WO2019246120A2 - Microarray and method of identifying interactions between compounds and rna - Google Patents

Abstract

The invention provides a microarray having a substrate coated with a gel and a plurality of compounds that are non-covalently adhered to the gel at discrete locations. The invention also provides a method of identifying binding interactions between a compound and an RNA by using a microarray. The invention also provides a method of treating a subject suffering from an RNA mediated disease by administering compounds identified has having a binding interaction with the RNA which mediate the disease. For example, the invention provides a method of preventing or reducing cancer metathesis in a subject by administering to the subject a topoisomerase inhibitor which binds with miR-21.

Description

MICROARRAY AND METHOD OF IDENTIFYING INTERACTIONS BETWEEN

COMPOUNDS AND RNA CLAIM OF PRIORITY

[0001] This application claims the benefit of priority to U.S. Application Serial No.

62/686,834, filed on June 19, 2018, which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under 5R01GM097455 awarded by the National Institutes of Health. The Government has certain rights in this invention. BACKGROUND

[0003] RNA is involved with a myriad of cellular roles beyond merely encoding and assembling proteins. The Encyclopedia of DNA Elements project and subsequent analyses showed that only 1-2% of our genome encodes for protein yet about 80% of it is transcribed into RNA (ENCODE, 2012). Although the majority of transcribed RNAs are non-coding, many non-coding RNAs are functionally involved in modulating cell activities and disease states. Such functional, non-coding RNAs represent a potential therapeutic target.

[0004] The development of therapeutics that target RNA has mostly centered on using oligonucleotides. Interactions between RNA and small molecule compounds are poorly understood which has led to the perception that RNA is“undruggable”. (Guan & Disney, 2012; Thomas & Hergenrother, 2008). However, small molecules have shown an ability to target RNA. For example, small molecules have been investigated for targeting the three- dimensional structure of ribosome, riboswitches, certain viral RNA and nucleotide repeat expansions. (Blount and Breaker, 2006; U.S. Patent No. 9,586,944 B2; U.S. Patent

Application Publication No.2016/0188791 A1)

[0005] A salient question is how to identify and better understand interactions between compounds and functional non-coding RNAs. An attractive and efficient approach is the use of microarrays in a massively parallel library-versus-library screening, dubbed two- dimensional combinatorial screening (2DCS) (Childs-Disney, et al., 2007; Disney, et al., 2008). Small molecule compound microarrays are powerful tools for identifying compounds that bind biomolecules. However, such microarrays have required that the arrayed compounds be modified to include reactive functional groups or covalent linkages which immobilize the compounds on the microarray substrate (U.S. Patent Application Publication No.2008/0188377 A1; Hergenrother, et al., 2000; MacBeath, et al., 1999; MacBeath and Schreiber, 2000).

SUMMARY OF THE INVENTION

[0006] The present disclosure provides a microarray, comprising a substrate coated with a gel; and a plurality of compounds that are non-covalently adhered to the gel at discrete locations. Methods of preparing and using the microarray and also provided herein.

[0007] The present disclosure further provides a method for identifying a binding interaction between a compound and an RNA, comprising: applying a plurality of labeled-RNAs and excess oligonucleotides to a microarray having a plurality of compounds non-covalently adhered at discrete locations of the microarray; incubating the microarray to induce binding between labeled-RNAs and adhered compounds; washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray; imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and characterizing the bound RNA to identify the binding interaction.

[0008] The present disclosure provides various methods of treatment. For example, the disclosure provides a method of treating a subject suffering from a miR-21 mediated disease, comprising administering to the subject an effective amount of a topoisomerase inhibitor. Methods of preventing or reducing cancer metastasis are also provided.

[0009] Advantages, some of which are unexpected, may be achieved by various

embodiments of the present disclosure. In various embodiments, the present invention can provide a microarray which allows for unmodified compounds, including FDA-approved drugs, to be probed for binding to RNA motif libraries in a massively parallel format. The present invention has advantages over microarrays which comprises compounds modified to include additional functional groups or compounds which covalently bind to the microarray. Specifically, the presence of additional functional groups or a covalently bound substrate can affect molecular recognition of the compound’s natural targets. Likewise, a compound which is covalently bound to a microarray will have conformational restrictions which may prevent or reduce binding. Unmodified compounds have the advantage of providing a more accurate picture of binding interactions during screening in comparison to versions of said compounds which were modified to contain binding functional groups or to covalently bind to the microarray. The method allows one to profile small molecules for the ability to bind RNA in an unbiased manner. Moreover, another advantage of the present invention resides in a microarray that can be assembled with compounds taken directly from a compound library without requiring intermediate steps to derivatize the compounds with covalent linkers. The microarray of the present invention thus may have the advantages of improved accuracy.

[0010] Various aspects and embodiments of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG.1 illustrates the development of the present microarrays and the RNA motif libraries used to study small molecule binding: (A) shows a representative image of the microarray after spotting mitoxantrone before and after washing; (B) shows a plot of the signal from mitoxantrone before and after washing; (C) shows secondary structures of the nucleic acids used in this study; (D) shows a representative image of the microarray used in a 2DCS screen (85.5 x 127.8 x 1.1 mm (width x height x thickness)).

[0012] FIG.2 shows how Dicer processing of pre-miR-21 is inhibited in vitro and in cells: (A) shows secondary structure of the miR-21 hairpin precursor; (B) shows locations of dicer processing and resulting fragments in illustration form with a representative gel image; (C) shows the effect of 1, 2, and 3 on mature levels in triple negative breast cancer cell line MDA-MB-231; (D) shows the effect of 1, 2, and 3 on pre-miR-21 levels in triple negative breast cancer cell line MDA-MB-231; and (E) shows the effect of 3, an LNA oligonucleotide targeting miR-21 (LNA-21; Exiqon, 4100689-101) and a control LNA (Scramble; Exiqon, 199006-102) on PTEN using a luciferase reporter assay. (* indicates p<0.5; ** indicates p<0.01, as determined by a two-tailed student t test).

[0013] FIG.3 shows the effect of compound 3 on DNA damage in MDA-MB-231 cells by VWDLQLQJ^IRU^Ȗ-H2AX foci, quantifying fluorescence and plotting against concentration of compound 3. (* indicates p<0.5; ** indicates p<0.01, as determined by a two-tailed student t test).

[0014] FIG.4 shows phenotypic effects of treatment with compound 3 on MDA-MB-231 cells incubated for 16 h at 37 °C through Matrigel basement membrane in a Boyden chamber assay, showing reduced invasion after miR-21 inhibition; a first plot shows quantification of cell migration shown in panel A (2 biological replicates, 4 fields of view); and a second plot shows effect on migration upon treatment with 3 (10 ^M) when pre-miR-21 is overexpressed via transfection of a miR-21 plasmid in MDA-MB-231 cells. (* indicates p<0.5; ** indicates p<0.01, as determined by a two-tailed student t test).

[0015] FIG.5 shows Chem-CLIP target validation studies of 3 in MDA-MB-231 cells; (A) shows structures of compound 10 (3-CA-Biotin) which is comprised of the RNA-binding module 3, a biotin purification module and a cross-linking chlorambucil (CA) module, and control compound 11 (Control CA-Biotin), which lacks the RNA-binding module; and (B) shows Chem-CLIP of pre-miR-^^^XVLQJ^^^^0^RI^^^^LQ^0'$-MB-231 cells and Competitive- Chem-CLIP (C-Chem-CLIP) of pre-miR-21 in MDA-MB-231 cells by using increasing concentrations of 3 to compete with 10 for binding (1 µM). (* indicates p<0.5; ** indicates p<0.01, as determined by a two-tailed student t test).

[0016] FIG.6 shows selectivity studies of 3; (A) shows pre-miRNAs that contain the same A and/or U bulge (A bulge and U bulges are indicated in boxes) found in the Dicer site of pre- miR-21; (B) shows selectivity of 3 for miR-21 over other miRNAs with the same target A and/or U bulge, as determined by measuring mature miRNA levels by RT-qPCR, where the numbers in parentheses after the miRNA name indicate relative expression levels as compared to miR-21; and (C) shows pull-down of other miRNAs that contain A and/or U bulges by 10, where the numbers in parentheses after the miRNA name indicates relative expression level as compared to miR-21. (* indicates p <0.05; ** indicates p<0.01, as determined by a two-tailed student t test; N.D. indicates mature miRNA levels were not detectable (C_t>32)).

[0017] FIG.7 shows representative fluorescent binding isotherms where a first plot shows binding isotherms of compounds 1, 2, and 3 to RNAs containing an A bulge, U bulge, or an A+U bulge; and a second plot shows binding isotherms of compounds 1, 2, and 3 to a base paired control RNA (data represents mean ± s.d.); and the secondary structures below show the A bulge, U bulge, A+U bulge, and base-paired control used for studying binding affinities of the tested compounds.

[0018] FIG.8 shows microscale thermophoresis binding analyses of compound 3; (A) shows a RNA construct containing both the A and U bulge displayed in the miR-21 hairpin precursor (miR-21 Hairpin Full); (B) shows a RNA construct containing only the A bulge displayed in the miR-21 hairpin precursor (miR-21 A Bulge); and (C) shows a base paired control RNA construct (miR-21 Base Pair).

[0019] FIG. 9 shows In vitro Dicer processing of pre-miR-21 wild type and pre-miR-21 A22 mutant; (A) shows pre-miR-21 RNA constructs used in this study. Pre-miR-21 A22 Mutant is the same as the Pre-miR-21 WT, but with the A bulge base paired to a U. Pre-miR-21 U27 Mutant is the same as Pre-miR-21 WT, but with the U bulge base paired to an A, where boxes represent the A or U bulge binding sites, and the S'-strand through to A22 represents the mature miR-21 product; (B) shows representative gels of in vitro Dicer processing of Pre- miR-21 WT and Pre-miR-21 A22 mutant with compound 3; and (C) shows quantification of the Dicer processing bands shown in B. (Data represents mean ± s.e.m. (n>3). * indicates p<0.5; *** indicates p<0.001, as determined by a two-tailed student t test).

[0020] FIG. 10 shows small molecule target validation in vitro; (A) shows in vitro Chem- CLIP of pre-miR-21; and (B) shows in vitro C-Chem-CLIP of pre-miR-21 by using increasing concentrations of 3 to compete with 10 for binding (1 pM). (* indicates p<0.5; ** indicates p<0.01, as determined by a two-tailed student t test).

[0021] FIG. 11 shows effects of Chem-CLIP compounds 10 and 11; (A) shows Chem-CLIP of pre-miR-21 using 1 mM of 10 and 11 in MDA-MB-231 cells; and (B) shows mature miR- 21 biogenesis is inhibited at 1 mM of 10. (** indicates p<0.01, as determined by a two-tailed student t test).

DETAILED DESCRIPTION OF THE INVENTION

[0022] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0023] In this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of“about 0.1% to about 5%” or“about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement“about X to Y” has the same meaning as“about X to about Y,” unless indicated otherwise. Likewise, the statement“about X, Y, or about Z” has the same meaning as“about X, about Y, or about Z,” unless indicated otherwise.

[0024] In this document, the terms“a,”“an,” or“the” are used to include one or more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive“or” unless otherwise indicated. The statement“at least one of A and B” has the same meaning as“A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. A comma can be used as a delimiter or digit group separator to the left or right of a decimal mark; for example, “0.000,1” is equivalent to“0.0001.”

[0025] In the methods described herein, the acts can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0026] The term“about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0027] The term“substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%.

[0028] The term“Nucleic acid”, as used herein, is meant to refer to RNA and DNA.

[0029]“RNA” or“RNAs”, as used herein, is meant to refer to ribonucleic acid molecules and oligomers. RNA includes mRNA, tRNA, rRNA, miRNA, siRNA, shRNA and the like.

[0030]“DNA”, as used herein, is meant to refer to deoxyribonucleic acid molecules and oligomers. [0031] The term“labeled-RNA”, as used herein refer to RNA which have been modified to contain a radiolabel, a fluorescent tag, a chromogenic tag or other detectable probe. Labeled RNAs may be provided or prepared from a RNA library.

[0032] The term“RNA library”, as used herein refer to a collection of RNA which may be screened in use of the present invention. Many institutions have RNA libraries and some may be commercially available. The RNA library includes a non-coding RNA library, a RNA motif library, a miRNA library, a viral RNA library, or any combination thereof. The RNA motif library may be an internal loop motif library, a hairpin loop motif library, a bulge motif library, a multibranch loop motif library, a pseudoknot motif library, or any combination thereof.

[0033] The term“RNA motif”, as used herein, is meant to refer to a targetable internal loop, hairpin loop, bulge, or other targetable RNA structural motifs. Examples of RNA motifs include symmetric internal loops, asymmetric internal loops, 1×1 internal loops, 1×2 internal loops, 1×3 internal loops, 2×2 internal loops, 2×3 internal loops, 2×4 internal loops, 3×3 internal loops, 3×4 internal loops, 4×4 internal loops, 4×5 internal loops, 5×5 internal loops, 1 base bulges, 2 base bulges, 3 base bulges, 4 base bulges, 5 base bulges, 4 base hairpin loops, 5 base hairpin loops, 6 base hairpin loops, 7 base hairpin loops, 8 base hairpin loops, 9 base hairpin loops, 10 base hairpin loops, multibranch loops, pseudoknots, etc. Examples of DNA motifs include symmetric internal loops, asymmetric internal loops, bulges, and hairpin loops. In some cases, the term“motif” includes RNA secondary structures generally, but also in cases may refer to a particular RNA structure that has already been identified.

[0034]“Chase oligonucleotides”, as used herein, are meant to include oligonucleotides that are designed to ensure that a screened compound interacts with the RNA motif (i.e., with the RNA motif library's variable region) and not with those nucleic acid regions that do not vary from member to member (e.g., invariant stem regions, invariant hairpin loop regions, etc.). The design of such stem chase and hairpin oligonucleotides may depend on the sequences used in the nucleic acid regions that do not vary from member to member. Chase nucleotides may sometimes include DNA chase oligonucleotides (i.e., oligonucleotides that are meant to ensure that the interactions are RNA specific). Example of suitable DNA chase

oligonucleotides include duplex AT decamers, duplex CG decamers, and combinations thereof. In certain embodiments, the one or more chase oligonucleotides includes stem chase oligonucleotides. In certain embodiments, the one or more chase oligonucleotides includes hairpin chase oligonucleotides. In certain embodiments, the one or more chase oligonucleotides includes DNA chase oligonucleotides. Combinations of these and other chase oligonucleotides can be employed, for example as in the case where the one or more chase oligonucleotides includes stem chase oligonucleotides, hairpin chase oligonucleotides, and DNA chase oligonucleotides.

[0035] The term“gel” as used herein, is meant to refer to a non-fluid colloidal network or polymer network that is expanded throughout its whole volume by a fluid. For example, the gel may, but is not required to, contain a covalently crosslinked polymer network; a polymer network formed through the physical aggregation of polymer chains, caused by hydrogen bonds, crystallization, helix formation, complexation, etc, that results in regions of local order acting as the network junction points; a polymer network formed through glassy junction points, e.g., one based on block copolymers. The gel may be a hydrogel.

[0036] The term“non-covalently adhered” or“adhered”, as used herein, is meant to refer to compounds adhered closely to a discrete area of a gel without forming covalent linkages between the compound and the gel. Adherence may be via absorption, e.g., into a fluid component of the gel solvent system, or may be via adsorption, e.g., to a surface of the gel, or a combination thereof. As another example, adherence may be the result of, e.g.,

thermodynamic and/or kinetic stabilization, van der Waals interactions, electrostatic interactions, solvation, or combinations thereof. In some cases, adherence may be the result of hydrogen bonding. In some cases, adherence may be described functionally or empirically, for example, adherence may be described by compounds which exhibit minimal diffusion, such that the compounds remain within discrete locations on a microarray gel. As another example, adherence may be described by compounds which remain substantially adhered when the microgel is washed or incubated. Groups of compounds may be considered non- covalently adhered when, as examples, at least 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9% are adhered to the gel without forming any covalent linkages. The above examples are not intended to limit the manner or extent that compounds adhere to the gel and are provided solely for illustrative purposes.

[0037] As used herein, non-covalently adhered does not include compounds which are coupled to the gel via a triazole linkage, e.g., formed via Huisgen cycloaddition of an azide and an alkyne. Adhered also does not include compounds which are spatially separated from the gel, such as where the gel has wells and compounds are dissolved or suspended in a solvent which is physically contained in the well. As another example, adhered does not include compounds which are contained with a discrete droplet which sits upon the gel but does not mix with the gel. For example, adhered does not include compounds which are immobilized as discrete droplets which are then contacted with the RNA motif library using aerosol deposition technology). Further, adhered does not include compounds which simply rest upon the microarray as a dry chemical microarray. For example, a dry chemical microarray has the disadvantage that, if subjected to washing, incubation, or both, the deposited compounds would mix or be removed completely from the discrete locations they were spotted to.

[0038] The term“binding interaction”, as used herein, is meant to refer to binding or other stabilized association between a small molecule and an RNA molecule or RNA motif. The association can be thermodynamically stabilized or kinetically stabilized or both, and the interaction can be the result of covalent bonding, hydrogen bonding, van der Waals interactions, electrostatic interactions, or combinations of these and/or other types of interactions.

[0039] The term“drug-like compound” refers to small molecule compounds having characteristics typical of FDA-approved small molecule drugs. For example, the compounds may have, but are not required to have, one or more of the following characteristics, no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, a molecular mass less than 500 g/mol, and an octanol-water partition coefficient log P not greater than 5. As another example, the compounds may have, but are not required to have, one or more of the following characteristics, no more than 3 hydrogen bond donors, no more than 3 hydrogen bond acceptors, a molecular mass less than 300 g/mol, and an octanol-water partition coefficient log P not greater than 3. As another example, the compound may be free of pharmacologically incompatible moieties. Other examples of drug-like compounds include small-molecule clinically-approved (e.g., FDA-approved) drugs and compounds which are derivatives thereof. A list of all FDA-approved drugs may be found at Drugs@FDA at www.accessdata.fda.gov/scripts/cder/daf/ or the FDA Orange Book which is incorporated by reference in its entirety.

[0040] The present invention provides a microarray comprising a substrate coated with a gel and a plurality of compounds that are non-covalently adhered to the gel at discrete locations.

[0041] The compounds of the microarray may be from a library of FDA-approved drugs, compounds used in phase I clinical trials, compounds used in phase II clinical trials, kinase inhibitors, topoisomerase inhibitors, mRNA splicing modulators, compounds predicted or known to have RNA-modulating activity, drug-like compounds, commercially-available bioactive compounds, or any combination thereof, and the compounds are unmodified therefrom. The compounds may be selected from a commercially-available bioactive compound library, or portion thereof, and the compounds may be unmodified therefrom. That is, the compounds may be unmodified forms of the compound for which a binding interaction with RNA is being evaluated. An advantage of the presently described microarray may be that it provides an array of compounds without requiring that the compounds be modified with functional groups or covalently coupled to the gel. In various embodiments, the microarray may be free of compounds which are covalently coupled to the gel. For example, the microarray may be free of modified forms of FDA-approved drugs, compounds used in phase I clinical trials, compounds used in phase II clinical trials, kinase inhibitors, topoisomerase inhibitors, mRNA splicing modulators, compounds predicted or known to have RNA-modulating activity, drug-like compounds, commercially-available bioactive compounds, or any combination thereof, where such compounds were modified to covalently couple to the gel.

[0042] The microarray of the present invention does not require that the arrayed compounds be subjected to immobilization chemistries. Thus, the compounds of the present invention can be free of the functionalization which was previously required to couple and thus immobilize compounds to a microarray. As an example, the compounds of the microarray may be free of any, or all, of azide moieties, alkyne moieties, silyl chloride moieties, maleimide moieties, thiol moieties. Likewise, the compounds may comprise a variety of functional groups provided they are free of a functional group which would irreversible and covalently bind to the gel. For example, the microarray may be free of azide and some of the compounds may comprise an alkyne.

[0043] The compounds may be adhered to the gel by being first deposited on the gel when the gel is partially dry and at least partially solvated so that the compounds may become incorporated into the gel. The gel may then be dried to result in a gel having compounds non- covalently adhered. The compounds may be adhered to the gel via absorption, e.g., into a fluid component of the gel solvent system. The compounds may be adhered to the gel via adsorption, e.g., to a surface of the gel. The compounds may exhibit minimal diffusion, such that the compounds remain within discrete locations on the microarray. The compounds may remain substantially adhered when the microgel is washed or incubated. [0044] At least 4 different compounds may be adhered to the gel. In various embodiments at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or at least 1000 different compounds may be adhered to the gel. Such compounds may be selected from any one or more of FDA-approved drugs, compounds used in phase I clinical trials, compounds used in phase II clinical trials, kinase inhibitors, topoisomerase inhibitors, mRNA splicing modulators, compounds predicted or known to have RNA-modulating activity, drug- like compounds, and commercially-available bioactive compounds. In various embodiments, such compounds may be structurally or functionally related. For example, all compounds deposited on the microarray may be from the same commercially available library of compounds, they may all be known topoisomerase inhibitors, or they may all be FDA- approved drugs.

[0045] The plurality of compounds may be adhered at the same loading, although this need not be the case. Suitable compound loadings may be from about 1 femptomole to about 100 nanomoles (such as from about 100 femptomoles to about 10 nanomoles and/or from about 1 picomole to about 1 nanomole) of compound per discrete location. In various embodiments, each compound may be present in the gel in an amount of about 1 pmol to about 1 µmol.

[0046] In various embodiments, at least one of the particular compounds may be present at two or more different loadings, the two or more different loadings being adhered at separate, discrete locations on the gel. In certain embodiments, each of the particular compounds may be present at two or more different loadings, the two or more different loadings being adhered at separate, discrete locations on the gel. In certain embodiments, at least one of the particular compounds may be present at four or more different loadings, the four or more different loadings being adhered at separate, discrete locations on the gel. In certain embodiments, each of the particular compounds may be present at four or more different loadings, the four or more different loadings being adhered at separate, discrete locations on the gel. Where two or more different loadings (e.g., 2, 3, 4, 5, 6, etc. different loadings) are employed for a particular compound, the different loadings can be effected by serial dilution (e.g., as in the case where the first loading is x, the second loading is x/2, the third loading is x/4, the fourth loading is x/8, the fifth loading is x/16, etc.).

[0047] Each compound may have a molecular weight less than about 1,000 g/mol. Each compound may have a molecular weight of less than, or greater than, 100 g/mol, 150 g/mol, 200 g/mol, 250 g/mol, 300 g/mol, 350 g/mol, 400 g/mol, 450 g/mol, 500 g/mol, 550 g/mol, 600 g/mol, 650 g/mol, 700 g/mol, 750 g/mol, 800 g/mol, 850 g/mol, 900 g/mol or 1,000 g/mol. The compounds may be within a range of 100 g/mol to 700 g/mol, 100 g/mol to 600 g/mol, 200 g/mol to 600 g/mol, 300 g/mol to 500 g/mol, or the like.

[0048] The gel of the microarray may comprise a polysaccharide, a polyacrylamide, or a combination thereof. The gel may comprise agarose or sepharose. The gel may comprise agar. The gel may comprise a polydextran. The gel may also comprise, for example, any of carrageenan, gellan, welan, alginate, amylose, rhamsan xanthan and guar gum

polysaccharides, and the like. The gel may be free of functionalized derivatives of polysaccharides or polyacrylamides. The gel may be free of functionalized agarose. For example, the gel may be agarose, which has not undergone any modification to further incorporate additional functional groups such as any azide or alkyne moieties.

[0049] The gel may be less than, may be greater than, and/or may be about equal to, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%,7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10% (w/v) gel.

[0050] The gel may be at least, may be less than, and/or may be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5.0% (w/v) agarose gel.

[0051] In various embodiments, the gel may be about 0.5% to about 5% (w/v) agarose gel.

[0052] The gel may be any shape and does not require, or may be free, of any particular molded structures or macroscopic architecture. For example, the gel may be free of macroscopic wells, channels or reservoirs. In various embodiments, the gel provides the appearance of being substantially flat, having a smooth outer surface. In various

embodiments, the gel has a substantially flat surface free of wells and the compounds may be incorporated into the gel mesophase or the gel fluid.

[0053] In various embodiments, the microarray may retain sufficient solvent or moisture such that deposited compounds exhibit at least some degree of diffusion and/or some freedom of movement such that the compounds may interact with binding partners freely. Further, the solvent with which the compounds are deposited on the microarray may be freely absorbed by the microarray. The microarray may constitute a solvent-based gel microarray or hydrogel microarray. The microarray may be other than a dry chemical microarray. In various embodiments, after being dried, the gel may remain a classical non-fluid colloidal network, or polymer network, that is expanded throughout its whole volume by a fluid. In other embodiments, after being dried, the gel may be a xerogel.

[0054] The substrate may be a rigid or semi-rigid body made of virtually any suitable, stable material including glass, polycarbonate, and the like.

[0055] The discrete locations on the gel may represent non-overlapping areas at which the deposited compounds are adhered. The discrete locations may be arranged in an array and may be each separated by at least 100 µm. The array may be a grid or other repeated pattern.

[0056] The microarray may further comprise chase oligonucleotides. In various

embodiments, the microarray may comprise stem chase oligonucleotides, hairpin chase oligonucleotides, DNA chase oligonucleotides, tRNA, phosphate buffer solution, sodium chloride, magnesium chloride, bovine serum albumin, or any combination thereof. See, FIG. 1 for examples of chase oligonucleotides. The oligonucleotides may constitute, e.g., any segment of RNA which is not being targeted for analysis. For example, oligonucleotides may constitute any segment of RNA which is conserved, or which is not varied, across the screened library of RNAs.

[0057] The present invention also provides a method of preparing the microarray, which may comprise:

applying a gel solution onto a substrate and partially drying the gel solution to form a solvated gel-coated substrate;

applying a plurality of aliquots of compounds directly to discrete locations of the solvated gel-coated substrate and drying to form a dried gel-coated substrate;

washing the dried gel-coated substrate with a buffer solution and water, and drying to form the microarray.

[0058] In various embodiments, the gel solution may be at least, may be less than, and/or may be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5.0% (w/v) gel.

[0059] In various embodiments, the gel solution may be at least, may be less than, and/or may be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5.0% (w/v) molten agarose gel.

[0060] The gel solution may comprise about 0.5% to about 5% (w/v) molten agarose.

[0061] In various embodiments, the partial drying may be performed for at least, for less than, or for about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 minutes. For example, the partial drying may be performed for between 10 minutes and 3 hours, 10 minutes and 2 hour, 30 minutes and 2 hours, or 30 minutes and 1.5 hours. Partial drying may, in some embodiments, be performed until the gel is dried of surface solvent but solvent expanded within and throughout the gel network remains; performed until the gel changes color; performed until the gel is transparent; performed until the gel does not drip; performed until the gel has the appearance of thickness and having defined geometry; or any

combination thereof. Partial drying may be performed by air drying, drying under inert gas, drying by blowing air over the surface of the gel, or by blowing inert gas over the surface of the gel. The air or inert gas may be at about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or any temperature between 10°C and 250°C. The air or inert gas may be room temperature.

[0062] The solvated gel-coated substrate may be a hydrated gel-coated substrate. The solvent may be water. The gel may be a hydrogel.

[0063] In various embodiments, the compounds may be applied in an array of discrete locations such that each application of compound may be separated by at least 100 µm. The compounds may be applied in an array of non-overlapping areas. The compounds may be applied in an array of discrete locations arranged in a grid or other repeated pattern.

[0064] Each aliquot may be about 0.1 mM to 100 mM solution of a compound. In various embodiments each aliquot may be about 0.1 mM to 1 mM, 0.1 mM to 10 mM, 1 mM to 10 mM, 1 mM to 100 mM or 10 mM to 100 mM solution of a compound. Each aliquot may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM solution of a compound.

[0065] The volume of each aliquot may be about 1 nL to about 400 nL. In various embodiments, the volume of each aliquot may be about 1 nL to about 10 nL, about 1 nL to about 100 nL, about 10 nL to about 100 nL, about 10 nL to about 200 nL, about 100 nL to about 200 nL, about 100 nL to about 300 nL, or about 100 nL to about 400 nL. The volume of each aliquot may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or 400 nL.

Method of Identifying Interactions Between Compounds And RNA

[0066] The present invention provides a way to evaluate the extent to which RNA and RNA- mediated diseases may be modulated with small molecule compounds. In some

embodiments, this may be achieved by probing known drugs for binding interactions with RNA involved in physiologically important pathways. For example, non-coding human RNAs may interact with approved drugs, but these interactions may previously have been unidentifiable or absent from the treatment course and patient population in which the approved drugs were used. Thus, in some embodiments, the invention relates to a method of screening RNAs against known small molecule drugs.

[0067] The present invention also provides a microarray and a method of assaying non- covalent, unmodified small molecule drugs, which may be probed for binding RNA motifs in a massively parallel library-versus-library screening approach dubbed two-dimensional combinatorial screening (2DCS). (Childs-Disney et al., 2007; Disney et al., 2008).

[0068] The present invention provides a method for identifying a binding interaction between a compound and an RNA, comprising:

applying a plurality of labeled-RNAs and excess oligonucleotides to the microarray described herein;

incubating the microarray to induce binding between labeled-RNAs and adhered compounds;

washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray; imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and

characterizing the bound RNA to identify the binding interaction.

[0069] The present invention also provides a method for identifying a binding interaction between a compound and an RNA, comprising:

applying a plurality of labeled-RNAs and excess oligonucleotides to a microarray having a plurality of compounds non-covalently adhered at discrete locations of the microarray;

incubating the microarray to induce binding between labeled-RNAs and adhered compounds;

washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray;

imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and

characterizing the bound RNA to identify the binding interaction.

[0070] In various embodiments, the method may comprise folding the labeled-RNAs and excess oligonucleotides, each separately, prior to applying the labeled-RNAs and excess oligonucleotides to the microarray.

[0071] In various embodiments, the method may further comprise treating the microarray with at least one of sodium phosphate buffer, sodium chloride, chelating agent, magnesium chloride and bovine serum album, prior to applying the labeled-RNAs and excess oligonucleotides to the microarray.

[0072] The plurality of labeled-RNAs and excess oligonucleotides may be applied so as to be evenly distributed across the gel surface. In various embodiments, the plurality of labeled- RNAs and excess oligonucleotides may be evenly distributed across the gel surface by use of a polymer film placed over the top surface of the gel.

[0073] Illustratively, in various embodiments, the method of the present invention may be used to identify an RNA motif which interacts with a compound, and an RNA motif library may be employed. For example, the RNA motif library can be an RNA internal loop library whose members differ from one another (i) in the identity of the bases in the RNA internal loop and/or (ii) in the identity of the base pairs adjacent to the RNA internal loop (the so- called loop closing base pairs). The RNA motif library can be, for example, a symmetric internal loop library, an asymmetric internal loop library, a 1×1 internal loop library, a 1×2 internal loop library, a 1×3 internal loop library, a 2×2 internal loop library, a 2×3 internal loop library, a 2×4 internal loop library, a 3×3 internal loop library, a 3×4 internal loop library, a 4×4 internal loop library, a 4×5 internal loop library, a 5×5 internal loop library, a 1 base bulge library, a 2 base bulge library, a 3 base bulge library, a 4 base bulge library, a 5 base bulge library, a 4 base hairpin loop library, a 5 base hairpin loop library, a 6 base hairpin loop library, a 7 base hairpin loop library, an 8 base hairpin loop library, a 9 base hairpin loop library, a 10 base hairpin loop library, a multibranch loop library, a pseudoknot library, etc. Combinations of these and other RNA motif libraries can be used. For completeness, it may be desirable to employ an RNA motif library which includes all possible combinations of bases (e.g., an 3×3 internal loop library containing 1600 different 3×3 internal loops). The members of the RNA motif library can further include (i.e., in addition to the variable RNA motif region) RNA regions that do not vary from member to member (e.g., invariant stem regions, invariant hairpin loop regions, etc.). Suitable RNA motif libraries can be prepared by conventional transcription techniques (e.g., those employing T7 RNA polymerase, as described, for example, in Milligan et al.,“Synthesis of Small RNAs Using T7 RNA

Polymerase,” Methods Enzymol.,180:51-62 (1989), which is hereby incorporated by reference) from DNA templates, such as DNA templates that are commercially available from Integrated DNA Technologies (Coralville, Iowa)).

[0074] In various embodiments, the plurality of adhered compounds can be contacted with the RNA library, or RNA motif library, by a variety of methods. For example, the RNA library can be dissolved or suspended in a suitable solvent, buffer, or buffer system, and the adhered compounds can be pre-equilibrated with a suitable hybridization buffer. The RNA library can then be applied to the adhered compounds, for example, by distributing the RNA library evenly over the array surface; and the adhered compounds and RNA library can be incubated with one another for a period of time and at a temperature effective for one or more members of the nucleic acid motif library to bind with the adhered compounds, such as, for example, at from about 15° C. to about 35° C. (e.g., at from about 20° C. to about 30° C. and/or at about room temperature) for from about 5 minutes to about 2 hours (e.g., from about 15 minutes to about 1 hour and/or for about 30 minutes). In various embodiments incubation may be conducted for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 minutes. In various embodiments incubation may be conducted for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 minutes. In various further embodiments, incubation may be conducted for at least In various embodiments incubation may be conducted for at most 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 minutes. In various other embodiments, incubation may be for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours. Incubation may be conducted over night, or over 1, 2 or 3 days. As another example, incubation may be conducted for about 30 min to about 2 h at about room temperature.

[0075] Even distribution can be effected by placing an inert film (e.g., a piece of

PARAFILM™, a type of plastic paraffin film) over the applied solution, and the film can be left in place during incubation. Thus, in various embodiments, incubation may be conducted with a polymer film placed over the top of the surface of the gel.

[0076] Characterizing the bound RNA may comprise:

a. harvesting the bound RNA;

b. performing reverse transcription on the harvested RNA;

c. performing PCR amplification; and

d. sequencing the amplified product.

[0077] In various embodiments, characterizing the bound RNA comprises mechanically harvesting the bound RNA and performing RNA-Seq on the harvested RNA.

[0078] As noted above, the methods of the present invention further include identifying the members of an RNA library, such as an RNA motif library, that are bound to an adhered compound. This can be carried out by harvesting members of the RNA library that are bound at the discrete location on the gel corresponding to the adhered compound. Harvesting can be carried out by any suitable technique, such as by direct excision. The harvested members of the nucleic acid motif library can then be cloned, RT-PCR amplified, and sequenced.

[0079] The methods of the present invention can include additional steps. For example, in certain embodiments, the method the present invention can further include incubating the plurality of compounds with one or more chase oligonucleotides. Excess oligonucleotides may comprise stem chase oligonucleotides, hairpin chase oligonucleotides, DNA chase oligonucleotides, or any combination thereof.

[0080] The chase oligonucleotides may include oligonucleotides that are designed to ensure that the compound interacts with the RNA motif (i.e., with the RNA motif library's variable region) and not with those nucleic acid regions that do not vary from member to member (e.g., invariant stem regions, invariant hairpin loop regions, etc.). The design of such stem chase and hairpin oligonucleotides may depend on the sequences used in the nucleic acid regions that do not vary from member to member.

[0081] Similarly, methods of the present invention may further include use of DNA chase oligonucleotides (i.e., oligonucleotides that are meant to ensure that the interactions are RNA specific). Example of suitable DNA chase oligonucleotides include duplex AT decamers, duplex CG decamers, and combinations thereof. In certain embodiments, the one or more chase oligonucleotides includes stem chase oligonucleotides. In certain embodiments, the one or more chase oligonucleotides includes hairpin chase oligonucleotides. In certain

embodiments, the one or more chase oligonucleotides includes DNA chase oligonucleotides. Combinations of these and other chase oligonucleotides can be employed, for example as in the case where the one or more chase oligonucleotides includes stem chase oligonucleotides, hairpin chase oligonucleotides, and DNA chase oligonucleotides.

[0082] Incubation with the one or more chase oligonucleotides can be carried out prior to the step identifying members of the RNA library that are bound to a particular adhered compound; and incubation with the one or more chase oligonucleotides can be carried out, for example, subsequent to and/or concurrently with the step of contacting the plurality of adhered compounds with the RNA library. The chase oligonucleotides may be employed at a concentration substantially greater than that of the RNA library, such as at a concentration that may be at least 10 times (e.g., at least 20 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, at least 1000 times, about 1000 times, etc.) the

concentration of the RNA library.

[0083] Using the aforementioned methods of the present invention, compounds which interact with particular RNA motifs can be identified. Since the nucleic acid sequences of many biologically important nucleic acid molecules are known, one can readily ascertain which biologically important nucleic acid molecules have the particular RNA motifs with which a particular compound interacts. Accordingly, the present invention can be used to identify compounds that bind or otherwise interact with biologically important RNAs. Such compounds can be used to target such biologically important RNAs, for example, for diagnostic or therapeutic purposes.

[0084] The information regarding compound-RNA motif interactions derived using the methods of the present invention can be assembled into a database. Such databases can then be used in methods for selecting, from a plurality of candidate compounds, one or more compounds that have increased likelihood of binding to a RNA having a particular RNA motif. Such methods can include providing a database which correlates various compounds’ abilities to bind to the particular RNA motif and various other RNA motifs (for example, in accordance with the methods of the present invention); comparing the candidate compounds’ ability to bind to the particular RNA motif; and choosing one or more compounds based on their ability to bind to the particular RNA motif.

[0085] The RNA library can include at least 4 members, such as in cases where the RNA library includes at least 10 members, at least 20 members, at least 40 members, at least 60 members, at least 80 members, at least 100 members, at least 200 members least 500 members, at least 1000 members, at least 2000 members, at least 3000 members, at least 4000 members, etc. In certain embodiments, the RNA library will contain members that differ from one another in the identities of the bases in a particular region of the RNA molecule (e.g., in a region containing from about 10 to about 40 bases, such as in a region containing from about 10 to about 35 bases, from about 10 to about 30 bases, from about 10 to about 25 bases, from about 10 to about 20 bases, from about 10 to about 15 bases, from 10 to 15 bases, 10 bases, 11 bases, 12 bases, 13 bases 14 bases, etc.), the remainder of the RNA molecule being the same or substantially the same amongst the members of the nucleic acid library. The variable region can, but need not, contain structural motif(s); and if the variable region does contain structural motif(s), the compound- RNA binding can, but need not occur at or otherwise involve the bases in the structural motif(s). As indicated above, the members of the RNA library can have invariant regions. In certain embodiments, the variable region contains more bases than the invariant region. In certain embodiments, the invariant region contains about 40 bases or fewer, such as about 30 bases or fewer, about 20 bases or fewer, 10 bases or fewer, between about 10 and about 40 bases, between about 10 and about 20 bases, etc. In certain embodiments, the nucleic acid library may be an X-mer RNA library, wherein X is from about 10 to about 40, such as from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 20, from about 10 to about 15, from 10 to 15, 10, 11, 12, 13, 14, etc.).

[0086] The labeled-RNAs may be RNAs which have been modified to contain a radiolabel, a fluorescent tag or a chromogenic tag.

[0087] The plurality of labeled-RNAs may be provided or prepared from a non-coding RNA library, a RNA motif library, a miRNA library, a viral RNA library, or any combination thereof.

[0088] The RNA motif library may be an internal loop motif library, a hairpin loop motif library, a bulge motif library, a multibranch loop motif library, a pseudoknot motif library, or any combination thereof.

[0089] The method may comprise comparing the frequency of the RNA bound to a small molecule to the frequency of the RNA in the starting library. In various embodiments, the method may further comprise comparing the frequency of the RNA motifs bound to a small molecule to the frequency of the RNA motifs in the starting library.

[0090] The method may further comprise identifying a compound that binds to an RNA, comprising comparing a query dataset of RNA secondary structures from the RNA, with a dataset of identified bound RNA motif-small molecule pairs, to thereby identify a compound that binds to the RNA. The method may further involve use of a computer system

comprising: one or more computer processors and storage configured to compare a structured query dataset describing RNA secondary structures of the RNA, and a structured dataset of identified RNA motif-small molecule pairs, to thereby identify a molecule that binds to the RNA.

[0091] The method may further comprise additional analysis steps, such as inform and/or StARTs, which may be performed as described in U.S. Patent Application Publication No. 2016/0188791 A1, which is hereby incorporated by reference in its entirety.

[0092] In various embodiments, the method may further comprise an informa approach to identify compounds which target RNA as applied to human microRNA (miRNA) precursors. The inforna methods provide an expedited route to identify small molecules that target the RNA product of those genes. The inforna methods not only speed up drug discovery, but also more accurately identify drug candidates that have a higher likelihood of having useful activity. The inforna methods utilize and compare datasets of information, providing an output of which RNA structural secondary structures will likely bind to which small molecule. Those datasets include (a) a dataset of RNA secondary structures to be queried; and (b) a dataset of identified RNA motif-small molecule interactions (e.g., as identified by two-dimensional combinatorial screening (2DCS)). For example, Sequences of all miRNA precursors in the human transcriptome may be downloaded from miRBase (Griffiths-Jones et al., Nucleic Acids Res.36, D154-158 (2008)) and their secondary structures predicted via RNAstructure (Mathews et al., Proc. Natl. Acad. Sci. U.S.A. 101, 7287-7292 (2004)). The secondary structural elements may be extracted from each query RNA and those secondary structures compared to a database of RNA motif-small molecule interactions identified by two-dimensional combinatorial screening (2DCS). Such a dataset can be generated, for example, by use of the microarray of the present invention in a two-dimensional

combinatorial screening (2DCS) process. See, e.g., U.S. Patent Application Publication No. 2008/0188377 A1; Childs-Disney et al., ACS Chem. Biol.2, 745-754 (2007); Disney et al., J. Am. Chem. Soc.130, 11185-11194 (2008), each of which is hereby incorporated by reference in its entirety.

[0093] A dataset of RNA secondary structures to be queried can be generated from one or more RNA sequences alone. For example, RNA secondary structures can be identified as the lowest free energy secondary structures formed by an RNA as it folds back upon itself to form double-stranded regions as well as single-stranded loops and mismatched‘bubbles’ in the double-stranded regions. Such low free energy secondary structures can be predicted by programs such as RNAstructure (Mathews et al., Proc. Natl. Acad. Sci. U.S.A 101, 7287-7292 (2004), which are specifically incorporated by reference in their entireties).

[0094] The output of RNA sequences and secondary structures that will likely bind to a small molecule can be further analyzed by other prediction processes and by chemical and biological assays (e.g., binding assays). For example, a StARTS statistical method can be used to further refine predictions. The StARTS method predicts the affinities and selectivities of RNA motif-small molecule interactions by comparing the rate of occurrence of small structural features (a guanine adjacent to an adenine, for example) in selected RNA motifs to its rate of occurrence in the entire RNA library. The StARTS method therefore facilitates identification of which RNA secondary structures and motifs are most unique or distinctive in populations of RNA molecules. StARTS is a statistical approach that can be paired with inforna to further evaluate the binding affinity of RNA secondary structures for the small molecule partner(s) identified by inforna. StARTS identifies features in RNA motifs that positively and negatively contribute to binding (see, Velagapudi et al., Angew. Chem. Int. Ed. Engl 49, 3816-3818 (2010); Velagapudi et al.,./. Am. Chem. Soc. 133, 10111-10118 (2011); Paul et al., Nucleic Acids Res. 37 (17): 5894-5907 (2009), each of which is incorporated by reference in its entirety).

[0095] In the StARTS approach, sequences of one or more KNA secondary structures identified as binding a small molecule are compiled, and the occurrence rate of each sequence feature in the RNA secondary structures may be compared to the occurrence rate of that feature in a larger population of RNA motifs. A sequence feature is any short RNA sequence (for example, a 5'GC step) that may or may not be different from the sequence features that are present in a larger population of RNA sequences. However, the sequence features are those sequences that are present in the population of RNA secondary structures that bind to a small molecule. By comparing these two populations, the relative enrichment for a specific feature in RNA secondary structure for binding to a small molecule can be computed. Thus, the StARTS method identifies which sequence features are more prevalent in a selected population of RNA sequences than in a larger population of RNA sequences.

[0096] Hie more distinctive sequence features may be assigned a statistical significance, or a Z-score and a corresponding two-tailed p-value. The Z scores can be determined by statistical analysis using a RNA Privileged Space Predictor (RNA-PSP) program that determines which features occur in the selected RNA secondary structures with greater than 95% confidence (see, Paul et al., Nucleic Acids Res. 37 (17): 5894-5907 (2009)). The confidence intervals are associated with a Z-score, where a larger value corresponds to a higher confidence level.

Each RNA secondary structure can have multiple features that contribute to it being different from a larger population of RNA motifs and a sum of the Z-scores for all features in an RNA secondary structure can be computed (åZ) as an indicator of the total structural

distinctiveness of an RNA motif.

[0097] To complete the StARTS analysis, the Z-scores can then plotted against the measured binding affinities of the RNA secondary structure for a compound, and this relationship can be fitted to an inverse first-order equation, which allows prediction of the affinity of a compound for a RNA library member.

[0098] In various embodiments, each compound may be present at two or more different loadings, said two or more different loadings being adhered at separate, discrete locations on the microarray. [0099] In various embodiments, the plurality of compounds comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 compounds and the plurality of labeled-RNAs comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, or 4000 labeled-RNAs.

[00100] In various embodiments, the microarray comprises a substrate coated with an agarose gel and the plurality of compounds are non-covalently adhered to the agarose gel at discrete locations.

[00101] The methods of the present invention found that various drug classes including kinase inhibitors, pre-mRNA splicing modulators, and topoisomerase inhibitors bound RNA avidly. Compounds 1-9 were identified as having binding interactions with RNA.

[00102]

[00103]

[00104]

[00105]

[00106] [00107]

[00108] [00109]

[00110]

[00111] By using an informa sequence-based lead identification strategy for RNA it was found that topoisomerase inhibitors bound the Dicer site of the microRNA (miR)-21 precursor (pre-miR-21). As further discussed in the Examples section, these compounds showed, in cells, reduced mature miR-21 levels and modulated a miR-21-mediated invasive phenotype, as miR-21 is oncogenic. Chemical Cross-Linking and Isolation by Pull-down studies revealed physical interactions between pre-miR-21 and tested compounds.

[00112] Thus, the present invention provides a method of treating a subject suffering from a miR-21 mediated disease, comprising administering to the subject an effective amount of a topoisomerase inhibitor. In various embodiments, the miR-21 mediated disease may be cancer or cancer metastasis. In various further embodiments, the miR-21 mediated disease may be a disease that is not related to, associated with, or comorbid with cancer. That is, the subject suffering the miR-21 mediated disease is not required to suffer from cancer and for example the subject may be, in various embodiments, other than a cancer patient.

[00113] The present invention also provides a method of preventing cancer metastasis in a subject suffering from pre-metastatic cancer, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

[00114] The present invention further provides a method of reducing cancer metastasis in a subject suffering from metastatic cancer, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

[00115] In various embodiments, the topoisomerase inhibitor may be:

or an enantiomer, diastereomer, salt, ester or prodrug thereof.

[00116] The present invention also provides a method of treating a subject suffering from an RNA mediated disease, comprising administering to the subject a compound having the structure

[00117]

[00118]

[00119]

[00120]

[00121]

[00122]

[00123]

_[00124]

2

[00125]

or an enantiomer, diastereomer, salt, ester or prodrug thereof.

[00126] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound having the structure:

enantiomer, diastereomer, salt, ester or prodrug thereof.

The present invention also provides a compound having the structure of Formula A or Formula B

Y

L¹

X ^Z

Formula A

Formula B

wherein

L1 may bea trivalent linker moiety comprising alkyl, amide, ether, amino and triazolyl linkages;

L2 may be a divalent linker moiety comprising alkyl, amide, ether, amino and triazolyl linkages;

X may be a purification moiety suitable to act as a binding partner in affinity purification, immunoprecipitation or pull-down assay;

Y may be an RNA-binding moiety; and

Z may be an RNA-reactive moiety comprising an alkylating group.

[00130] In various embodiments, the purification moiety may be a biotin group; the RNA-binding moiety may be a topoisomerase inhibitor, a kinase inhibitor, a RNA splicing modulator, an compound predicted or known to have RNA-modulating activity, or any of compound 1-9. The RNA-reactive moiety may be bis(2-chloroethyl)amine-containing moiety.

[00131] The compound of Formula A may have the structure:

.

[00133] The compound of Formula B may have the structure:

.

[00135] In various embodiments, the compounds of Formula A and Formula B may be useful for performing Chemical Cross-Linking and Isolation by Pull-down studies. The compounds of Formula B may be modified, e.g., via Huisgen cycloaddition of a suitable azide, to generate a compound of Formula A. Where such azide corresponds to a modified form of a screened compound, the resulting compound may be useful in confirmation studies using Chemical Cross-Linking and Isolation by Pull-down.

Examples

[00136] Various embodiments of the present invention can be better understood by reference to the following Examples, which are offered by way of illustration. The present invention is not limited to the Examples given herein.

Table 1. Material Sources and Identifiers

Cell Model

[00137] All cells were grown at 37 ^oC and 5% CO2. MDA-MB-231 (ATCC: HTB-26) cells were cultured in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 1% penicillin/streptomycin, 1% glutagro and 10% fetal bovine serum (FBS) (complete growth medium). Cells were directly purchased from ATCC but were not authenticated. RNA Isolation and RT-qPCR Protocol

[00138] MDA-MB-231 cells were cultured as described at“Cell Model” hereinabove and grown to 80% confluency in 24-well plates. Cells were treated with compound of interest for 24-48 h. Total RNA was then extracted with a Quick-RNA MiniPrep Kit (Zymo

Research) per the manufacturer’s protocol. Approximately 200 ng of total RNA was used for reverse transcription reactions (RT), which were completed by using a miScript II RT Kit (Qiagen) per the manufacturer’s protocol. RT-qPCR was completed using a 7900HT Fast Real Time PCR System (Applied Biosystems), using Power SYBR Green Master Mix (Applied Biosystems). Primers for RT-qPCR were purchased from IDT or Eurofins (Table 2) and used without further purification. Expression levels of RNAs were normalized to U6 small nuclear RNA or 18S rRNA. Sequences of primers used for RT-qPCT are shown below. Table 2: Sequences of primers used for RT-qPCR.

Compound Libraries

[00139] The 727-member NIH Clinical Collection (NIH-CC) was obtained from Scripps Molecular Screening Center. The NIH-CC is a small molecule library comprised of drugs that have been in Phase I or II clinical trials. Thus, compounds were selected for drug- likeness, bioavailability, and stability. The 95-member Kinase library (SYNLibrary-95, catalog # SYN-2103) was purchased from Synkinase. SYNLibrary-95 contains a kinase inhibitor library that recognizes 57 targets. Three pre-mRNA splicing regulators were obtained from the California Institute for Biomedical Research (Calibr). The 201-member RNA-focused compound library was obtained from Scripps Molecular Screening Center. These compounds were selected by performing chemical similarity search based on molecules that possess bis-benzimidazole or similar cores (Rzuczek, et al., 2015). Hit compounds were obtained in larger quantities for testing. Compound 1 was purchased from Cayman Chemicals, while 2 and 3 were purchased from Biotang, Inc. Compounds 8 and 9 were obtained from Scripps Molecular Screening Center. Structures of certain exemplified compounds are shown below.

Topoisomerase inhibitors

[00141] Compound 2

[00142] Compound 3 Kinase inhibitors

[00143] Compound 4

[00144] Compound 5

[00145] Compound 6 RNA splicing regulator

Benzimidazole-related compounds

General Nucleic Acids

[00149] All DNA oligonucleotides were purchased from Integrated DNA

Technologies, Inc. (IDT) and used without further purification. The RNA competitor oligonucleotides and pre-miR-21 constructs for ITC were purchased from Dharmacon and de-protected according to the manufacturer’s standard procedure. Competitor

oligonucleotides were used to ensure that RNA-small molecule interactions were confined to the randomized region of 3×2 nucleotide internal loop pattern). All aqueous solutions were made with nanopure water. The RNA library was transcribed by in vitro transcription from the corresponding DNA template.

Example 1. Construction of Microarray

[00150] Microarrays were constructed by pouring 15 mL of 1% molten agarose solution onto a glass slide (85.5 x 127.8 x 1.1 mm (width x height x thickness)). The agarose was then air dried for 1 h at room temperature to form a thick gel surface. A 200 nL aliquot of compounds (10 mM in DMSO) were then pinned into the agarose gel from a 384-well plate (Greiner; catalog #781201-906) using a Biomek NXP Laboratory Automation

Workstation equipped with a 100 nL 384-pin floating head with diameter 0.787 mm and inter-pin distance of 4.5 mm. The compound-spotted slides were allowed to air dry completely to form a thin, invisible agarose layer. After drying, slides were washed three times with 1u PBST (1u PBS+0.1% Tween) followed by washing three times for 5 min with nanopure water and air dried completely. 2DCS of the final hit compounds were completed on microscopic slides (Fisher Scientific, 12550016; 75 x 25 x 1 mm (width x height x thickness)). Quantification of compound spotting before and after washing was analyzed using Quantity One (BioRad).

[00151] The compounds spotted on the microarray were compounds from a library of pharmacologically active compounds used in human clinical trials (NIH-CC, n=727); a library of kinase inhibitors known to modulate RNA biology but not known to directly engage RNA (SYNlibrary95, n=95); a library of RNA-focused compounds (n=201)

(Rzuczek, et al., 2015); and compounds that modulate the alternative splicing of survival motor neuron 2 (SMN2, n=3) (Naryshkin, et al., 2014; Palacino, et al., 2015). The compounds were used without further modification. As many of the compounds in these studies have been used in clinical trials, screening them provided a potentially rich source of drugs that can be re-purposed to target RNA.

Results

[00152] After testing various conditions for constructing the microarray, it was found that unmodified small molecules can surprisingly be adhered onto a agarose-coated microarray when spotted onto a solvated gel surfaces which is subsequently dried.

Unexpectedly, the compounds exhibited minimal diffusion and no inter-spot diffusion from initial spotting of the wet gel and through mutiple washing steps. The compounds sufficiently adhered to the gel so as to be retained after washing and incubation. (See, FIG. 1).

[00153] As discussed hereinabove, existing microarrays employ compounds which are modified with functional groups that allow for covalent attachment to the microarray substrate in order to immobilize the compounds. Existing microarrays used such modification and covalent coupling so the compounds would not diffuse and to permit washing the microarray and incubation. However, such modification and coupling of compounds can affect molecular compound binding interactions. Thus, a microarray comprising modified or coupled compounds may not accurately reflect the binding interactions of the original, unmodified compounds.

[00154] The present invention thus describes an improved microarray which can comprise compounds pulled directly from commercially available chemical libraries, i.e., without requiring installation of a functional group for immobilization, and thus provides a platform which more accurate reflects the binding profile of the tested compounds. The present invention also has the advantage of not requiring that the microarray substrate be modified. The observation that compounds could be adhered onto a gel-coated microarray surface sufficiently to withstand washes and incubation steps is surprising. The microarray of the present invention thus can be used for screenings involving incubation (hybridization) steps or other steps involving solvent without the microarray suffering from significant diffusion or loss of compound. The microarray has the advantage of substantially maintaining concentration of compounds at the areas where spotted, e.g., due to minimal diffusion and minimal compound being washed away.

[00155] Preparing the microarrays according to other conditions may not result in compounds adhering and may result in compounds diffusing or having reduced concentration where spotted. For example, the compounds do not adhere if deposited directly onto a fully dried gel coating, or deposited directly onto a completely undried gel coating. Similarly, the compounds do not adhere if deposited directly on to a solid substrate, such as a glass slide, without a gel coating.

Example 2. Identification Of Compounds That Bind RNA

[00156] The microarrays of Example 1 were probed for binding using a 2DCS approach (Disney et al., 2008; U.S. Patent Application Publication No.2008/0188377 A1).

[00157] All oligonucleotides, including a 5’-end ³²P-labeled RNA library [representing a motif library, prepared as described previously (Disney et al., 2008)], competitor chase oligonucleotides and tRNA (See, FIG. 1), were folded separately in 1u Hybridization Buffer (HB, 8 mM Na2HPO4, pH 7.0, 185 mM NaCl, and 1 mM EDTA) by heating at 90 °C for 2 min followed by cooling to room temperature on the bench top.

[00158] The 5’-end ³²P-labeled folded RNAs (~50,000 counts by Geiger counter) were mixed together and MgCl₂ and bovine serum albumin (BSA) were added at 1 mM and 40 µg/mL final concentrations, respectively, in a total volume of 3000 µL. [00159] Prior to incubation, microarrays were pre-equilibrated with 3000 µL of 1u HB supplemented with 1 mM MgCl2 and 40 µg/mL BSA (1u HB2) for 10 min at room temperature to prevent non-specific binding.

[00160] After the slides were pre-equilibrated, HB2 was removed and the mixture of folded RNAs was applied to the microarray surface and distributed evenly across the array surface with a custom-cut piece of Parafilm (solution height ~2-3 mm).

[00161] The slide was incubated for approximately 45 min at room temperature. After incubation, the Parafilm was removed, and the slide was washed by submersion in 30 mL of HB for 5 s with gentle agitation three times. Excess buffer was removed completely from the slide, and the slide was dried at room temperature for 30 min.

[00162] While some compounds maybe have exhibited minor diffusion from their initial spot, significant inter-spot diffusion of the RNA-compound complexes was not observed. Similarly, although diffusion rates differ depending on the physical/chemical properties of each compound, incubation and washing did not significantly result in concentration loss of any particular compounds at spotted areas. Washing of the plates was done before and after incubation to remove any complexes that diffused out of the spotted area. The microarray was exposed to a phosphorimager screen and imaged using a Molecular Devices Typhoon phosphorimager. Then, the image was used as a template to identify spotted compounds that bound RNAs and to guide harvesting bound RNAs from the microarray surface as described in Example 3.

Results

[00163] The microarrays of Example 1 were successfully used for probing unmodified compounds for binding to radiolabeled RNA, specifically binding to radiolabeled RNA motifs in a motif library via a 2DCS approach, and successfully identified the spotted compounds which engaged in binding (See, FIG.1). The platform was thus validated. For additional information regarding conducting a generic 2DCS approaching, see, U.S. Patent Application No.2008/0188377 A1, which his incorporated by reference in its entirety.

[00164] This experiment identified compounds and compound classes as RNA binders including topoisomerase inhibitors, kinase inhibitors, and splicing modulators. It was unexpectedly found that compounds 1-9 have RNA binding activity. Compounds 8 and 9, as RNA binding compounds, represent novel pharmacophores. The ³²P-labeled RNA library used displayed randomized nucleotides in the pattern of a 3u2 nucleotide internal loop (3u2 ILL). The 1,024 member 3u2 ILL was chosen as it contains asymmetric internal loops and bulges present in various cellular RNAs that are of high importance, as described previously via a transcriptome-wide RNA structural analysis (Liu, et al., 2016). Incubation was completed in the presence of excess oligonucleotides that include tRNA, DNA, and mimics of the regions common to all library members, thus ensuring interaction of the small molecules with the randomized region. (See, FIG. 1) The results of identifying a compound bound to RNA thus identifies the compound as having a binding interaction with RNA and, more specifically, a binding interaction with the randomized RNA region which was screened.

Example 3. Identification of RNA - Harvesting bound RNAs, Reverse Transcription, PCR Amplification to Install Barcodes for RNA-seq, and RNA-seq.

[00165] The phosphorimage of Example 2 was used as a template to guide harvesting of bound RNAs from the microarray surface. To harvest bound RNAs, 1 µL of nanopure water was added to each spot. After 30 s, the buffer was absorbed and the agarose gel at that spot was excised using a toothpick.

[00166] The excised agarose was placed into a thin-walled PCR tube with 18 µL of water, 2 µL of 10x RQ1 DNase I buffer and 2 units of RQ1 RNase-free DNase (Promega). The solution was incubated at 37 °C for 2 h and then quenched by addition of 2 µL of 10x DNase stop solution (Promega). Samples were incubated at 65 °C for 10 min to completely inactivate the DNase and then subjected to RT-PCR amplification to install a unique barcode.

[00167] Reverse transcription reactions were completed in 1u RT buffer, 1 mM dNTPs, 5 µM RT primer (5’-CCTCTCTATGGGCAGTCGGT- GATCCTTGCGGATCCAAT), 200 µg/mL BSA, 4 units of AMV reverse transcriptase, and 20 µL of DNase-treated selected RNAs. Samples were incubated at 60 °C for 1 h. A 20 µL aliquot of the RT reaction was added to 6 µL of 10u PCR Buffer, 4 µL of 100 µM forward primer including barcode (5’- CCATCTCATCCCTGCGTGTCTCCGACTCAG- XXXXXXXXXX-GATGGGAGAGGGTTTAAT where X represents unique barcode, GAT is the barcode adapter), 2 µL of 100 µM reverse primer, 0.6 µL of 250 mM MgCl2, and 2 µL of Taq DNA polymerase. Two-step PCR was performed at 95 °C for 1 min and 72 °C for 1 min. Aliquots of the RT-PCR product were checked every two cycles starting at cycle 10 on a non-denaturing 8% polyacrylamide gel stained with SYBR Green to ensure that background spots (excised from the array where compound was not delivered) were not amplified. RT- PCR products encoding selected RNAs were purified on a non-denaturing 8%

polyacrylamide gel. Purity was assessed using a Bioanalyzer and samples were mixed in equal amounts and sequenced using an Ion Proton deep sequencer using PI chips (60-80 million reads).

Results [00168] The RNA motifs from 3u2 ILL that bound each compound were thus harvested from the array surface and sequenced.

[00169] The sequencing results were analyzed by applying an approach called High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS) which scored the relative affinities of the selected RNA motifs (Velagapudi, et al., 2017). For additional details regarding StARTS-type analysis, see, U.S. Patent Application No.

2016/0188791 A1, which is incorporated by reference in its entirety.

[00170] In HiT-StARTS, the frequency of each RNA bound to a small molecule is compared to its frequency in the starting library, as determined by RNA-seq analysis. This pooled population comparison quantified the statistical significance of enrichment, or the parameter Z_obs, and quickly identified binding and non-binding RNA motifs for a specific small molecule while minimizing false negatives and positives. Binding assays were completed between selected RNA motif-small molecule pairs, revealing high affinity and selective binding when Zobs >8 (Table 3), and thus confirming the predictive value of HiT- StARTS. Fitness Scores are assigned for RNA binders by normalizing Zobs values to the highest Z_obs for a given selection, with the best score assigned a value of 100.

Table 3. Global analysis of selected RNA-motif small molecule interactions. Kd’s are reported in nM.

Example 4. Inforna Analysis

[00171] The RNA motifs identified in Example 3 were mined against the RNA motifs in all human miRNA precursors (Velagapudi, et al., 2014) to identify RNA targets.

Specifically, RNA motifs identified as having binding interactions with compounds 2 and 3 to identify potential oncogenic miRNA targets. For additional details regarding using an informa analysis, see, U.S. Patent Application No.2016/0188791 A1, which is incorporated by reference in its entirety.

Results

[00172] The Inforna search query identified additional miRNAs that are upregulated in cancer with high fitness score (>50) which can be targeted by topoisomerase inhibitors 2 and 3. Compounds 2 and 3 were predicted to bind Drosha or Dicer processing sites of pre- miRNAs (Table 4).

Table 4. Inforna search identifies that topoisomerase inhibitors 2 and 3 can target miRNAs that are upregulated in cancers with high fitness score (>50)

[00173] To further refine the RNA targets, the relative expression profiles of the potential miRNA hits (Table 4) were compared from publicly available databases using miRmine (Panwar, et al., 2017). miR-21 was found to be expressed 30-4,000-fold higher expression compared to other potential miRNA targets. As the oncomiR is upregulated in most cancers, contributes to various cancer phenotypes, and has been a focus of various small molecule targeting efforts, miR-21 was further studied.

[00174] Inforna identified that topoisomerase inhibitors 1, 2 and 3 bind the A bulge in the Dicer site of the miR-21 hairpin precursor . This result corroborates with previous observations that compounds 1 and 3 stabilize single-stranded regions of RNA by NMR. Additional Assays

[00175] To confirm identified binding interactions and elucidate activity of compounds of interest, additional assays were performed.

Example 5. Fluorescent Binding Affinity Measurements [00176] Dissociation constants were determined using an in-solution fluorescence- based assay. The RNA of interest was folded in 1u Assay Buffer (8 mM Na₂HPO₄, pH 7.0, 190 mM NaCl, 1 mM EDTA and 40 µg/mL BSA) by heating at 60 °C for 5 min and slowly cooling to room temperature. Small molecules were added to a final concentration of 250 nM for 3, or 2000 nM for 1 and 2. Serial dilutions (1:2) were then completed in 1u Assay Buffer supplemented with 250 nM of 3, or 2000 nM of 1 and 2. The solutions were incubated for 30 min at room temperature and then transferred to a 96-well plate and fluorescence intensity was measured. The change in fluorescence intensity as a function of RNA concentration was fit to equation 2:

[00177] ^(^) = ^^^^ × ([^^^]^{^}/(^^{^}

ௗ + [^^^]^{^}) (2) [00178] where Bmax, is maximum specific binding; [RNA], is RNA concentration; h, is hill slope.

[00179] Example 6. Microscale Thermophoresis (MST) Binding Measurements

[00180] MST measurements were performed on a Monolith NT.115 system

(NanoTemper Technologies) using the intrinsic fluorescent signal from compound 3. The samples were prepared in MST buffer containing 50 mM Tris-HCl, pH 7.4, 150mM NaCl,5 mM MgCl2 and 0.05 (v/v) % Tween-20. The concentration of compound was kept constant at 150 nM. The following RNA constructs were ordered from GE Healthcare Dharmacon for use in MST studies: Pre-miR-21 Full: GUUGACUGUUGAAUCUCAUGGCAAC; Pre-miR- 21 A bulge: GUUGACUGUUGAAUCUCAAUGGCAAC; Pre-miR-21 Base Paired:

GUUGACUGUUGAAUCUCAAUGGUCAAC. RNA was titrated in 1:1 dilutions beginning at 20 µM and then samples were filled into in premium-coated capillaries. The measurement was performed at 40 % LED and 20 to 80 % MST power, with a Laser-On time of 30 sec and Laser-Off time of 5 sec, detecting fluorescence at Ex: 605–645 nm, Em: 680–685 nm. The data were analyzed by thermophoresis analysis, and fitted by quadratic binding equation in MST analysis software (NanoTemper Technologies). The dissociation constant was then determined using a single-site model to fit the curve.

Example 7. In Vitro Dicer Processing Assay

[00181] The miR-21 precursor (pre-miR-21) was 5’-HQG^ODEHOHG^ZLWK^>Ȗ-32P] ATP and T4 polynucleotide kinase as previously described (Velagapudi, et al., 2014). The RNA was then folded in 1u Reaction Buffer (Genlantis) by heating at 60 °C for 5 min and slowly cooling to room temperature, where it was then supplemented with 1 mM ATP and 2.5 mM MgCl2. Compound was added to the reaction mixture and the samples were allowed to incubate at room temperature for 15 min. Recombinant human Dicer enzyme (Genlantis) was added to a final concentration of 0.01 U/µL and the samples were incubated for an additional 30 min at 37 °C. Reactions were stopped by adding in 2u Gel Loading Buffer (8 M urea, 50 mM EDTA, 0.05% (w/v) bromophenol blue, 0.05% (w/v) xylene cyanol). To generate sequencing markers, pre-miR-21 was digested with RNase T1 (0.125 U/µL) in T1 Buffer (25 mM sodium citrate, pH 5, 7 M urea, and 1 mM EDTA) for 20 min at room temperature. An RNA hydrolysis ladder was prepared by incubating RNA in 1u RNA Hydrolysis Buffer (50 mM NaHCO₃, 1 mM EDTA, pH 9.4) at 95 °C for 5 min. Cleavage products were resolved on a denaturing 15% polyacrylamide gel, which was imaged using a Molecular Dynamics Typhoon phosphorimager and quantified with Bio-Rad’s QuantityOne software.

Results and Discussion of Examples 5-7

[00182] We thus measured the affinity of each compound to the A bulge of miR-21 affording K_d’s of 58 ± 7.6 nM, 24 ± 8.1 nM, and 33 ± 3.3 nM for 1, 2, and 3, respectively. Notably, binding was not saturable for compounds 1 and 3 to a fully base paired RNA up to 5000 nM; binding of 2 to the fully paired RNA was 28-fold weaker than to the A bulge, with a Kd of 1080 ± 231 nM (See, Table 5 & FIG. 7).

Table 5. Binding affinities of topoisomerase inhibitors to the A bulge in the Dicer site of pre- miR-21 and the corresponding fully paired RNA.

[00183] Values in Table 5 correspond to K_d’s and are reported in nM. Representative binding plots are shown in FIG. 7.

[00184] Interestingly, 3 also binds the U bulge adjacent to the Dicer site with high affinity, as predicted by Inforna, with a dissociation constant of 46 ± 7.1 nM. Increased avidity of 3 is observed to an RNA that contains both the A and U bulges (Kd = 22 ± 8.3 nM).

[00185] Additional binding analyses using microscale thermophoresis (MST) (Moon, et al., 2018; Seidel, et al., 2013) indicated the binding affinity of 3 to a miR-21 hairpin containing both the A and U bulge (miR-21 Hairpin; FIG. 8) and a miR-21 hairpin containing only the A bulge (miR-21 A Bulge; FIG. 8) as 500 and 300 nM, respectively. Notably, no binding was observed to a miR-21 base pair control without the A and U bulges (miR-21 Base Pair; FIG. 8). Ka for the miR-21 Full (miR-21 hairpin) was 500 ± 146 nM; and Ka for the miR-21 A bulge was 274 ± 122 nM.

[00186 J As 3 showed high affinity for both the A and U bulges found in miR-21,

Compound 3 was also tested for inhibition of pre-miR-21 Dicer processing in vitro.

[00187] A dose response was observed with an IC50 of ~3 pM (FIG. 2). The wild type pre-miR-21 (Pre-miR-21 WT) and a construct of pre-miR-21 without the U bulge (Pre-miR- 21 U27 mutant) both had significant inhibition of in vitro Dicer processing with the addition of 3 pM of 3 (FIG. 9). No significant in vitro Dicer processing of pre-miR-21 without an A bulge (Pre-miR-21 A22 mutant) was observed at 3 pM, indicating that compound 3 can more effectively inhibit biogenesis of the wild type pre-miR-21 (Pre-miR-21 WT) and of the pre- miR-21 construct without the A bulge (Pre-miR-21 U27 Mutant), and does not globally inhibit Dicer enzyme activity (FIG. 9).

Example 8. PTEN Luciferase Assay

[00188] MDA-MB-231 cells were grown in 48-well plates to ~60% confluency in complete growth medium. The cells were transiently co-transfected with 200 ng of a plasmid encoding the 3’ untranslated region (UTR) of PTEN fused to luciferase and 40 ng of a plasmid encoding Renilla luciferase using Lipofectamine 2000 per the manufacturer's protocol. At 5 h post-transfection, compounds were added in complete growth medium, and the cells were incubated for 48 h. Luciferase assays were completed based on a previously described protocol (Hampf and Gossen, 2006). Cells were lysed in 40 pL lx Lysis Buffer (100 mM potassium phosphate buffer (pH 7.8), 0.2% Tween 20) for 10 min at room temperature. Next, 150 pL of lx Firefly Luciferase Buffer (200 mM Tris-HCl (pH 8), 15 mM magnesium sulfate, 0.1 mM EDTA, 25 mM dithiothreitol, ImM ATP, 200 pM coenzyme A, and 200 pM luciferin) and incubated for 2 min at room temperature.

Luminescence signal was measured on a Biotek FlxSOO plate reader. To measure Renilla luciferase activity, 150 pL of lx Renilla Luciferase Buffer (25 mM sodium pyrophosphate, 10 mM sodium acetate, 15 mM EDTA, 500 mM sodium sulfate, 500 mM sodium chloride, 50 pM of 4-(6-Methyl-2-benzothiazolyl)benzeneamine (CAS# 92-36-4), and 4 pM benzyl- coelenterazine, pH 5.0) was added and incubated for 2 min at room temperature.

Luminescence signal was measured on a Biotek FlxSOO plate reader. Example 9. Imaging for DNA Damage:

[00189] 7KH^Ȗ-H2AX immunofluorescence assay was used to assess DNA double strand breaks in cells. MDA-MB-231 cells were grown to ~80% confluence in a Mat-Tek 96- well glass bottom plate in growth medium. Cells were treated with 3 in complete growth medium for 24 hours. Cells were washed with 1x DPBS then fixed with 4%

paraformaldehyde in 1u DPBS at 37 °C and 5% CO₂ for 10 minutes. Cells were washed 5 times with 1u DPBS and three times with 0.1% Triton X-100 in 1u DPBS for 5 min at 37 °C and 5% CO₂. Cells were then washed with 30% formamide in 2u SSC buffer for 10 min at room temperature and 2u SSC for 30 min at 37 °C and 5% CO₂. Cells were incubated with a 1:500 dilution of anti-phospho-Histone-H2A.X (Ser139) (clone JBW301, EMD Millipore) in 2u SSC for 1 h at 37 °C and 5% CO2 then washed three times with 0.1% Triton X-100 in 1x DPBS for 5 min at 37 °C and 5% CO₂. Cells were the incubated with a 1:200 dilution of goat anti-mouse IgG-DyLight 488 conjugate (Thermo Fisher) in 2u SSC for 1 h at 37 °C and 5% CO2 then washed three times with 0.1% Triton X-100 in 1u DPBS for 5 min at 37 °C and 5% CO2. Cells were washed three times with 1u DPBS and then stained using a 1 µg/µL solution of DAPI in 1u DPBS for 5 min at 37 °C and 5% CO2. Cells were washed five times in 1x DPBS and then imaged in 1u DPBS using an Olympus FluoView 1000 confocal microscope at 40u magnification. Anti-Ȗ-H2AX fluorescence intensity was quantified using Olympus Fluoview software version 3.0. Average Anti-Ȗ-H2AX fluorescence intensity measurements represent the average fluorescence intensity across eight images.

Example 10. In Vitro Topoisomerase Inhibition Assay

[00190] Topoisomerase II inhibitory activity was measured using a Topoisomerase II Drug Screening Kit (TopoGEN, Inc.) per the manufacturer’s protocol. Dilutions of 1, 2, and 3 in water were added to 300 ng of DNA in 1u Complete Buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM MgCl₂, 0.5 mM dithiothreitol, 30 µg/mL BSA, and 2 mM ATP), followed by addition of 7.5 U of Topoisomerase II enzyme. Samples were incubated at 37 °C for 30 min and stopped with 2 µL of 10% sodium dodecyl sulfate (SDS). Proteinase K (50 µg/mL) was added and incubated at 37 °C for 15 min. Topoisomers were separated on 1% agarose gels with or without 0.5 µg/mL ethidium bromide. Gels prepared with ethidium bromide were run in 1u TAE running buffer supplemented with 0.5 µg/mL ethidium bromide. Gels run without ethidium bromide were post stained with 0.5 µg/mL ethidium bromide. Both gels were destained in 1u TAE for 15 min and DNA products were visualized using a Bio-Rad Gel Doc XR+ imaging system.

Results and Discussion of Examples 8-10

[00191] Compounds 1, 2, and 3 are readily taken up into cells and localize in both the cytoplasm and nucleus.

[00192] Therefore, the efficacy of the topoisomerase inhibitors for inhibiting biogenesis of miR-21 was tested in the triple negative breast cancer cell line MDA-MB-231 by measuring mature and pre-miR-21 levels by RT-qPCR. Indeed, all three compounds reduced levels of the mature miR-21 (FIG. 2) and increased levels of pre-miR-21 (FIG. 2). ^{The IC50’s for 1, 2, and 3 for reducing mature miR-21 levels are approximately 5, 10, and 1} _{PM, respectively. As 3 most significantly increased pre-miR-21 levels (FIG. 2), it was} further characterized for de-repressing a downstream protein target. Indeed, 3 de-repressed the downstream effect of miR-21 on the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN), using a previously validated luciferase reporter

(O'Donnell, et al., 2005) (FIG.2).

[00193] The most widely accepted mode of action for 3 is DNA intercalation, leading to cross-links and strand breaks. Therefore the concentration required to reduce mature miR- 21 levels which also causes DNA damage was studied. DNA damage was assessed by imaging Ȗ-H2AX foci that form in response to DNA double strand breaks. These studies showed that both DNA damage and a reduction of miR-21 levels were observed at the active concentration of 1 PM (FIG. 3). The effect of 3 on topoisomerase II activity in vitro was tested and showed an inhibitory effect of 3 at 1 µM.

Example 11. Boyden Chamber Invasion Assay

[00194] Matrigel (Corning) was thawed overnight at 4 °C. Matrigel was diluted to 3 mg/mL of basement protein and then 100 µL was plated out into 24 well tissue culture inserts with 8 µm pore sizes (Greiner). Matrigel was incubated at 37 °C for 15 min and then placed back into 4 °C overnight. The following day, cells were cultured as described above and allowed to migrate towards complete growth media in the bottom well for 16 h. The media was vacuum aspirated and both hanging cell culture insert and bottom wells were washed twice with 1u PBS, gently shaking to mix. Excess liquid and cells inside the insert were removed with cotton swabs, after which 400 µL of 4% paraformaldehyde in 1u PBS was placed into the bottom well and incubated for 20 min at room temperature. The wells and inserts were washed twice with lx PBS and then treated with 400 mL of 0.1% crystal violet solution in lx PBS for 20 min at room temperature. Then wells and inserts were washed twice with water. Then wells and inserts were washed once with lx PBS and dried with cotton swabs to remove extra stain and cells inside the insert. The inserts were air-dried and then analyzed by microscopy using a Leica DMI3000 B upright fluorescent microscope. Four different fields of view from each captured image were counted for crystal violet stained or unstained cells. Alternatively, MDA-MB-231 cells were transfected with a plasmid containing pre-miR-21 (Addgene) using Lipofectamine 2000 according to the manufacturer’s protocol and the experiment was performed as described above.

Results and Discussion of Example 11

[00195] Compound 3 inhibits a miR-21-mediated invasive phenotype. As previous studies have shown that miR-21 contributes to a migratory and invasive phenotype (Huang, et al., 2009; Zhu, et al., 2008), inhibition of miR-21 by 3 was studied next and it was sufficient to induce a reversal of phenotype in MDA-MB-231 cells by using a Boyden chamber assay. Indeed, dose dependent inhibition of invasive phenotype was observed (FIG. 4). Importantly, the activity of 3 against an invasive phenotype is ablated by overexpression of pre-miR-21 (FIG. 4), indicating the effect is via a miR-21 -mediated circuit.

Example 12. In vitro Chem-CLIP and C-Chem-CLIP

[00196] Approximately 50000 counts of ³²P 5’ -end labeled precursor miR-21 was added to inactivated DMEM growth medium and folded at 95 °C for 45 s, then cooled on ice for 5 min. Compound 10 or 11 were added to folded RNA and incubated at 37 °C overnight. For C-Chem-CLIP, nucleic acids were pre-treated with dilutions of 3 for 15 min at room temperature before Chem-CLIP probes were added. Streptavidin-agarose beads (Sigma- Aldrich, SI 638; >15 pg/mL binding capacity) were washed three times with lx PBS and resuspended in lx PBS. Beads were added to the samples and incubated for 1 h at room temperature. Samples were centrifuged and the supernatant was transferred to a new tube. Beads were washed three times with lx PBS with 0.1% (v/v) Tween-20 and centrifuged, with each wash being added to the supernatant tube. Radioactivity in the beads and the

supematant/wash tubes were quantified using a Beckman Coulter LS6500 Liquid

Scintillation Counter.

Example 13. Cell-based Chem-CLIP and C-Chem-CLIP [00197] The MDA-MB-231 cells were grown to ~70% confluency as monolayers in 100 mm dishes. The cells were treated with 1 µM of 10 or 11 for 8 h. Total RNA was extracted using a Quick-RNA MiniPrep Kit (Zymo Research) per the manufacturer’s protocol. Approximately 30 µg of total RNA was then incubated with 100 µL of streptavidin- agarose beads (Sigma-Aldrich) and shaken for 1 h at room temperature. The solution was removed and beads washed six times with 300 µL of 1u PBS. The RNA bound to beads was released by heating at 65 °C for 20 min in 1u Elution Buffer (95% formamide, 10 mM EDTA, pH 8.2). Eluted RNA was then purified with a Quick-RNA MiniPrep Kit (Zymo Research) and used for subsequent RNA isolation and RT-qPCR as described above (RNA isolation and RT-qPCR).

[00198] Normalized miRNA Enrichment of the measured RNA before and after pulldown was measured using equation 1:

[00199] Normalized miRNA Enrichment:

[00200]

[00201] where“ǻC_t before pulldown” is the difference between the C_t values for the RNA of interest and a housekeeping gene (U6 small nuclear RNA) in total RNA isolated from before pulldown cell lysate RNA and“ǻCt after pulldown” is the difference between the C_t values for the RNA of interest and the same housekeeping gene after pulldown. Data was normalized to the levels of mature miR-21 measured after treatment with Chem-CLIP probe at the appropriate concentration.

Results and Discussion of Examples 12 and 13

[00202] Compound 3 binds pre-miR-21 in cells as determined by Chem-CLIP. To further evaluate whether 3 directly engages pre-miR-21 in cells, Chem-CLIP, a small molecule-RNA profiling approach, was used. (Guan and Disney, 2013; Su, et al., 2014). A Chem-CLIP probe was synthesized by appending a biotin and a chlorambucil cross-linking module onto 3 to afford compound 10 (See, FIG.5). Compound 10 cross-links with its cellular RNA targets, and the resulting small molecule-RNA conjugates are harvested by biotin capture. Both in vitro and in cells, 10 reacted with pre-miR-21 (See, FIG. 5; FIG. 10).

[00203] Importantly, co-addition of parent 3 and 10 in cells (Competitive Chem-CLIP [C-Chem-CLIP]) decreased the extent of pull-down of pre-miR-21 in a dose-dependent manner, thus proving that 3 binds its RNA target in cells. Chem-CLIP was also performed on 11, which lacks the RNA-binding module; no enrichment of miR-21 observed in the pulled down fractions, whether in vitro or in cells, as expected (See, FIG.5; FIG. 10; FIG. 11). Enrichment levels of miR-21 were normalized to levels of mature miR-21 after treatment with 10 or 11 (See, FIG. 11).

[00204] Selectivity of 3. The selectivity of 3 for inhibiting miR-21 was measured by using RT-qPCR, in particular by studying its effect on miRNAs that contain the same motifs in pre-miR-21’s Dicer site, or RNA isoforms. Previous studies have identified that the expression levels of miRNAs will most likely be affected if the small molecule binds in a biologically important site, such as Dicer or Drosha processing sites (Velagapudi, et al., 2014). Therefore, a database of miRNA secondary structures was queried to identify precursor miRNAs that contain the same A and/or U bulges found in pre-miR-21’s Drosha or Dicer processing sites (FIG.6). Despite the presence of the A and/or U bulges in Dicer processing sites of other pre-miRNAs, only levels of miR-21 were affected (FIG. 6).

[00205] To determine if 3 does not inhibit levels of the other miRNAs studied because it does not bind the target in cells, Chem-CLIP studies were performed for the RNA isoforms (FIG.6). Binding is observed to only two miRNAs, let-7e and miR-25; however, binding is not sufficient to reduce their mature levels. The binding site for 3 is located in the Dicer site of both pre-miRNAs, however the lack of activity of 3 against let-7e and miR-25 could be traced to the fact that both let-7e and miR-25 are expressed at much lower levels compared to miR-21 (7% and 3%, respectively compared to miR-21). Collectively, these results suggest that the enhanced anti-miR-21 activity of 3 is due to the presence of two binding sites in pre- miR-21 (only a singular binding site is present in pre-let-7e and pre-miR-25) and its comparatively much higher expression level.

Synthesis of Compounds 10 and 11

Example 11. Synthesis of N1-(2-(prop-2-yn-1-yloxy)ethyl)ethane-1,2-diamine (Fragment 3a)

Scheme 1. Synthesis of Fragment 3a

[00206] Both 2-(prop-2-yn- 1 -y loxy )ethan- 1 -ol (3aa), 2-(prop-2-yn-l-yloxy)ethyl 4- methylbenzenesulfonate (3ab) were prepared according to reference (Cserep, et al., 2014; McConnell, et al., 2010). To a solution of 2-(prop-2-y n- 1 -y loxy )ethy 14- methylbenzenesulfonate. (3ab) (5400 mg, 21 mmol) in MeOH (30 mL) ethylenediamine was added (20 mL, 300 mmol) dropwise at 0 °C. After the addition of ethylenediamine, the reaction mixture was stirred overnight. Then, 10% K2CO3 was added to the reaction mixture and extracted with DCM. The combined oiganic layers were dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by distillation (85 °C/ < 1 mmHg) to afford 1.6 g of 3a (53% yield), a colorless oil (Scheme SI). ¹H NMR(400 MHz, CDCb)d4.17-4.16 (m, 2H), 3.65 (t, J= 5.1 Hz, 2H), 2.85-2.80 (m, 4H), 2.69 (t, J= 7.8, 2H), 2.44-2.43 (m, 1H) 1.34 (br s, 3H). ¹³C NMR(100 MHz, CDCb)541.8, 49.1, 52.5, 58.4, 69.5, 74.4, 79.7; MS m/z (ESI); calcd for C7H15N2O (M + H)⁺ 143.1; found 143.3

Example 15. Synthesis of Mitoxantrone Derivative (Fragment 3b)

Scheme 2. Synthesis of Mitoxantrone Derivative (Fragment 3b)

[00207] The compounds 1 ,4-difluoro-5,8-dihydroxy anthracene-9, 10-d ione (3ba) and 1 -fluoro-5, 8-dihy droxy -4-((2-((2-hy droxyethyl)amino)ethyl)amino)-anthracene-9, 10-dione (3bb) were prepared according to Liu et al. and Mansour et al (Liu, et al., 2009; Mansour, et al., 2010). To a solution of 3bb (50 mg, 0.14 mmol) in pyridine (4 mL) was added 3a (36 mg, 0.25 mmol). Then the mixture was stirred at 100 °C under argon for 2 h. The reaction mixture was cooled to room temperature. BoczO (283 mg, 1.3 mmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was evaporated and the residue was purified by silica gel column chromatography (15-60% ethyl acetate in hexane) to afford 50 mg of 3b (43% yield) as a dark blue powder (Scheme S2).¹H NMR(CD30D, 700 MHz)d 7.51-7.49 (m, 1H), 7.46-7.40 (m, 2H), 7.38-7.27 (m, 1H), 4.21-4.19 (m, 2H), 3.68-3.62 (m, 8H), 3.50-3.47 (m, 6H), 3.38-3.36 (m, 2H), 2.88 (s, 1H), 1.58 (s, 18H), 1.48 (s, 9H), 1.40- 1.39 (m, 9H). ¹³C NMR(175 MHz, CD₃OD)6 181.9, 157.3, 157.2, 157.1, 152.8, 149.3, 147.3, 147.2, 147.1, 129.2, 128.7, 125.2, 125.0, 124.9, 124.8, 111.1, 111.0, 84.6, 81.6, 81.5, 81.4, 81.3, 80.7, 76.2, 69.8, 69.6, 61.5, 61.3, 59.2, 52.1, 51.3, 41.8, 41.3, 28.8, 28.7, 28.2. HRMS m/z (ESI): calcd for C45H63N4O14 (M + H)⁺ 883.4341, found 883.4375.

Example 16. Synthesis of the Peptoid backbone f Fragment 3c!

Scheme 3. Synthesis of Peptoid backbone (Fragment 3c).

[00208] Rink amide resin (400 mg, 0.23 mmol) was swollen in DMF at room temperature for 10 min and then deprotected with a solution of 20% piperidine in DMF (5 mL, 2 x 20 min). The resin was washed with DMF (3 x 5 mL). To the resin were added 1.1 mL of 1.0 M bromoacetic acid in DMF, DIC (0.3 mL, 1.2 mmol) and oxyma (163 mg, 1.1 mmol) and the mixture was shaken at room temperature for 2 h. The resin was washed with DCM (5 x 6 mL) and DMF (5 x 6 mL). Then to the resin was added a solution of N-(2- aminoethyl) biotinamide (384 mg, 1.0 mmol) in DMF (2 mL) and DIPEA (0.2 mL, 1.2 mmol) and the mixture was shaken at room temperature for 1.5 h. The resin was washed with DCM (5 x 6 mL) and DMF (5 x 6 mL). Then to the resin were added 1.1 mL of 1.0 M bromoacetic acid in DMF, DIC (0.3 mL, 1.2 mmol) and oxyma (163 mg, 1.1 mmol) and the mixture was shaken at room temperature for 2 h. The resin was washed with DCM (5 x 6 mL) and DMF (5 x 6 mL). Then to the resin were added DMF (2 mL) and 3-azidopropan-1- amine (230 mg, 2.3 mmol). The mixture was shaken at room temperature overnight. The mixture was washed with DCM (5 x 6 mL) and DMF (5 x 6 mL). Then to the resin were added 1.1 mL of 1.0 M bromoacetic acid in DMF, DIC (0.3 mL, 1.2 mmol) and oxyma (163 mg, 1.1 mmol) and the mixture was shaken at room temperature for 2 h. The resin was washed with DCM (5 x 6 mL) and DMF (5 x 6 mL). Then to the resin were added DMF (2 mL), N-Boc-ethylenediamine (184 mg, 1.2 mmol) and DIEA (0.2 mL, 1.2 mmol) and the mixture was shaken at room temperature for 1 h. The beads were washed with DCM (5 x 6 mL) and DMF (5 x 6 mL) followed by addition of DMF (4 mL), acetic anhydride (0.12 mL 1.3 mmol) and DIEA (0.4 mL, 2.3 mmol). The mixture was shaken at room temperature for 1 h followed by washing the resin with DCM (5 x 6 mL) and DMF (5 x 6 mL). Then to the resin was added 30% TFA in DCM (2 mL), and the mixture was shaken at room temperature for 30 min. The mixture was filtered and the filtrate was evaporated (Scheme S3). The crude product was purified by preparative HPLC (linear gradient of 0% to 100% CH3OH in H2O with 0.1% (v/v) TFA over 60 min) to afford compound 3c as a colorless oil (8 mg, 4.8% yield). HRMS m/z (ESI): calcd for C₂₅H₄₄N₁₁O₆S (M + H)⁺ 626.3197, found 626.3216.

Example 17. Synthesis of 3-CA-Biotin (10) Boc

Scheme 4. Synthesis of 3-CA-Biotin (10)

[00209] To a solution of 3b (57 mg, 0.065 mmol) and 3c (51 mg, 0.082 mmol) in DMSO (0.6 mL) were added Cu(I)-catalyst (7.5 mg, 0.0048 mmol) and DIEA (0.5 mL, 2.9 mmol). The reaction mixture was stirred at 60 °C overnight. Then saturated NaHCCO₃ was added, the aqueous layer was extracted with DCM, and the organic layer was evaporated. The residue was purified by silica gel column chromatography (0-5% MeOH with 1% NH₃ aq in DCM) to give 70 mg of 3d (partially purified).

[00210] To a solution of 3d (24 mg, 0.016 mmol) in DCM were added chlorambucil (42 mg, 0.14 mmol), HOAt (19 mg, 0.14 mmol), HATU (53 mg, 0.14 mmol) and DIEA (40 uL, 0.23 mmol). The reaction mixture was stirred at room temperature overnight and then evaporated. To the residue was added 4ATHC1 dioxane (3 mL) and the mixture was stirred at room temperature for 2 h followed by evaporation of the suspension (Scheme S4). The residue was diluted with water (3 mL) and the aqueous layer was washed with DCM and ethyl acetate. The aqueous layer was directly purified by preparative HPLC with a linear gradient of 0-100% acetonitrile in H2O with 0.1% TFA over 60 min. Fractions containing the compound were concentrated in vacuo and the residue was dissolved in 200 pL of DMSO. The concentration of the DMSO stock solution (1.69 mM, 200 mL, 2.1% yield) was determined with 10 mM Tris-HCl (pH 7.4) and molecular extinction coefficient of mitoxantrone hydrochloride (19200 M^-1 cm^-1 at 608 nm). Purity was analyzed on an analytical HPLC with a linear gradient of 0-100% acetonitrile in water with 0.1% TFA.

[00211] HRMS m/z (ESI): calcd for C₆₄H₉₁Cl₂N₁₆O₁₃S (M + H)⁺ 1393.6049, found 1393.6036.

Conclusion

[00212] We have shown that various drug classes have the capacity to bind RNA, including topoisomerase inhibitors that affect a key non-coding, oncogenic miRNA. Thus, our studies may provide an example of compounds whose activity can be traced to affecting multiple pathways, and the present compounds affect pathways which were not previously recognized. (Swift, et al., 2006). Other classes of small molecules have been shown to target RNA in addition to their previously known target. Aggressive activity in repurposing known drugs has revealed many additional targets across all types of biomolecules. It is likely that many drugs affect multiple pathways, some known and others unknown, to provide a therapeutic effect.

[00213] In this study, it was found that compound 3 inhibited levels of mature miR-21, concomitantly increased levels of pre-miR-21, and reversed the invasive phenotype caused by elevated expression of miR-21 in triple negative breast cancer cells. These effects are ablated upon overexpression of pre-miR-21, giving further support to 3’s mechanism of action.

[00214] These compounds may also offer some specialized utility as chemical probes to study miR-21, although in cells they their use may be challenging due to the multiple pathways which these compounds are involved.

[00215] Also, of interest is the observation that kinase inhibitors are a key compound class that target RNA. These molecules could be medicinally optimized to drug RNA and reach a clinical end point.

[00216] In conclusion, the present invention provides a microarray-based method to identify the preferred RNA motifs for unmodified small molecules. That is, the compounds do not require installation of a functional group for immobilization. The microarray was applied to the NIH Clinical Collection, a library of RNA-focused small molecules, RNA splicing modulators, and a library of kinase inhibitors. Indeed, these drugs, in particular topoisomerase inhibitors, kinase inhibitors, and RNA splicing modulators bind to RNAs. Use of 2DCS and HiT-STARTS identified privileged motifs for each small molecule, and overlap was identified with oncogenic miR-21. One topoisomerase inhibitor, 3, selectively inhibited miR-21 biogenesis, de-repressed a downstream protein, and reversed an invasive phenotype in MDA-MB-231 cells. These studies suggest that many known drugs have affinity for RNA and RNA may indeed be one of their cellular targets and known drugs may be rationally re- purposed based on new targets.

Additional Embodiments.

[00217] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[00218] Embodiment 1 provides a microarray, comprising:

a substrate coated with a gel; and

a plurality of compounds that are non-covalently adhered to the gel at discrete locations.

[00219] Embodiment 2 provides the microarray of Embodiment 1, wherein the compounds are selected from a library of FDA-approved drugs, compounds used in phase I clinical trials, compounds used in phase II clinical trials, kinase inhibitors, topoisomerase inhibitors, mRNA splicing modulators, RNA-modulating compounds, drug-like compounds, commercially-available bioactive compounds, or any combination thereof, and the compounds are unmodified therefrom.

[00220] Embodiment 3 provides the microarray of anyone of Embodiments 1-2, wherein the compounds are selected from a commercially-available bioactive compound library, or portion thereof, and the compounds are unmodified therefrom.

[00221] Embodiment 4 provides the microarray of anyone of Embodiments 1-3, wherein each compound has a molecular weight less than 1,000 g/mol.

[00222] Embodiment 5 provides the microarray of anyone of Embodiments 1-4, wherein the compounds are non-covalently adhered to the gel via adsorption or absorption.

[00223] Embodiment 6 provides the microarray of anyone of Embodiments 1-5, wherein each compound is present in the gel in an amount of about 1 pmol to about 1 µmol.

[00224] Embodiment 7 provides the microarray of anyone of Embodiments 1-6, wherein the gel comprises a polysaccharide or a polyacrylamide.

[00225] Embodiment 8 provides the microarray of anyone of Embodiments 1-7, wherein the gel is an about 0.5% to about 5% (w/v) agarose gel. [00226] Embodiment 9 provides the microarray of anyone of Embodiments 1-8, wherein the gel has a substantially flat surface free of wells and the compounds are incorporated into the gel mesophase.

[00227] Embodiment 10 provides the microarray of anyone of Embodiments 1-9, wherein the discrete locations on the gel are arranged in an array and are each separated by at least 100 µm.

[00228] Embodiment 11 provides the microarray of anyone of Embodiments 1-10, further comprising stem chase oligonucleotides, hairpin chase oligonucleotides, DNA chase oligonucleotides, tRNA, phosphate buffer solution, sodium chloride, magnesium chloride, bovine serum albumin, or any combination thereof.

[00229] Embodiment 12 provides a method of preparing a microarray, comprising: applying a gel solution onto a substrate and partially drying the gel solution to form a hydrated gel-coated substrate;

applying a plurality of aliquots of compounds directly to discrete locations of the hydrated gel-coated substrate and drying to form a dried gel-coated substrate; and

washing the dried gel-coated substrate with a buffer solution and water, and

drying to form the microarray.

[00230] Embodiment 13 provides the method of Embodiment 12, wherein the gel solution comprises about 0.5% to about 5% (w/v) molten agarose.

[00231] Embodiment 14 provides the method of anyone of Embodiments 12-13, wherein the partial drying is performed at about room temperature in air for about 1 hour.

[00232] Embodiment 15 provides the method of anyone of Embodiments 12-14, wherein the compounds are applied in an array of discrete locations such that each application of compound is separated by at least 100 µm.

[00233] Embodiment 16 provides the method of anyone of Embodiments 12-15, wherein each aliquot is a 0.1 mM to 100 mM solution of a compound.

[00234] Embodiment 17 provides the method of anyone of Embodiments 12-16, wherein the volume of each aliquot is about 1 nL to about 400 nL.

[00235] Embodiment 18 provides a method for identifying a binding interaction between a compound and an RNA, comprising: applying a plurality of labeled-RNAs and excess oligonucleotides to the microarray of any one of Embodiments 1-11;

incubating the microarray to induce binding between labeled-RNAs and adhered compounds; washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray;

imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and

characterizing the bound RNA to identify the binding interaction.

[00236] Embodiment 19 provides a method for identifying a binding interaction between a compound and an RNA, comprising:

applying a plurality of labeled-RNAs and excess oligonucleotides to a microarray having a plurality of compounds non-covalently adhered at discrete locations of the microarray;

incubating the microarray to induce binding between labeled-RNAs and adhered compounds; washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray;

imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and

characterizing the bound RNA to identify the binding interaction.

[00237] Embodiment 20 provides the method of Embodiments 19, further comprising folding the labeled-RNAs and excess oligonucleotides, each separately, prior to applying the labeled-RNAs and excess oligonucleotides to the microarray.

[00238] Embodiment 21 provides the method of anyone of Embodiments 19-20, further comprising treating the microarray with at least one of sodium phosphate buffer, sodium chloride, chelating agent, magnesium chloride and bovine serum album, prior to applying the labeled-RNAs and excess oligonucleotides to the microarray.

[00239] Embodiment 22 provides the method of anyone of Embodiments 19-21, wherein the plurality of labeled-RNAs and excess oligonucleotides are applied so as to be evenly distributed across the gel surface.

[00240] Embodiment 23 provides the method of anyone of Embodiments 19-22, wherein the plurality of labeled-RNAs and excess oligonucleotides are evenly distributed across the gel surface by use of a polymer film which is placed over the top surface of the gel. [00241] Embodiment 24 provides the method of anyone of Embodiments 19-23, wherein incubation is conducted for about 30 min to about 2 h at about room temperature.

[00242] Embodiment 25 provides the method of anyone of Embodiments 19-24, wherein incubation is conducted with a polymer film placed over the top of the surface of the gel.

[00243] Embodiment 26 provides the method of anyone of Embodiments 19-25, wherein characterizing the bound RNA comprises:

harvesting the bound RNA;

performing reverse transcription on the harvested RNA;

performing PCR amplification; and

sequencing the amplified product.

[00244] Embodiment 27 provides the method of anyone of Embodiments 19-26, wherein characterizing the bound RNA comprises mechanically harvesting the bound RNA and performing RNA-Seq on the harvested RNA.

[00245] Embodiment 28 provides the method of anyone of Embodiments 19-27, wherein the excess oligonucleotides comprise stem chase oligonucleotides, hairpin chase oligonucleotides, DNA chase oligonucleotides, or any combination thereof.

[00246] Embodiment 29 provides the method of anyone of Embodiments 19-28, wherein the labeled-RNAs are RNAs which have been modified to contain a radiolabel, a fluorescent tag or a chromogenic tag.

[00247] Embodiment 30 provides the method of anyone of Embodiments 19-29, wherein the plurality of labeled-RNAs is provided or prepared from a non-coding RNA library, a RNA motif library, a miRNA library, a viral RNA library, or any combination thereof.

[00248] Embodiment 31 provides the method of anyone of Embodiments 19-30, wherein the RNA motif library is an internal loop motif library, a hairpin loop motif library, a bulge motif library, a multibranch loop motif library, a pseudoknot motif library, or any combination thereof.

[00249] Embodiment 32 provides the method of anyone of Embodiments 19-31, further comprising comparing the frequency of the RNA bound to a small molecule to the frequency of the RNA in the starting library. [00250] Embodiment 33 provides the method of anyone of Embodiments 19-32, further comprising comparing the frequency of the RNA motifs bound to a small molecule to the frequency of the RNA motifs in the starting library.

[00251] Embodiment 34 provides the method of anyone of Embodiments 19-33, wherein each compound is present at two or more different loadings, said two or more different loadings being adhered at separate, discrete locations on the microarray.

[00252] Embodiment 35 provides the method of anyone of Embodiments 19-34, wherein the plurality of compounds comprises at least 10 compounds and the plurality of labeled-RNAs comprises at least 10 labeled-RNAs.

[00253] Embodiment 36 provides the method of anyone of Embodiments 19-35, wherein the microarray comprises a substrate coated with an agarose gel and the plurality of compounds are non-covalently adhered to the agarose gel at discrete locations.

[00254] Embodiment 37 provides a method of treating a subject suffering from a miR- 21 mediated disease, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

[00255] Embodiment 38 provides a method of preventing cancer metastasis in a subject suffering from pre-metastatic cancer, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

[00256] Embodiment 39 provides a method of reducing cancer metastasis in a subject suffering from metastatic cancer, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

[00257] Embodiment 40 provides the method of anyone of Embodiments 37-39, wherein the topoisomerase inhibitor is:

, ,

;

or an enantiomer, diastereomer, salt, ester or prodrug thereof.

[00258] Embodiment 41 provides a method of treating a subject suffering from an RNA mediated disease, comprising administering to the subject an effective amount of a compound having the structure

,

or an enantiomer, diastereomer, salt, ester or prodrug thereof.

[00259] Embodiment 42 provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound having the structure:

. [00260] Embodiment 43 provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound having the structure:

. [00261] Embodiment 44 provides a compound having the structure of Formula A or Formula B

Y

L¹

X ^Z

Formula A

Formula B

wherein L1 is a trivalent linker moiety comprising alkyl, amide, ether, amino and triazolyl linkages; L2 is a divalent linker moiety comprising alkyl, amide, ether, amino and triazolyl linkages; X is a purification moiety suitable to act as a binding partner in affinity purification, immunoprecipitation or pull-down assay;

Y is an RNA-binding moiety; and

Z is an RNA-reactive moiety comprising an alkylating group. [00262] Embodiment 45 provides the compound of Embodiment 44, wherein:

the purification moiety is a biotin group;

the RNA-binding moiety is a topoisomerase inhibitor, a kinase inhibitor, a RNA splicing modulator, or a compound according to any one of claims 41-44; and

the RNA-reactive moiety is bis(2-chloroethyl)amine-containing moiety. [00263] Embodiment 46 provides the compound of Embodiment 44, having the structure:

. [00264] Embodiment 47 provides the compound of any one of Embodiments 44-45, having the structure:

.

References

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Claims

1. A microarray, comprising:

a substrate coated with a gel; and

a plurality of compounds that are non-covalently adhered to the gel at discrete locations.

2. The microarray of claim 1, wherein the compounds are selected from a library of FDA- approved drugs, compounds used in phase I clinical trials, compounds used in phase II clinical trials, kinase inhibitors, topoisomerase inhibitors, mRNA splicing modulators, RNA- modulating compounds, drug-like compounds, commercially-available bioactive compounds, or any combination thereof, and the compounds are unmodified therefrom.

3. The microarray of claim 1, wherein the compounds are selected from a commercially- available bioactive compound library, or portion thereof, and the compounds are unmodified therefrom.

4. The microarray of claim 1, wherein each compound has a molecular weight less than 1,000 g/mol.

5. The microarray of claim 1, wherein the compounds are non-covalently adhered to the gel via adsorption or absorption.

6. The microarray of claim 1, wherein each compound is present in the gel in an amount of about 1 pmol to about 1 µmol.

7. The microarray of claim 1, wherein the gel comprises a polysaccharide or a

polyacrylamide.

8. The microarray of claim 1, wherein the gel is an about 0.5% to about 5% (w/v) agarose gel.

9. The microarray of claim 1, wherein the gel has a substantially flat surface free of wells and the compounds are incorporated into the gel mesophase.

10. The microarray of claim 1, wherein the discrete locations on the gel are arranged in an array and are each separated by at least 100 µm.

11. The microarray of claim 1, further comprising stem chase oligonucleotides, hairpin chase oligonucleotides, DNA chase oligonucleotides, tRNA, phosphate buffer solution, sodium chloride, magnesium chloride, bovine serum albumin, or any combination thereof.

12. A method of preparing a microarray, comprising:

applying a gel solution onto a substrate and partially drying the gel solution to form a hydrated gel-coated substrate;

applying a plurality of aliquots of compounds directly to discrete locations of the hydrated gel-coated substrate and drying to form a dried gel-coated substrate; and

washing the dried gel-coated substrate with a buffer solution and water, and drying to form the microarray.

13. The method of claim 12, wherein the gel solution comprises about 0.5% to about 5% (w/v) molten agarose.

14. The method of claim 12, wherein the partial drying is performed at about room temperature in air for about 1 hour.

15. The method of claim 12, wherein the compounds are applied in an array of discrete locations such that each application of compound is separated by at least 100 µm.

16. The method of claim 12, wherein each aliquot is a 0.1 mM to 100 mM solution of a compound.

17. The method of claim 12, wherein the volume of each aliquot is about 1 nL to about 400 nL.

18. A method for identifying a binding interaction between a compound and an RNA, comprising:

applying a plurality of labeled-RNAs and excess oligonucleotides to the microarray of claim 1;

incubating the microarray to induce binding between labeled-RNAs and adhered compounds;

washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray;

imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and

characterizing the bound RNA to identify the binding interaction.

19. A method for identifying a binding interaction between a compound and an RNA, comprising:

applying a plurality of labeled-RNAs and excess oligonucleotides to a microarray having a plurality of compounds non-covalently adhered at discrete locations of the microarray;

incubating the microarray to induce binding between labeled-RNAs and adhered compounds;

washing the microarray with a buffer solution, removing excess buffer solution and drying the microarray;

imaging the microarray to detect labeled-RNA which has bound to an adhered compound; and

characterizing the bound RNA to identify the binding interaction.

20. The method of claim 19, further comprising folding the labeled-RNAs and excess oligonucleotides, each separately, prior to applying the labeled-RNAs and excess

oligonucleotides to the microarray.

21. The method of claim 19, further comprising treating the microarray with at least one of sodium phosphate buffer, sodium chloride, chelating agent, magnesium chloride and bovine serum album, prior to applying the labeled-RNAs and excess oligonucleotides to the microarray.

22. The method of claim 19, wherein the plurality of labeled-RNAs and excess oligonucleotides are applied so as to be evenly distributed across the gel surface.

23. The method of claim 22, wherein the plurality of labeled-RNAs and excess

oligonucleotides are evenly distributed across the gel surface by use of a polymer film which is placed over the top surface of the gel.

24. The method of claim 19, wherein incubation is conducted for about 30 min to about 2 h at about room temperature.

25. The method of claim 19, wherein incubation is conducted with a polymer film placed over the top of the surface of the gel.

26. The method of claim 19, wherein characterizing the bound RNA comprises:

harvesting the bound RNA;

performing reverse transcription on the harvested RNA;

performing PCR amplification; and

sequencing the amplified product.

27. The method of claim 19, wherein characterizing the bound RNA comprises mechanically harvesting the bound RNA and performing RNA-Seq on the harvested RNA.

28. The method of claim 19, wherein the excess oligonucleotides comprise stem chase oligonucleotides, hairpin chase oligonucleotides, DNA chase oligonucleotides, or any combination thereof.

29. The method of claim 19, wherein the labeled-RNAs are RNAs which have been modified to contain a radiolabel, a fluorescent tag or a chromogenic tag.

30. The method of claim 19, wherein the plurality of labeled-RNAs is provided or prepared from a non-coding RNA library, a RNA motif library, a miRNA library, a viral RNA library, or any combination thereof.

31. The method of claim 30, wherein the RNA motif library is an internal loop motif library, a hairpin loop motif library, a bulge motif library, a multibranch loop motif library, a pseudoknot motif library, or any combination thereof.

32. The method of claim 19, further comprising comparing the frequency of the RNA bound to a small molecule to the frequency of the RNA in the starting library.

33. The method of claim 19, further comprising comparing the frequency of the RNA motifs bound to a small molecule to the frequency of the RNA motifs in the starting library.

34. The method of claim 19, wherein each compound is present at two or more different loadings, said two or more different loadings being adhered at separate, discrete locations on the microarray.

35. The method of claim 19, wherein the plurality of compounds comprises at least 10 compounds and the plurality of labeled-RNAs comprises at least 10 labeled-RNAs.

36. The method of claim 19, wherein the microarray comprises a substrate coated with an agarose gel and the plurality of compounds are non-covalently adhered to the agarose gel at discrete locations.

37. A method of treating a subject suffering from a miR-21 mediated disease, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

38. A method of preventing cancer metastasis in a subject suffering from pre-metastatic cancer, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

39. A method of reducing cancer metastasis in a subject suffering from metastatic cancer, comprising administering to the subject an effective amount of a topoisomerase inhibitor.

40. The method of any one of claims 37-39, wherein the topoisomerase inhibitor is:

;

or an enantiomer, diastereomer, salt, ester or prodrug thereof.

41. A method of treating a subject suffering from an RNA mediated disease, comprising administering to the subject an effective amount of a compound having the structure

,

or an enantiomer, diastereomer, salt, ester or prodrug thereof.

42. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound having the structure:

.

43. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound having the structure:

.

44. A compound having the structure of Formula A or Formula B

Formula A Formula B

wherein

L1 is a trivalent linker moiety comprising alkyl, amide, ether, amino and triazolyl linkages;

L2 is a divalent linker moiety comprising alkyl, amide, ether, amino and triazolyl linkages;

X is a purification moiety suitable to act as a binding partner in affinity purification, immunoprecipitation or pull-down assay;

Y is an RNA-binding moiety; and

Z is an RNA-reactive moiety comprising an alkylating group.

45. The compound of claim 44, wherein:

the purification moiety is a biotin group;

the RNA-binding moiety is a topoisomerase inhibitor, a kinase inhibitor, a RNA splicing modulator, or a compound according to any one of claims 41-44; and

the RNA-reactive moiety is bis(2-chloroethyl)amine-containing moiety.

46. The compound of claim 44, having the structure:

.

47. The compound of claim 44, having the structure:

.