (21) and a mini-gene of interest (cTNT (21) or PLEKHH2 (40)) were used. Approximately 5 h post-transfection, the transfection cocktail was removed and replaced with growth medium containing the compound of interest. After 16–24 h, the cells were lysed in the well, and total RNA was harvested with a Qiagen RNAEasy kit. An on-column DNA digestion was completed per the manufacturer’s recommended protocol.

A sample of RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) as previously described (40) except 5 units of AMV Reverse Transcriptase from Life Sciences were used. Approximately 300 ng were reverse transcribed, and 150 ng were subjected to PCR using a radioactively labeled forward primer. RT-PCR products were observed after 25–30 cycles of: 95 °C for 1 min; 55 °C for 1 min; 72 °C for 2 min and a final extension at 72 °C for 10 min. The products were separated on a denaturing 5% polyacrylamide gel and imaged using a Typhoon phosphorimager.

Control experiments were also completed in which HeLa cells were transfected with a plasmid encoding a mini-gene with five CTG repeats in the 3′ UTR or with a mini-gene that encodes a pre-mRNA whose splicing is not controlled by MBNL1 (PLEKHH2; (40)). The effect of the compound on the splicing of endogenous mRNAs not regulated by MBNL1 (TTC8 and CAMKK2) was also determined as previously described.(32) Differences in alternative splicing were evaluated by a t test. Please see the Supporting Information for a list of the primers used for each gene.

Disruption of Nuclear Foci Using Fluorescence In Situ Hybridization (FISH) (32)

HeLa cells were grown as monolayers in Mat-Tak glass-bottomed, 96-well plates. After the cells reached 90–95% confluency, they were transfected with 200 ng of a plasmid encoding a DM1 mini-gene (21) using Lipfoectamine 2000 per the manufacturer’s standard protocol. The transfection cocktail was removed 5 h post-transfection, and the compound of interest was added in growth medium.

After 16–24 h, the cells were washed with 1X DPBS and fixed with 4% paraformaldehyde in 1X DPBS for 10 min at 37 °C/5% CO2. After washing with 1X DPBS, the cells were permeabilized with 1X DPBS + 0.1% Triton X-100 for 10 min at room temperature. The cells were washed with 1X DPBS + 0.1% Triton X-100 and then with 30% formamide in 2X SSC Buffer (30 mM sodium citrate, pH 7.0, 300 mM NaCl) for 10 min at room temperature.

The cells were incubated in 1X FISH Buffer (30% formamide, 2X SSC Buffer, 66 μg mL−1 bulk yeast tRNA, 2 μg mL−1 BSA, 2 mM vanadyl complex (New England Bio Labs) and 1 ng μL−1 DY547-2′OMe-(CAGCAGCAGCAGCAGCAGC)) for 2 h at 37 °C. They were then washed with 30% formamide in 2X SSC for 30 min at 42 °C, 1X SSC for 30 min at 37 °C, and 1X DPBS + 0.1% Triton X-100 for 5 min at room temperature. Finally, nuclei were stained by incubating the cells with 1 μg mL−1 DAPI for 5 min at room temperature. The cells were washed with 1X DPBS + 0.1% Triton X-100, and 100 μL of 1X DPBS were added to each well. The cells were imaged using an Olympus FluoView 1000 Confocal Microscope at 60X magnification.

Treatment in mice

All experimental procedures, mouse handling, and husbandry were completed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care. A mouse model for DM1, HSALR in line 20b,(20) was used to investigate if 4K-2-DR9 improves splicing defects in animals. HSALR mice express human skeletal actin RNA with r(CUG)exp in the 3′ UTR. Age- and gender-matched HSALR mice were injected intraperitoneally with 80 mg/kg 4K-2-DR9 in saline or saline alone once per day for 7 days. Mice were sacrificed one day after the last injection. The vastus muscle was removed, and the RNA was extracted. cDNA was synthesized as previously described.(44) PCR amplification was carried out for 22–24 cycles with the following primer pairs: Clcn1 forward: 5′-TGAAGGAATACCTCACACTCAAGG and reverse: 5′-CACGGAACACAAAGGCACTG; Atp2a1 forward: 5′-GCTCATGGTCCTCAAGATCTCAC and reverse: 5′-GGGTCAGTGCCTCAGCTTTG. The PCR products were separated by polyacrylamide gel electrophoresis, and the gel was stained with SYBR Green I (Invitrogen). The gel was imaged with a laser fluorimager (Typhoon, GE Healthcare) and the products quantified using ImageQuant. A t test was used to determine the statistical significance of differences between two groups.

Supplementary Material



We thank S. Matosevic for assistance with confocal microscopy and M. Lee for preliminary studies. This work was funded by the National Institutes of Health (3R01GM079235-02S1 and 1R01GM079235-01A2 to MDD; AR049077 and U54NS48843 to CAT), by the Muscular Dystrophy Association (Grant# 158552 to MDD), and by The Scripps Research Institute. MDD is a Camille & Henry Dreyfus New Faculty Awardee, a Camille & Henry Dreyfus Teacher-Scholar, and a Research Corporation Cottrell Scholar.


Supporting Information Available: This material is available free of charge via the Internet at


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