Laboratory Projects

Research Overview

Chemical probes of cellular function have unraveled many of biology’s mysteries, for example the use of antibiotics to elucidate the mechanism of translation.1 In addition, precise compounds that target RNAs can serves as important lead therapeutics because there is a close association with specific RNA structures and various diseases.  The potential impact of chemical probes to study RNA function is immense. Indeed, it is estimated there are 30,000 non-redundant human mRNAs2 and >700 novel conserved RNA structures, almost all with unknown function, in the mammalian transcriptome.3 However, there is a dearth of probes that augment or disable RNA function, and indeed RNA has been considered as undruggable. Although an extraordinarily challenging problem, the development of methods to selectively target RNA structures with small molecules will help define the many unknown roles of RNA. Our research group has developed transformative technologies that allow one to design highly selective and potent small molecules from RNA sequence to define and control RNA function. We apply these technologies to develop lead therapeutics against disease to which there is a poor prognosis such as various cancers or to orphan diseases to which there is no known cure. By using these approaches we have elucidated a novel, RNA-mediated mechanism of gene silencing in Fragile X Syndrome, the most common cause of Autism,4 have defined the roles of a specific miRNA precursor in various cancers including hard to treat triple negative breast cancer,5 and have modulated the functions of disease-related mRNAs6-12 and translation of RNAs that lack a canonical start codon for diseases that range from ALS, the most common causes of adult on-set muscular dystrophy, and a myriad of other diseases.13

Decoding RNA Structure with Small Molecules


Figure 1:  The identification and annotation of selective RNA motif-small molecule interactions using 2DCS and StARTS, methods that were developed in our laboratory.  Briefly, a small molecule library is immobilized onto a microarray and hybridized with an RNA motif library.  The selected RNAs are sequenced and analyzed to form a chemical code for selectively targeting RNA. The selected RNA motif-small molecule partners (bottom panel) are then mined against the transcriptome to agnostically identify RNAs that can be targeted with these small molecules. We have developed software, termed Inforna, which identifies lead compounds using 2DCS and StARTS.

RNA folds are composites of secondary structural elements (i.e., motifs) such as internal loops, bulges and hairpins. These motifs, comprised of non-canonically paired regions (outside of normal GC or AU base pairs), are potential binding sites for small molecules. Heretofore it was not possible to translate RNA structure to develop small molecule probes that augment or disable RNA functions.  Ideally, the design of small molecules that selectively target RNA should be programmable and predictable, analogous to Watson-Crick base pairing rules used in oligonucleotide design. To accomplish this, a “code” is required to define which small molecules most avidly and selectively bind to a given RNA structure. We reasoned that advances in RNA secondary structure determination (phylogeny or experimental) would allow for the design of probes solely based on RNA sequence.  To define this code, we developed a selection-based approach coined Two-Dimensional Combinatorial Screening (2DCS) that allows one to rapidly identify small molecule-RNA motif partners in which the motifs are likely to be found within cellular RNAs present.14  In 2DCS, a library of small molecules is immobilized onto a microarray surface, and the array is hybridized with a library of discrete motifs, to select RNA motifs that a small molecule prefers to bind (Fig. 1).  These preferred interactions are used to design and synthesize chemical probes that manipulate RNA function. Along with 2DCS, we developed an approach termed Structure-Activity Relationship Through Sequencing (StARTS), which allows for facile annotation of 2DCS data by scoring the relative affinity and selectivity of RNA motifs that bind to a small molecule (a metric of small molecule “fitness”).15  Both technologies have been essential to our success in designing highly selective small molecules that modulate RNA function.4-7, 10, 16

Using Computation to Identify “Druggable” RNA Targets from RNA Sequence (the Transcriptome)  – Inforna: a bioinformatics approach to design selective small molecule chemical probes from sequence


Figure 2:  The Inforna approach applied to design chemical probes of miRNA function without target bias. A, schematic of Inforna; B, profiling 149 miRNAs expressed in MCF7 cells. Compound 1 only affects the pri-miRNA precursor of miR-96, while an oligonucleotide antagomir to miR-96 affects many; C, the small molecule selectively induces apoptosis in MCF7 breast cancer cells by inhibiting the miR-96-FOXO1 pathway.

To design small molecules that specifically target disease-associated RNAs from sequence, we developed a computational approach termed Inforna.5 Inforna integrates RNA motif-small molecule interactions identified by 2DCS, the fitness of these interactions as determined by StARTS, and structural data on the RNA target of interest, whether determined by experiment, phylogeny, or computation. Specifically, Inforna generates lead compounds for an RNA of interest by comparing the motifs found in its structure to the motifs in our chemical code.  The output is the targetable motifs, the corresponding lead small molecules for the RNA of interest, and the fitness of the predicted RNA-small molecule interaction.  Lead compounds are then used to study RNA biology.

Inforna was used, in a transcriptome-wide fashion, to design lead compounds that bind microRNA (miRNA) precursors (pri-miRNA) that inhibit their processing by binding to nuclease sites (Fig. 2).  MiRNAs are 21-25 nucleotides and negatively regulate gene expression via translational repression or cleavage of a target mRNA. MiRNAs have fundamental importance and yet, there are gaps in our understanding of their function.  Further, miRNA dysfunction contributes to disease, including cancer, immune dysfunction and infectious disease.17

The outputs from Inforna are the targetable motifs in each miRNA precursor and the corresponding lead small molecules that can bind to them. The lead interactions were then refined by restricting the targetable motif to Dicer or Drosha processing sites, to inhibit processing to the mature form, and to miRNAs that were validated to contribute to disease.  Collectively, Inforna identified 27 lead interactions, of which 44% were bioactive without lead optimization. The most fit of these interactions (as determined StARTS analysis) was between the benzimidazole 1, and the Drosha site in the miR-96 precursor. As predicted, 1 reduces levels of mature miR-96 and pre-miR-96 (product after Drosha cleavage) and boosts levels of pri-miR-96.

Importantly, 1 allowed us to probe several aspects of miRNA biology.5 First, miR-96, -182, and -183 precursors are transcribed from a single pri-miRNA transcript,18 and we found that 1 inhibits Drosha processing of miR-96 but not that of miR-182 or -183. Thus, there is no cooperativity between Drosha processing sites.  We will determine if this lack of cooperativity is a general phenomenon for other pri-miRNAs, which is likely governed by spatial effects.  Second, these analyses established selective effects of miR-96 on downstream targets and pathways. One target of miR-96 is FOXO1, a pro-apoptotic transcription factor.  Although FOXO1 is targeted by several miRNAs (miR-96, -182, and -27a),19 we showed that inhibiting miR-96 is sufficient to selectively upregulate FOXO1 and promote apoptosis of cancer cells. We also showed that 1 selectively modulates apoptosis via the miR-96-FOXO1 pathway as siRNA directed against FOXO1 mRNA ablated the apoptotic effect of 1.

Finally, we established the selectivity of 1 by profiling 149 other miRNAs in MCF7 cells (Fig. 2B).  Our data show that 1 only significantly affects miR-96 biogenesis, providing an unparalleled level of compound selectivity. Moreover, 1 is more selective than an oligonucleotide antagomir that targets sequence (Fig. 2B)!5 Collectively, these studies illustrate that small molecules targeting RNAs can be extremely selective and affect downstream processes such as apoptosis.

As our chemical code expands, our ultimate goal is to develop highly selective probes of all miRNAs, so we can answer fundamental questions regarding the function and regulation miRNA circuits in physiology and disease.  We are well on our way in these campaigns, where we have also targeted two hypoxia-associated miRNAs associated with metastatic and chemo-resistant behavior20 as well as an oncogenic miRNA.12  These studies, completed by interrogating effects on the proteome and transcriptome, will reveal the singular effects of miRNA, and of its effects on a given miRNA-mRNA complex in a cell.  Moreover, this approach can also be applied to a whole organism.

Lead Therapeutics and Chemical Probes for Large Classes of Rare, Orphan Diseases – Designer chemical probes to study the biology of repeating transcripts


Figure 3:  We assess the cellular biology of repeating transcripts using in-house chemical genetic probes developed in our laboratory. A, although downstream effects of expanded repeating transcripts is varied, each RNA folds into a hairpin structure with non-canonically paired nucleotides in the stem.  Repeating transcripts cause toxicity by binding and sequestering proteins involved in RNA biogenesis (gain-of-function), by producing toxic proteins via non-canonical start codons, and/or by gene silencing via novel RNA-mediated mechanisms.  Small molecules designed to target repeats specifically target the repeating structure of these aberrant RNAs. B, In cellulo click approaches developed to target repeats. Precisely positioned alkyne and azide moieties react with one other upon binding to a repeating RNA target.  By using a monomer in which a reactive handle is replaced with biotin, the cellular target(s) that enabled polymerization can be identified.  Further, the extent of polymerization in cellulo can be determined.

There are over 20 known microsatellite disorders that cause human neurological diseases, including myotonic dystrophy (DM), fragile X-associated tremor ataxia syndrome (FXTAS), Huntington’s Disease (HD), and amyotrophic lateral sclerosis (Lou Gehrig’s Disease; ALS).21 Though largely unknown, the effects of repeating transcripts on biological processes include alterations in alternative splicing, formation of nuclear foci/inclusions, and reduced translation of the repeat-containing transcript (Fig. 3A).22  More recently, it was discovered that expanded RNA repeats are translated without the use of a start codon (a.k.a., repeat-associated non-ATG [RAN] translation).23 Although their exact function is unclear, the resulting RAN proteins contribute to toxicity in microsatellite disorders,.23, 24  The interplay between these mechanisms is also poorly understood.  Small molecules can be used to target each mode of toxicity to dissect their effects in cells.  For example, do RAN peptides solely induce apoptosis in neurons to provoke disease?  RAN peptides are translated from multiple reading frames; are all RAN peptides equally toxic?  What are the functions and targets of RAN peptides?  Are alterations in alternative splicing the principle cause of cytotoxicity and disease?

We have developed chemical probes that target numerous expanded RNA repeats to interrogate their effects in cells and animals.6-11, 16, 25-29 Our general approach to target repeating transcripts utilizes our chemical code to identify small molecules that bind non-canonically paired structures embedded in the repeating transcript hairpin. The small molecule modules are then linked together to construct precise multivalent compounds that simultaneously bind multiple motifs (Fig. 3A). Both the nature of the RNA-binding module and the spacing between modules affects compound affinity and selectivity.30 Importantly, this approach engenders compounds with greater affinity and selectivity than proteins that bind to these RNAs.  Indeed, these compounds are the highest affinity and most selective RNA binding probes known to date!  Using these designer small molecules, we have shown that they can: (i) inhibit non-canonical translation by targeting the repeat RNA;13 (ii) manipulate RNA localization;6 (iii) unveil novel mechanisms of RNA-mediated gene silencing;4 and (iv) knockout pre-mRNA splicing regulators. Conversely, we have also designed agents that can induce repeat disorder pathology in healthy cells or exacerbate it in diseased cells.27 We will continue these investigations to develop high-resolution maps of the cellular biology of repeats.

We recently developed a transformative approach that synthesizes low nanomolar inhibitors of RNA repeat dysfunction in cellulo (Fig. 3B).31 That is, the disease-affected cell is used as a reaction flask and the repeating transcript is used as a catalyst to synthesize its own inhibitor!31  Here small molecule modules bind to adjacent sites in repeating transcripts and present reactive groups (azide and alkynes) to each other so cell-stable triazole moieties are formed.  By replacing a single azide or alkyne with biotin, the cellular targets of the oligomerized compound are then isolated with streptavidin beads and sequenced. This approach can be applied to any repeating transcript, and affords exquisitely selective, nM afiinity modulators of function!31

Importantly, one of our designed probes allowed the discovery of a novel mechanism of gene silencing that causes fragile X syndrome (FXS).4 Specifically, an RNA-DNA hybrid forms between r(CGG)exp and the fragile X mental retardation 1 (FMR1) gene, silencing transcription via induction of chromatin remodeling. Our small molecule thermodynamically stabilizes the r(CGG)exp, repeat and blocks its binding to the DNA target. This mechanism of gene silencing revealed new targets for chemical biology probes – the d(CCG) repeating DNA in FMR1 that is bound by r(CGG)exp and the RNA-DNA hybrid complex that silences the gene transcription. By specifically targeting each with a small molecule we can define their roles in gene silencing. As we have developed compounds that target other repeats, we will probe the generality of RNA-mediated gene silencing in other repeat expansion disorders. Indeed, it was reported that the r(G4C2) repeat that is associated with ALS/FTD may also involve a DNA-RNA hybrid.32

As noted above, toxic RAN peptides contribute to disease pathogenesis in both FXTAS and ALS/FTD.24, 33, 34  We have shown that our small molecules inhibit RAN translation of r(CGG)exp (the causative agent of FXTAS) in cell models, and of r(G4C2)exp (associated with ALS/FTD) in induced pluripotent stem cells (iPSCs) derived from ALS patients.13  Importantly, our compounds do not affect the translation of proteins encoded by canonical open reading frames, even when the repeat is embedded in the 5’UTR of the mRNA.  These compounds open new avenues to explore the mechanism by which proteins are translated without a canonical start codon.  In fact, proteome-wide profiling in viruses and human cell lines have shown that many peptides are translated without the use of start codons,35, 36 and these have been suggested to function as signaling molecules. Our methods are the first that allow one to manipulate the production of these novel translation products to define their roles in biology and disease These investigations are particularly important as compounds that modulate non-canonical translational events may have broad applicability, where these probes could define the mechanism of RAN protein production as well as their physiological roles.

Developing Technologies to Identify RNAs that Bind Small Molecules is Cells – ChemCLIP (Chemical Cross-Linking Isolated by Pull-down):  An approach to identify the RNA targets of small molecules and metabolites in cells.


Figure 4:  ChemCLIP identifies the cellular RNA targets of small molecules. In this approach, small molecules targeting RNA are appended with cross-linking and a purification tag.  Application of this compound to cells cross-links the small molecule to its cellular targets.  The biotin tag allows these targets to be purified from cells.  Analysis via seq. or qRT-PCR shows the targets bound by the small molecules.

Decoding RNAs that are bound by small molecules in cellulo is difficult due to the dearth of techniques to probe these interactions. We have developed a novel approach, termed ChemCLIP, to react small molecules with their cellular RNA targets in cellulo and in vivo, which allows facile identification of binding partners (or, in vivo 2DCS; Fig. 4). We will apply this approach to identify the cellular targets of the RNA-directed chemical probes noted above to validate targets and to enable more potent ways to manipulate and study their biological function.

In ChemCLIP, small molecules are appended with a reactive module and biotin. When added to cells, the small molecule reacts with its cellular targets and forms a covalent bond (Fig. 4).  The s mall molecule-target adducts are then isolated and purified using streptavidin resin. To control for non-specific reaction of reactive modules, we will perform competition studies using the unreactive parent agent.  Cellular targets specifically are depleted in sequencing data as a function of concentration of the parent compound.  ChemCLIP also has the potential to identify small molecule binding sites within an RNA via Chem-CLIP-Map. Indeed, we used to approach to validate binding of our designed small molecules to expanded repeating RNAs in cellulo.25


A myriad of roles for RNA in cellular biology have been uncovered, yet chemical probes to study their function are lacking. The impact of such probes, however, could be transformative, where these tools can be used to define the roles and regulatory pathways controlled by regulatory small RNAs (e.g., miRNAs), the effects of long noncoding RNAs, RAN peptides and disease-associated repeat RNAs on translation, transcription, gene silencing, and upon cell fate, proliferation, survival and transformation. The Disney lab has developed novel and transformative technologies that enable the selective targeting of any RNA molecule by chemical probes to define their role in biology and disease.  Importantly, proof-of-principle studies have established that this new generation of RNA-targeting chemical probes have tremendous potential to also transform medicine, for the treatment of a broad class of disorders and diseases.


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[4] Colak, D., Zaninovic, N., Cohen, M. S., Rosenwaks, Z., Yang, W. Y., Gerhardt, J., Disney, M. D., and Jaffrey, S. R. (2014) Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome, Science 343, 1002-1005.

[5] Velagapudi, S. P., Gallo, S. M., and Disney, M. D. (2014) Sequence-based design of bioactive small molecules that target precursor microRNAs, Nat Chem Biol 10, 291-297.

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[12] Velagapudi, S. P., and Disney, M. D. (2013) Design of a pre-microRNA-10b inhibitor enabled by 2DCS studies on the RNA motifs that bind molecular transporters, Chem Commun, submitted.

[13] Su, Z., Zhang, Y., Yang, W.-Y., Fostvedt, E., Petrucelli, L., and Disney, M. D. (2014) Designer small molecule modulators of r(GGGGCC) RNA toxicity in ALS/FTD, submitted.

[14] Disney, M. D., Labuda, L. P., Paul, D. J., Poplawski, S. G., Pushechnikov, A., Tran, T., Velagapudi, S. P., Wu, M., and Childs-Disney, J. L. (2008) Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners, J Am Chem Soc 130, 11185-11194.

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[20] Haga, C. L., Velagapudi, S. P., Phinney, D. G., and Disney, M. D. (2013), unpublished data.

[21] Brouwer, J. R., Willemsen, R., and Oostra, B. A. (2009) Microsatellite repeat instability and neurological disease, BioEssays : news and reviews in molecular, cellular and developmental biology 31, 71-83.

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[26] Guan, L., and Disney, M. D. (2013) Small molecule-mediated cleavage of RNA in living cells, Angew Chem Int Ed Engl 52, 1462-1465.

[27] Childs-Disney, J. L., Stepniak-Konieczna, E., Tran, T., Yildirim, I., Park, H., Chen, C. Z., Hoskins, J., Southall, N., Marugan, J. J., Patnaik, S., Zheng, W., Austin, C. P., Schatz, G. C., Sobczak, K., Thornton, C. A., and Disney, M. D. (2013) Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules, Nat Commun 4, 2044.

[28] Strack, R. L., Disney, M. D., and Jaffrey, S. R. (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA, Nat Methods 10, 1219-1224.

[29] Tran, T., Childs-Disney, J. L., Liu, B., Guan, L., Rzuczek, S., and Disney, M. D. (2014) Targeting the r(CGG) repeats that cause FXTAS with small molecules and oligonucleotides, ACS Chem Biol, submitted.

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[36] Slavoff, S. A., Mitchell, A. J., Schwaid, A. G., Cabili, M. N., Ma, J., Levin, J. Z., Karger, A. D., Budnik, B. A., Rinn, J. L., and Saghatelian, A. (2013) Peptidomic discovery of short open reading frame-encoded peptides in human cells, Nat Chem Biol. 9, 59-64. doi: 10.1038/nchembio.1120. Epub 2012 Nov 1018.