Compositions and methods for the delivery of nucleic acids

Abstract

Disclosed herein are methods of delivering nucleic acids to the cytosol of a cell using a bifacial peptide nucleic acid.

Description

[0001] Compositions and Methods for the Delivery of Nucleic Acids

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under Grant No. R01 GM143543 and R01 GM111995 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims benefit of priority of U.S. Provisional Application No. 63 / 643,863, filed May 7, 2024, which is incorporated herein by reference in its entirety.

[0006] BACKGROUND

[0007] Introducing nucleic acids into living cells is an important process in modern biological research, industry, and medicine. Efficient delivery of a functional nucleic acid into a living cell is an indispensable component of genetic engineering, recombinant protein production, and medical technologies known as gene therapy.

[0008] For example, gene therapy involves the transfer of normal, functional genetic material into specific cells to correct an abnormality due to a deficient or defective gene product. A variety of methods have been developed to facilitate both in vivo, in vitro, or ex vivo gene transfer.

[0009] Nucleic acid therapies involve the transfer of natural or synthetic oligonucleotides and polynucleotides into normal and / or pathological cells with the purpose of correcting or eliminating the diseased cells. For example, antisense oligonucleotides and interfering RNAs such as siRNAs and shRNAs are used to block undesirable pathways of protein expression in the cells. Plasmids, e.g., plasmids that comprise one or more protein-encoding sequences, may be introduced into cells to correct a cellular defect associated with a defective or absent gene or gene product, or to induce tumor cell death. Polynucleotide inductors of immunity, such as poly(I, C) or oligo- and polynucleotides having methylated GC pairs are used to increase the patients' defense against pathogens such as viruses or cancer cells. Ribozymes are ribonucleic acids that catalyze selective degradation of other polynucleotides in the diseased cells, for example, in cancer or virus-infected cells. Because oligo- and polynucleotides generally have low permeability through cell membranes, and are quickly eliminated from the body, there is the need for oligo / polynucleotide delivery vehicles that would allow enhanced intracellular delivery and protection from degradation and / or elimination from the body.

[0010] Many strategies for delivering nucleic acids to cells have been explored, including, for example, the encapsulating nucleic acids in liposomes and lipid nanoparticles. However, there remains a need for improved methods for delivering nucleic acids to cells.

[0011] SUMMARY

[0012] Described herein is the RNA transport function of bifacial Peptide Nucleic Acids (bPNAs). Selective binding of U-rich internal loops (URILs) in RNA by bPNA yields a hybrid complex with increased mammalian cell (e.g., HEK-293T) permeability. Notably, identical RNAs lacking a URIL were not transported, nor were URIL-RNAs transported in the absence of bPNA. This strategy for rendering RNAs cell-permeable may be applied to any RNA or oligonucleotide therapeutic, including siRNAs, miRNAs and antisense oligos (ASOs).

[0013] This methodology allows for the transport of fully synthetic bPNA-RNA complexes, which elevates the impact of URIL-tagging approaches. URIL-tagging of RNAs also allows for functionalized, synthetic RNAs to reach cellular targets, opening up a number of therapeutic avenues. In particular, bPNAs modified with E3-ligase ligands can be complexed with synthetic URIL-RNAs that mimic disease-relevant, dysregulated intracellular RNAs and ribonucleoprotein complexes (RNPs). Cellular delivery of the bPNA-RNA complex can allow the URIL-RNA complex to take the place of dysregulated RNA in the targeted RNP, thus driving endogenous degradation of the toxic RNP. This is similar to Proteolysis Targeting Chimeras (PROTACs), but uses RNA in place of a protein ligand to direct degradation to RNPs.

[0014] Accordingly, provided herein are methods of delivering a nucleic acid to a cell (e.g., to the cytosol of a cell). These methods can comprise contacting the nucleic acid with a bifacial Peptide Nucleic Acid (bPNA) comprising a triplex hybrid forming moiety, wherein the bPNA binds to a non-canonical base pairing site in the nucleic acid via triplex hybridization, forming a bPNA-nucleic acid complex; and contacting the cell with the bPNA-nucleic acid complex. The nucleic acid can comprise, for example, an RNA. The nucleic acid can comprise any suitable nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide therapeutic. In some embodiments, the nucleic acid an siRNA, an miRNAs, or an antisense oligo (ASO).

[0015] In some embodiments, the bPNA binds to a non-canonical base pairing site in the nucleic acid (e.g., RNA) via triplex hybridization. In certain embodiments, the non- canonical base pairing site comprises a U-rich internal loop (URIL). In certain embodiments, the URIL is a loop that includes at least four non-canonical base pairs, wherein at least 50% of the non-canonical base pairs comprise U-U pairs. In certain embodiments, the URIL comprises from 4-8 non-canonical base pairs.

[0016] The bifacial peptide nucleic acid (bPNA) can include a triplex hybrid forming moiety (e.g., a plurality of binding motifs disposed along a peptidyl backbone) optionally conjugated to a prosthetic group. The triplex hybrid forming moiety can bind to a non- canonical base pairing site in a target nucleic acid via triplex hybridization. In this way, a variety of prosthetic groups (e.g., dyes, labels, reactive groups, etc.) can be selectively delivered to a target nucleic acid (e.g., in vivo, in vitro, or ex vivo).

[0017] For example, provided herein are bifacial peptide nucleic acid probes defined by Formula I below

[0018] Formula I wherein

[0019] A is absent, or represents a prosthetic group;

[0020] X is absent, or represents a first bivalent linking group;

[0021] L is absent, or represents a second bivalent linking group; n is, individually for each occurrence, an integer selected from 1 and 2; m is an integer selected from 2, 3, and 4;

[0022] Z represents, individually for each occurrence, a binding motif selected from one of the following

[0023]

[0024] Q1and Q2individually represent -O- or -NRA-;

[0025] Y is N, -CRB-,

[0026] R1is, individually for each occurrence, selected from one of the following

[0027]

[0028] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0029] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0030] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0031] In some embodiments, RArepresents H for each occurrence (e.g., Q1and Q2individually represent -O- or -NH-). In some embodiments, Q1and Q2represent -O- for each occurrence. In other embodiments, Q1and Q2represent -NH- for each occurrence.

[0032] In some embodiments, R2represents H for each occurrence.

[0033] In some embodiments, R1is, individually for each occurrence, selected from one of the following

[0034] In certain embodiments, R1is, individually for each occurrence, -H or -CH3. In some embodiments, m is 2. In other embodiments, m is 3.

[0035] In some embodiments, n is 1 in all occurrences. In other embodiments, m is 3 and n is 1 in two occurrences and n is 2 in one occurrence.

[0036] In some embodiments, X represents a first bivalent linking group. In some embodiments, the first bivalent linking group comprises from 3 to 20 atoms, such as from 3 to 16 atoms or from 3 to 12 atoms. In certain embodiments, the first bivalent linking group comprises an alkylene linker or a heteroalkylene linker. In some embodiments, L represents a second bivalent linking group. In some embodiments, the second linking group comprises from 3 to 36 atoms, such as from 3 to 24 atoms or from 3 to 16 atoms. In certain embodiments, the second bivalent linking group comprises an alkylene linker or a heteroalkylene linker.

[0037] In some embodiments, in at least one occurrence, Z represents the binding motif shown below wherein

[0038] Q1and Q2individually represent -O- or -NRA-;

[0039] Y is N, -CRB-,

[0040] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0041] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0042] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0043] In certain embodiments, m is 2 and Z represents the binding motif shown below in two occurences wherein

[0044] Q1and Q2individually represent -O- or -NRA-;

[0045] Y is N, -CRB-,

[0046] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0047] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0048] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl. In certain embodiments, m is 3 and Z represents the binding motif shown below in two occurences wherein

[0049] Q1and Q2individually represent -O- or -NRA-;

[0050] Y is N, -CRB-,

[0051] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0052] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0053] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0054] In certain examples, the compound is defined by Formula IA below

[0055] Formula I A wherein A , L, Y, Q1, Q2, X, n, and m are as defined above

[0056] In certain examples, the compound is defined by Formula IB

[0057] Formula IB wherein

[0058] A, L, R1, m, and n are as defined above; and a is, individually for each occurrence, an integer selected from 3, 4, 5, and 6.

[0059] In some embodiments, the prosthetic group is not cyanine 5 (Cy5), cyanine 3 (Cy3), or carboxyfluorescein (Cbf).

[0060] In some embodiments, the prosthetic group can be absent. In other embodiments, the prosthetic group can be present (optionally connected via a linker L).

[0061] In some embodiments, the prosthetic group is selected from the group consisting of a fluorogenic dye, a protein ligand, a redox-active center, an ROS -generating center, a photoreactive center, a spin label, a group transfer agent, a therapeutic agent, a catalytic center, a click motif, or an NMR active label.

[0062] In some embodiments, the prosthetic group is a fluorogenic dye. In certain embodiments, the fluorogenic dye is selected from the group consisting of thiazole derived dyes such as thiazole orange, dimethylindole red and derivatives thereof, symmetric cyanine dyes, asymmetric cyanine dyes, fluoresceins, rhodamines, fluorogenic variants of asymmetric cyanine dyes and fluoresceins such as JF635 and JF646, arsenate dyes such as F1ASH and ReASH, malachite green and derivatives thereof, courmarin dyes, and hydroxybenzylidene dyes. In certain embodiments, the fluorogenic dye is thiazole orange.

[0063] In some embodiments, the prothetic group is a protein ligand. In certain embodiments, the protein ligand is selected from the group consisting of a ubiquitin ligase ligand, a ligand for a translational activator, a ligand for a translational inhibitor, a ligand for a transcription activator, a ligand for a transcription inhibitor, a ligand for a nuclease, or a ligand for a cell surface protein. In certain embodiments, the protein ligand is a ubiquitin ligase ligand that binds to an E3 ligase selected from the group consisting of XIAP, VHL, cereblon, and MDM2.

[0064] Also provided herein are methods for selectively degrading a target ribonucleoprotein complex in a cell. These methods can comprise contacting a nucleic acid that mimics a dysregulated intracellular RNA present in the target ribonucleoprotein complex with a bifacial peptide nucleic acid (bPNA) comprising a triplex hybrid forming moiety conjugated to a ubiquitin ligase ligand to form a nucleic acid-bPNA complex, wherein the bifacial peptide nucleic acid binds to a non-canonical base pairing site in the nucleic acid via triplex hybridization; and contacting the cell with the nucleic acid-complex.

[0065] Also provided herein are methods of delivering a nucleic acid to a cell that comprise covalently linking a transporter motif to the nucleic acid to form a transporter motif-nucleic acid conjugate; and contacting the cell with the transporter motif-nucleic acid conjugate. The transporter motif can be defined by Formula II below

[0066] Formula II wherein R2represents H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; Li represents, individually for each occurrence, a first bivalent linking group of from one to six atoms in length; L2 represents a second bivalent linking group; and E represents, individually for each occurrence, a 5- to 10-membered carbocyclic ring or a 5- to 10-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0067] In some embodiments, the transporter motif can be defined by Formula IIA below

[0068] Formula IIA wherein E, Li, and R2are as defined above with respect to Formula II, L3 is absent, or represents a third bivalent linking group; X is absent, or represents a fourth bivalent linking group; R1is, individually for each occurrence, an amino acid sidechain, such as one of the following

[0069] n is, individually for each occurrence, an integer selected from 1 and 2; and m is an integer selected from 2, 3, and 4.

[0070] In some embodiments of Formula II and IIA, Li is, individually for each occurrence, a bivalent linking group of from one to three atoms in length. In certain embodiments, Li is, individually for each occurrence, an alkylene linker or -NH-CH2-CH2-.

[0071] In some embodiments of Formula II and IIA, E represents, individually for each occurrence, a 6-membered carbocyclic ring or a 6-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In some embodiments, E represents, individually for each occurrence, a 6-membered aryl ring or a 6-membered heteroaryl ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a phenyl ring or a napthyl ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a triazine ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a 6-membered N-containing heterocyclic ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, the 6- membered N-containing heterocyclic ring comprises a piperidine ring, a piperazine ring, a pyridine ring, a triazine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, an indole ring, a quinoline ring, or a purine ring.

[0072] In some embodiments, covalently linking the transporter motif to the nucleic acid comprises performing a click chemistry reaction between a transporter motif molecule bearing a first click motif and a nucleic acid bearing a second click motif. In other embodiments, covalently linking the transporter motif to the nucleic acid comprises performing solid phase nucleic acid synthesis using a transporter motif molecule bearing a phosphoramidite moiety.

[0073] Also provided herein are transporter motif molecules that can be used to covalently functionalize a nucleic acid with a transporter motif (e.g., by reaction of the transporter motif molecule with a nucleic acid, for example, via click chemistry or via solid phase nucleic acid synthesis). For example, provided herein are transporter motif molecules defined by Formula III below

[0074] Formula III wherein R2represents H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; Li represents, individually for each occurrence, a first bivalent linking group of from one to six atoms in length; L2 represents a second bivalent linking group; E represents, individually for each occurrence, a 5- to 10-membered carbocyclic ring or a 5- to 10-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group; and G represents a reactive functional group. In some embodiments, L2 comprises an oligopeptide linker or a pseudopeptide linker.

[0075] In some embodiments, the transporter motif is defined by Formula IIIA below

[0076] Formula II A wherein E, Li, G, and R2are as defined above with respect to Formula II, L3 is absent, or represents a third bivalent linking group; X is absent, or represents a fourth bivalent linking group; R1is, individually for each occurrence, an amino acid sidechain, such as one of the following n is, individually for each occurrence, an integer selected from 1 and 2; and m is an integer selected from 2, 3, and 4. In some embodiments of Formula III and IIIA, Li is, individually for each occurrence, a bivalent linking group of from one to three atoms in length. In certain embodiments, Li is, individually for each occurrence, an alkylene linker or -NH-CH2-CH2-.

[0077] In some embodiments of Formula III and IIIA, E represents, individually for each occurrence, a 6-membered carbocyclic ring or a 6-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In some embodiments, E represents, individually for each occurrence, a 6-membered aryl ring or a 6-membered heteroaryl ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a phenyl ring or a napthyl ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a triazine ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a 6-membered N-containing heterocyclic ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, the 6- membered N-containing heterocyclic ring comprises a piperidine ring, a piperazine ring, a pyridine ring, a triazine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, an indole ring, a quinoline ring, or a purine ring.

[0078] In some embodiments of Formula III and IIIA, G can comprise any suitable functional group that can participate in a reaction with a functional group present on a nucleic acid so as to form a covalent bond between the transporter motif and the nucleic acid (so as to form the transporter motif-nucleic acid conjugate). In certain embodiments, G comprises a click motif. In certain embodiments, G comprises a phosphoramidite moiety. DESCRIPTION OF DRAWINGS

[0079] Figure 1 is an illustration of RNA motif centered PROTACs. Bifacial peptide nucleic acids (bPNAs) bind to U-rich internal loops (URILs) in RNA via base-triple formation between melamine and uracil and present an E3-ligase ligand. Following RNP formation, the E3 -ligase ligand recruits an E3 -ligase, resulting in ubiquitinylation of the RBP and subsequent degradation by the proteasome.

[0080] Figure 2A illustrates the design of an exampe RNA-PROTACs reagent. An E3- ligase ligand is coupled via a linker to bPNA. Examples of established linker and ligands are shown below, which target two distinct E3-ligases, Cereblon / CNBR and VHL.

[0081] Figure 2B shows the structure of a bPNA used in Example 1, with triplex hybridization to a URIL below.

[0082] Figure 3 shows the synthetic RNA, SL2-URIL6, that mimics stemloop 2 of UlsnRNA, bearing Alexa Fluor 488 at the 5’ terminus. Cy5-modified bPNA is shown.

[0083] Figure 4 shows Confocal fluorescence of HEK-293T cells showing cellular uptake of the RNA-bPNA complex (green and red fluorescence, respectively, Top Row). Remaining rows are control experiments lacking SL2-URIL6 RNA (2nd Row), Cy5-bPNA (3rd Row) and untreated cells (4th row).

[0084] Figure 5 illustrates the mechanism by which gene silencing by RNAi occurs.

[0085] Figure 6 illustrates a hypothesis by which hybridization with bPNA functions with synthetic siRNA variets targeting ApoB.

[0086] Figure 7 illustrates example knockdown studies showing functional silencing delivery of a nucleic acid using bPNA.

[0087] Figure 8 shows example bPNA variants and URIL binding.

[0088] Figure 9 shows (Left) a generalized bPNA scaffold where typical repeat number is n=(2-3) and (Right) a generalized STA scaffold with similar length range and possible substituents X indicated in the table at left. In both transporter scaffolds, Ri is the linkage to an oligonucleotide, examples of which are shown in Figure 10.

[0089] Figure 10 shows examples of linkages between transporter scaffolds and oligonucleotide. (Top) Glycol-based linkage to the transporter scaffold displays a phosphoramidate reactive site for terminal incorporation in solid phase oligonucleotide synthesis. (Middle) Modified phosphoramidate monomer for incorporation of the transporter at internal sites in the sequence. Thymine (unprotected) is shown as an example for A, T, G, C modified monomers. (Bottom) Azide-terminated transporter for click linkage to oligonucleotide. Figure 11 shows (Top) an example of a strain-driven copper free click coupling of modified RNA with transporter peptides and (Below) example ESC+ modifications used in each strand.

[0090] Figure 12 shows ApoB silencing in HepG2 cells. Concentration dependent knockdown of ApoB mRNA upon treatment with (Left) Anti -ApoB siRNA conjugated with benzyl STA (n=2, X=H) after 48 hr incubation and (Right) anti-ApoB siRNA conjugated with bPNA (n=2) after 96 hr incubation.

[0091] DETAILED DESCRIPTION

[0092] Definitions

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0094] At various places in the present specification, divalent linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups.

[0095] The term “n-membered” where n is an integer typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0096] As used herein, the phrase “optionally substituted” means unsubstituted or substituted. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. It is to be understood that substitution at a given atom is limited by valency.

[0097] Throughout the definitions, the term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include Ci-4, Ci-6, and the like. As used herein, the term “Cn-m alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, / / -propyl, isopropyl, / / -butyl, tert-butyl, isobutyl, ec-butyl; higher homologs such as 2-methyl-l -butyl, / / -pentyl, 3 -pentyl, / / -hexyl, 1,2,2- trimethylpropyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0098] As used herein, “Cn-m alkenyl” refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons. Example alkenyl groups include, but are not limited to, ethenyl, / / -propenyl, isopropenyl, / / -butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0099] As used herein, “Cn-m alkynyl” refers to an alkyl group having one or more triple carbon-carbon bonds and having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-l-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0100] As used herein, the term “Cn-m alkylene”, employed alone or in combination with other terms, refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethan-l,2-diyl, propan- 1,3 -diyl, propan-1, 2- diyl, butan-l,4-diyl, butan-l,3-diyl, butan-l,2-diyl, 2-methyl -propan- 1,3 -diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0101] As used herein, the term “Cn-m alkoxy”, employed alone or in combination with other terms, refers to a group of formula -O-alkyl, wherein the alkyl group has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., / / -propoxy and isopropoxy), te / 7-butoxy, and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0102] As used herein, the term “Cn-m alkylamino” refers to a group of formula -NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0103] As used herein, the term “Cn-m alkoxycarbonyl” refers to a group of formula -C(O)O-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-m alkylcarbonyl” refers to a group of formula -C(O)- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0104] As used herein, the term “Cn-m alkylcarbonylamino” refers to a group of formula -NHC(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0105] As used herein, the term “Cn-m alkylsulfonylamino” refers to a group of formula -NHS(O)2-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0106] As used herein, the term “aminosulfonyl” refers to a group of formula -S(O)2NH2.

[0107] As used herein, the term “Cn-m alkylaminosulfonyl” refers to a group of formula -S(O)2NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0108] As used herein, the term “di(Cn-m alkyl)aminosulfonyl” refers to a group of formula -S(O)2N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0109] As used herein, the term “aminosulfonylamino” refers to a group of formula - NHS(O)2NH2.

[0110] As used herein, the term “Cn-m alkylaminosulfonylamino” refers to a group of formula -NHS(0)2NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0111] As used herein, the term “di(Cn-m alkyl)aminosulfonylamino” refers to a group of formula -NHS(O)2N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0112] As used herein, the term “aminocarbonylamino”, employed alone or in combination with other terms, refers to a group of formula -NHC(0)NH2.

[0113] As used herein, the term “Cn-m alkylaminocarbonylamino” refers to a group of formula -NHC(O)NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0114] As used herein, the term “di(Cn-m alkyl)aminocarbonylamino” refers to a group of formula -NHC(0)N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0115] As used herein, the term “Cn-m alkylcarbamyl” refers to a group of formula -C(O)- NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0116] As used herein, the term “thio” refers to a group of formula -SH.

[0117] As used herein, the term “Cn-m alkylsulfinyl” refers to a group of formula -S(O)- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0118] As used herein, the term “Cn-m alkyl sulfonyl” refers to a group of formula -S(O)2- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0119] As used herein, the term “amino” refers to a group of formula -NH2.

[0120] As used herein, the term "aryl," employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term "Cn-m aryl" refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g., phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, the aryl group is a substituted or unsubstituted phenyl.

[0121] As used herein, the term “carbamyl” to a group of formula -C(O)NH2.

[0122] As used herein, the term “carbonyl”, employed alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O).

[0123] As used herein, the term “di(Cn-m-alkyl)amino” refers to a group of formula -N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0124] As used herein, the term “di(Cn-m-alkyl)carbamyl” refers to a group of formula - C(O)N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0125] As used herein, the term “halo” refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br. In some embodiments, a halo is F or Cl.

[0126] As used herein, “Cn-m haloalkoxy” refers to a group of formula -O-haloalkyl having n to m carbon atoms. An example haloalkoxy group is OCF3. In some embodiments, the haloalkoxy group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0127] As used herein, the term “Cn-m haloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+l halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to

[0128] 4, or 1 to 3 carbon atoms.

[0129] As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbons including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4,

[0130] 5, 6, 7, 8, 9, or 10 ring-forming carbons (C3-10). Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O) or C(S)). Cycloalkyl groups also include cycloalkylidenes. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbomyl, norpinyl, norcamyl, and the like. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, the cycloalkyl has 6-10 ring-forming carbon atoms. In some embodiments, cycloalkyl is adamantyl. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring.

[0131] As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-10 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six- membereted heteroaryl ring. A five-membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five-membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4- oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary sixmembered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

[0132] As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Example heterocycloalkyl groups include pyrrolidin-2-one, l,3-isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)2, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moi eties that have one or more aromatic rings fused (z.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

[0133] At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an azetidine ring may be attached at any position of the ring, whereas a pyridin-3-yl ring is attached at the 3 -position. The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0134] Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0135] In some embodiments, the compounds described herein can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, enantiomerically enriched mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures (e.g., including (R)- and (5)-enantiomers, diastereomers, (D)-isomers, (Z)-isomers, (+) (dextrorotatory) forms, (-) (levorotatory) forms, the racemic mixtures thereof, and other mixtures thereof). Additional asymmetric carbon atoms can be present in a substituent, such as an alkyl group. All such isomeric forms, as well as mixtures thereof, of these compounds are expressly included in the present description. The compounds described herein can also or further contain linkages wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring or double bond (e.g., carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds). Accordingly, all cis / trans and E / Z isomers and rotational isomers are expressly included in the present description. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms of that compound.

[0136] Optical isomers can be obtained in pure form by standard procedures known to those skilled in the art, and include, but are not limited to, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw- Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972), each of which is incorporated herein by reference in their entireties. It is also understood that the compounds described herein include all possible regioisomers, and mixtures thereof, which can be obtained in pure form by standard separation procedures known to those skilled in the art, and include, but are not limited to, column chromatography, thin-layer chromatography, and high-performance liquid chromatography.

[0137] Unless specifically defined, compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. Unless otherwise stated, when an atom is designated as an isotope or radioisotope (e.g., deuterium, [nC], [18F]), the atom is understood to comprise the isotope or radioisotope in an amount at least greater than the natural abundance of the isotope or radioisotope. For example, when an atom is designated as “D” or “deuterium”, the position is understood to have deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium).

[0138] All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g. hydrates and solvates) or can be isolated.

[0139] In some embodiments, preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts.

[0140] Example acids can be inorganic or organic acids and include, but are not limited to, strong and weak acids. Some example acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, -toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0141] Example bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some example strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides and arylamines, wherein; alkoxides include lithium, sodium and potassium salts of methyl, ethyl and t-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, / .w-propyl, n-butyl, tert-butyl, trimethyl silyl and cyclohexyl substituted amides.

[0142] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[0143] The expressions, “ambient temperature” and “room temperature” or “rt” as used herein, are understood in the art, and refer generally to a temperature, e.g. a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 20 °C to about 30 °C.

[0144] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0145] The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0146] Methods of Use

[0147] Provided herein are methods of delivering a nucleic acid to a cell (e.g., to the cytosol of a cell). These methods can comprise contacting the nucleic acid with a bifacial Peptide Nucleic Acid (bPNA) comprising a triplex hybrid forming moiety, wherein the bPNA binds to a non-canonical base pairing site in the nucleic acid via triplex hybridization, forming a bPNA-nucleic acid complex; and contacting the cell with the bPNA-nucleic acid complex.

[0148] The nucleic acid can comprise, for example, an RNA. The nucleic acid can comprise any suitable nucleic acid. In some embodiments, the nucleic acid comprises an oligonucleotide therapeutic. In some embodiments, the nucleic acid an siRNA, an miRNAs, or an antisense oligo (ASO).

[0149] In some embodiments, the bPNA binds to a non-canonical base pairing site in the nucleic acid (e.g., RNA) via triplex hybridization. In certain embodiments, the non- canonical base pairing site comprises a U-rich internal loop (URIL). In certain embodiments, the URIL is a loop that includes at least four non-canonical base pairs, wherein at least 50% of the non-canonical base pairs comprise U-U pairs. In certain embodiments, the URIL comprises from 4-8 non-canonical base pairs.

[0150] The bPNA can also localize any suitable prosthetic group with a target nucleic acid of interest (which itself can be associated with other biomolecules in vivo, such as nucleic acids, and / or delivered to a target cell). Depending on the nucleic acid and the nature of the prosthetic group, the prosthetic group can then be utilized for some functionality (e.g., for labeling the target nucleic acid, for reacting with the target nucleic acid, for labeling a protein associated with the target nucleic acid, and / or for reacting with a protein associated with the target nucleic acid).

[0151] For example, provided herein are methods of associating a prosthetic group with a target nucleic acid. These methods can comprise contacting the nucleic acid with a bifacial peptide nucleic acid comprising a triplex hybrid forming moiety conjugated to a prosthetic group. The bifacial peptide nucleic acid can bind to a non-canonical base pairing site in the target nucleic acid via triplex hybridization, thereby associating the prosthetic group with the target nucleic acid.

[0152] Also provided herein are methods of associating a prosthetic group with a target protein. These methods can comprise contacting a nucleic acid with a bifacial peptide nucleic acid probe comprising a triplex hybrid forming moiety conjugated to a prosthetic group. The bifacial peptide nucleic acid probe can bind to a non-canonical base pairing site in the target nucleic acid via triplex hybridization. The nucleic acid can form a ribonucleoprotein complex with the target protein, thereby associating the prosthetic group with the target protein.

[0153] Also provided herein are methods for selectively degrading a target ribonucleoprotein complex in a cell. These methods can comprise contacting a nucleic acid that mimics a dysregulated intracellular RNA present in the target ribonucleoprotein complex with a bifacial peptide nucleic acid (bPNA) comprising a triplex hybrid forming moiety conjugated to a ubiquitin ligase ligand to form a nucleic acid-bPNA complex, wherein the bifacial peptide nucleic acid binds to a non-canonical base pairing site in the nucleic acid via triplex hybridization; and contacting the cell with the nucleic acid-complex.

[0154] Also provided herein are methods of delivering a nucleic acid to a cell that comprise covalently linking a transporter motif to the nucleic acid to form a transporter motif-nucleic acid conjugate; and contacting the cell with the transporter motif-nucleic acid conjugate. The transporter motif can be defined by Formula II below

[0155] Formula II wherein R2represents H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; Li represents, individually for each occurrence, a first bivalent linking group of from one to six atoms in length; L2 represents a second bivalent linking group; and E represents, individually for each occurrence, a 5- to 10-membered carbocyclic ring or a 5- to 10-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0156] In some embodiments, the transporter motif can be defined by Formula IIA below

[0157] Formula IIA wherein E, Li, and R2are as defined above with respect to Formula II, L3 is absent, or represents a third bivalent linking group; X is absent, or represents a fourth bivalent linking group; R1is, individually for each occurrence, an amino acid sidechain, such as one of the following n is, individually for each occurrence, an integer selected from 1 and 2; and m is an integer selected from 2, 3, and 4. In some embodiments of Formula II and IIA, Li is, individually for each occurrence, a bivalent linking group of from one to three atoms in length. In certain embodiments, Li is, individually for each occurrence, an alkylene linker or -NH-CH2-CH2-.

[0158] In some embodiments of Formula II and IIA, E represents, individually for each occurrence, a 6-membered carbocyclic ring or a 6-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In some embodiments, E represents, individually for each occurrence, a 6-membered aryl ring or a 6-membered heteroaryl ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a phenyl ring or a napthyl ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a triazine ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, E represents a 6-membered N-containing heterocyclic ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group. In certain embodiments, the 6- membered N-containing heterocyclic ring comprises a piperidine ring, a piperazine ring, a pyridine ring, a triazine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, an indole ring, a quinoline ring, or a purine ring.

[0159] In some embodiments, covalently linking the transporter motif to the nucleic acid comprises performing a click chemistry reaction between a transporter motif molecule bearing a first click motif and a nucleic acid bearing a second click motif. In other embodiments, covalently linking the transporter motif to the nucleic acid comprises performing solid phase nucleic acid synthesis using a transporter motif molecule bearing a phosphoramidite moiety.

[0160] Bifacial Peptide Nucleic Acids The methods described herein can utilize bifacial peptide nucleic acids (bPNAs).

[0161] These bPNAs can include a triplex hybrid forming moiety (e.g., a plurality of binding motifs disposed along a peptidyl backbone) optionally conjugated to a prosthetic group. The triplex hybrid forming moiety can bind to a non-canonical base pairing site in a target nucleic acid via triplex hybridization. In some embodiments, the bifacial peptide nucleic acid can be defined by Formula I below

[0162] Formula I wherein

[0163] A is absent, or represents a prosthetic group;

[0164] X is absent, or represents a first bivalent linking group;

[0165] L is absent, or represents a second bivalent linking group; n is, individually for each occurrence, an integer selected from 1 and 2; m is an integer selected from 2, 3, and 4;

[0166] Z represents, individually for each occurrence, a binding motif selected from one of the following

[0167]

[0168] Q1and Q2individually represent -O- or -NRA-;

[0169] Y is N, -CRB-,

[0170] R1is, individually for each occurrence, selected from one of the following

[0171] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0172] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0173] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0174] In some embodiments, RArepresents H for each occurrence (e.g., Q1and Q2individually represent -O- or -NH-). In some embodiments, Q1and Q2represent -O- for each occurrence. In other embodiments, Q1and Q2represent -NH- for each occurrence.

[0175] In some embodiments, R2represents H for each occurrence.

[0176] In some embodiments, R1is, individually for each occurrence, selected from one of the following

[0177] In certain embodiments, R1is, individually for each occurrence, -H or -CH3. In some embodiments, m is 2. In other embodiments, m is 3.

[0178] In some embodiments, n is 1 in all occurrences. In other embodiments, m is 3 and n is 1 in two occurrences and n is 2 in one occurrence.

[0179] In some embodiments, X represents a first bivalent linking group. In some embodiments, the first bivalent linking group comprises from 3 to 20 atoms, such as from 3 to 16 atoms or from 3 to 12 atoms. In certain embodiments, the first bivalent linking group comprises an alkylene linker or a heteroalkylene linker.

[0180] In some embodiments, L represents a second bivalent linking group. In some embodiments, the second linking group comprises from 3 to 36 atoms, such as from 3 to 24 atoms or from 3 to 16 atoms. In certain embodiments, the second bivalent linking group comprises an alkylene linker or a heteroalkylene linker. In some embodiments, in at least one occurrence, Z represents the binding motif shown below wherein

[0181] Q1and Q2individually represent -O- or -NRA-;

[0182] Y is N, -CRB-,

[0183] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0184] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0185] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0186] In certain embodiments, m is 2 and Z represents the binding motif shown below in two occurences wherein

[0187] Q1and Q2individually represent -O- or -NRA-;

[0188] Y is N, -CRB-,

[0189] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0190] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0191] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluorom ethyl.

[0192] In certain embodiments, m is 3 and Z represents the binding motif shown below in two occurences wherein

[0193] Q1and Q2individually represent -O- or -NRA-;

[0194] Y is N, -CRB-,

[0195] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0196] RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0197] RBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0198] In certain examples, the compound is defined by Formula IA below

[0199] Formula I A wherein A , L, Y, Q1, Q2, X, n, and m are as defined above

[0200] In certain examples, the compound is defined by Formula IB

[0201] Formula IB wherein

[0202] A, L, R1, m, and n are as defined above; and a is, individually for each occurrence, an integer selected from 3, 4, 5, and 6.

[0203] Linking Groups In the compounds above, the linking groups (e.g., the first linking group and / or the second linking group), when present, can be any suitable group or moiety which can function as a bivalent linker connecting the prothetic moiety to the peptidyl backbone. The linking group can be composed of any assembly of atoms, including oligomeric and polymeric chains. In some cases, the total number of atoms in the linking group can be from 3 to 200 atoms (e.g., from 3 to 150 atoms, from 3 to 100 atoms, from 3 and 50 atoms, from 3 to 25 atoms, from 3 to 15 atoms, or from 3 to 10 atoms).

[0204] In some embodiments, the linking group can be, for example, an alkyl, alkoxy, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, or polyamino group. In some embodiments, the linking group can comprise one of the groups above joined to one or both of the moieties to which it is attached by a functional group. Examples of suitable functional groups include, for example, secondary amides (-CONH-), tertiary amides (-CONR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, or -NRCONR-), carbinols ( -CHOH-, - CROH-), ethers (-O-), and esters (-COO-, -CH2O2C-, CHRO2C-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. For example, in some embodiments, the linking group can comprise an alkyl group (e.g., a C1-C12 alkyl group, a Ci-Cs alkyl group, or a Ci-Ce alkyl group) bound to one or both of the moieties to which it is attached via an ester (-COO-, -CH2O2C-, CHRO2C-), a secondary amide (-CONH-), or a tertiary amide (- CONR-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. In certain embodiments, the linking group can be chosen from one of the following: where m is an integer from 1 to 12 and R1is, independently for each occurrence, hydrogen, an alkyl group, an aryl group, or a heterocyclic group.

[0205] In some embodiments, the linking group can be , where m is an integer from 1 to 12 (e.g., an integer from 1 to 6, or an integer from 1 to 3). In certain embodiments, the linking group can be , where m is 1.

[0206] If desired, the linker can serve to modify the solubility of the compounds described herein. In some embodiments, the linker is hydrophilic. In some embodiments, the linker can be an alkyl group, an alkylaryl group, an oligo- or polyalkylene oxide chain (e.g., an oligo- or polyethylene glycol chain), or an oligo- or poly(amino acid) chain.

[0207] Prosthetic Groups

[0208] In some embodiments, the prosthetic group can be absent. In other embodiments, the prosthetic group can be present (optionally connected via a linker L). When present, the prosthetic group can comprise any functional moiety which can be delivered as cargo using the bifacial peptide nucleic acid.

[0209] For example, the prosthetic group can comprise a fluorogenic dye (e.g., a thiazole derived dye such as thiazole orange, dimethylindole red or a derivative thereof, a symmetric cyanine dye, an asymmetric cyanine dye, a fluorescein, a rhodamine, a fluorogenic variant of an asymmetric cyanine dye or fluorescein such as JF635 and JF646, an arsenate dye such as F1ASH and ReASH, malachite green or a derivative thereof, a courmarin dye, or a hydroxybenzylidene dye), a protein ligand (e.g., a ligand for a protein which facilitates / drives association of a protein of interest with a target nucleic acid bound to the bifacial peptide nucleic acid probe and / or other proteins bound to the target nucleic acid, such as aaubiquitin ligase ligand (e.g., a ligand that binds to an E3 ligase such as XIAP, VHL, cereblon, and MDM2), a ligand for a translational activator, a ligand for a translational inhibitor, a ligand for a transcription activator, a ligand for a transcription inhibitor, a ligand for a nuclease, or a ligand for a cell surface protein), a redox-active center, an ROS-generating center (e.g., Cu-Phen or Fe-EDTA), a photoreactive center (e.g., a photoreactive organic moiety (e.g., benzophenone) that generate radicals upon activation with actinic radiation, an ethylenically unsaturated moiety that induces crosslinking), a spin label (e.g., a paramagnetic metal center or stable radical that assists in spectroscopic analysis of a target nucleic acid bound to the bifacial peptide nucleic acid probe and / or other proteins bound to the target nucleic acid), a group transfer agent (e.g., an active ester that transfers to a protein bound to the target nucleic acid to which the bifacial peptide nucleic acid probe is bound, thereby deactivating the proteint), a therapeutic agent (e.g., a therapeutic, diagnostic, or prophylactic agent), a catalytic center, a click motif (e.g., a functional group that can participate in a click chemistry to allow for subsequent conjugation of another moiety bearing a complementary click motif to the bifacial peptide nucleic acid probe), or an NMR active label.

[0210] In some embodiments, the prosthetic group is not cyanine 5 (Cy5), cyanine 3 (Cy3), or carboxyfluorescein (Cbf).

[0211] Transporter Motif Molecules

[0212] Also provided herein are transporter motif molecules that can be used to covalently functionalize a nucleic acid with a transporter motif (e.g., by reaction of the transporter motif molecule with a nucleic acid, for example, via click chemistry or via solid phase nucleic acid synthesis). For example, provided herein are transporter motif molecules defined by Formula III below

[0213] Formula III wherein R2represents H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; Li represents, individually for each occurrence, a first bivalent linking group of from one to six atoms in length; L2 represents a second bivalent linking group; E represents, individually for each occurrence, a 5- to 10-membered carbocyclic ring or a 5- to 10-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group; and G represents a reactive functional group.

[0214] In some embodiments, L2 comprises an oligopeptide linker or a pseudopeptide linker.

[0215] In some embodiments, the transporter motif is defined by Formula IIIA below

[0216] Formula II A wherein E, Li, G, and R2are as defined above with respect to Formula II, L3 is absent, or represents a third bivalent linking group; X is absent, or represents a fourth bivalent linking group; R1is, individually for each occurrence, an amino acid sidechain, such as one of the following n is, individually for each occurrence, an integer selected from 1 and 2; and m is an integer selected from 2, 3, and 4.

[0217] In some embodiments of Formula III and IIIA, Li is, individually for each occurrence, a bivalent linking group of from one to three atoms in length. In certain embodiments, Li is, individually for each occurrence, an alkylene linker or -NH-CH2-CH2-.

[0218] In some embodiments of Formula III and IIIA, E represents, individually for each occurrence, a 6-membered carbocyclic ring or a 6-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0219] In some embodiments, E represents, individually for each occurrence, a 6-membered aryl ring or a 6-membered heteroaryl ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0220] In certain embodiments, E represents a phenyl ring or a napthyl ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0221] In certain embodiments, E represents a triazine ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0222] In certain embodiments, E represents a N-containing heterocyclic ring. The term "N-containing heterocyclic ring", as used herein means, unless otherwise stated, a monocyclic non-aromatic 3-, 4-, 5-, 6- or 7- membered heterocyclic ring containing a nitrogen atom and optionally one or more heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. In certain embodiments, E represents a 6-membered N-containing heterocyclic ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

[0223] In certain embodiments, the 6-membered N-containing heterocyclic ring comprises a piperidine ring, a piperazine ring, a pyridine ring, a triazine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, an indole ring, a quinoline ring, or a purine ring.

[0224] In some embodiments of Formula III and IIIA, G can comprise any suitable functional group that can participate in a reaction with a functional group present on a nucleic acid so as to form a covalent bond between the transporter motif and the nucleic acid (so as to form the transporter motif-nucleic acid conjugate). In certain embodiments, G comprises a click motif. In certain embodiments, G comprises a phosphoramidite moiety.

[0225] Linking Groups

[0226] In the compounds above, the linking groups (e.g., the first linking group and / or the second linking group), when present, can be any suitable group or moiety which can function as a bivalent linker connecting the prothetic moiety to the peptidyl backbone. The linking group can be composed of any assembly of atoms, including oligomeric and polymeric chains. In some cases, the total number of atoms in the linking group can be from 3 to 200 atoms (e.g., from 3 to 150 atoms, from 3 to 100 atoms, from 3 and 50 atoms, from 3 to 25 atoms, from 3 to 15 atoms, or from 3 to 10 atoms).

[0227] In some embodiments, the linking group can be, for example, an alkyl, alkoxy, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, or polyamino group. In some embodiments, the linking group can comprise one of the groups above joined to one or both of the moieties to which it is attached by a functional group. Examples of suitable functional groups include, for example, secondary amides (-CONH-), tertiary amides (-CONR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, or -NRCONR-), carbinols ( -CHOH-, - CROH-), ethers (-O-), and esters (-COO-, -CH2O2C-, CHRO2C-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. For example, in some embodiments, the linking group can comprise an alkyl group (e.g., a C1-C12 alkyl group, a Ci-Cs alkyl group, or a Ci-Ce alkyl group) bound to one or both of the moieties to which it is attached via an ester (-COO-, -CH2O2C-, CHRO2C-), a secondary amide (-CONH-), or a tertiary amide (- CONR-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. In certain embodiments, the linking group can be chosen from one of the following:

[0228] where m is an integer from 1 to 12 and R1is, independently for each occurrence, hydrogen, an alkyl group, an aryl group, or a heterocyclic group.

[0229] In some embodiments, the linking group can be , where m is an integer from 1 to 12 (e.g., an integer from 1 to 6, or an integer from 1 to 3). In certain embodiments, the linking group can be , where m is 1.

[0230] If desired, the linker can serve to modify the solubility of the compounds described herein. In some embodiments, the linker is hydrophilic. In some embodiments, the linker can be an alkyl group, an alkylaryl group, an oligo- or polyalkylene oxide chain (e.g., an oligo- or polyethylene glycol chain), or an oligo- or poly(amino acid) chain.

[0231] Click Chemistry and Click Motifs

[0232] In some embodiments, the transporter motif molecules described above can include a reactive functional group (G) that can participate in a click reaction with a functional group present on a nucleic acid so as to form a covalent bond between the transporter motif and the nucleic acid (so as to form the transporter motif-nucleic acid conjugate). Reactive functional groups that can participate in a click chemistry reaction are referred to as click motifs.

[0233] Click chemistry refers to a class chemical reaction (referred to as a “click reaction”) between two click groups that exhibit good yields, wide functional group tolerance, and are highly selective even in the presence of a complex mixture of biological molecules. These characteristics allow the click reactions to proceed even in vivo. Example click motif pairs used as the first click motif and the second click motif include, but not limited to, azide with phosphine; azide with cyclooctyne; nitrone with cyclooctyne; nitrile oxide with norbornene; oxanorb ornadiene with azide; trans-cyclooctene with s-tetrazine; quadricyclane with bis(dithiobenzil)nickel(II).

[0234] In some embodiments, the second click motif comprises an alkene, e.g., a cyclooctene, e.g., a transcyclooctene (TCO) or norbornene (NOR), and the first click motif comprises a tetrazine (Tz). In other embodiments, the second click motif comprises an alkyne, e.g., a cyclooctyne such as dibenzocyclooctyne (DBCO), and the first click motif comprises an azide (Az). In some embodiments, the second click motif comprises a Tz, and the first click motif comprises an alkene such as transcyclooctene (TCO) or norbomene (NOR). Alternatively or in addition, the first click motif comprises an Az, and the second click motif comprises a cyclooctyne such as dibenzocyclooctyne (DBCO). TCO reacts specifically in a click chemistry reaction with a tetrazine (Tz) moiety. DBCO reacts specifically in a click chemistry reaction with an azide (Az) moiety. Norbornene reacts specifically in a click chemistry reaction with a tetrazine (Tz) moiety.

[0235] Exemplary click chemistry reactions (and by extension click motifs) are shown below. For example, copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) comprises using a Copper (Cu) catalyst at room temperature. The Azide-Alkyne Cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3- tri azole.

[0236] Another example of click chemistry includes Staudinger ligation, which is a reaction that is based on the classic Staudinger reaction of azides with triarylphosphines. It launched the field of bioorthogonal chemistry as the first reaction with completely abiotic functional. The azide acts as a soft electrophile that prefers soft nucleophiles such as phosphines. This is in contrast to most biological nucleophiles which are typically hard nucleophiles. The reaction proceeds selectively under water-tolerant conditions to produce a stable product. Phosphines are completely absent from living systems and do not reduce disulfide bonds despite mild reduction potential. Azides had been shown to be biocompatible in FDA- approved drugs such as azidothymidine and through other uses as cross linkers. Additionally, their small size allows them to be easily incorporated into biomolecules through cellular metabolic pathways.

[0237] Copper-free click chemistry is a bioorthogonal reaction first developed by Carolyn Bertozzi as an activated variant of an azide alkyne cycloaddition. Unlike CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity. Instead of copper, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC). It was developed as a faster alternative to the Staudinger ligation, with the first generations reacting over sixty times faster. The incredible bioorthogonality of the reaction has allowed the Cu-free click reaction to be applied within cultured cells, live zebrafish, and mice. Cyclooctynes were selected as the smallest stable alkyne ring which increases reactivity through ring strain which has calculated to be 19.9 kcal / mol.

[0238] Copper-free click chemistry also includes nitrone dipole cycloaddition. Copper-free click chemistry has been adapted to use nitrones as the 1,3-dipole rather than azides and has been used in the modification of peptides.

[0239] This cycloaddition between a nitrone and a cyclooctyne forms N-alkylated isoxazolines. The reaction rate is enhanced by water and is extremely fast with second order rate constants ranging from 12 to 32 M1-s ', depending on the substitution of the nitrone. Although the reaction is extremely fast, incorporating the nitrone into biomolecules through metabolic labeling has only been achieved through post-translational peptide modification.

[0240] Another example of click chemistry includes norbornene cycloaddition. 1,3 dipolar cycloadditions have been developed as a bioorthogonal reaction using a nitrile oxide as a 1,3-dipole and a norbornene as a dipolarophile. Its primary use has been in labeling DNA and RNA in automated oligonucleotide synthesizers.

[0241] Norbornenes were selected as dipolarophiles due to their balance between strain- promoted reactivity and stability. The drawbacks of this reaction include the cross-reactivity of the nitrile oxide due to strong electrophilicity and slow reaction kinetics.

[0242] Another example of click chemistry includes oxanorbornadiene cycloaddition. The oxanorb ornadiene cycloaddition is a 1,3-dipolar cycloaddition followed by a retro-Diels Alder reaction to generate a triazole-linked conjugate with the elimination of a furan molecule. This reaction is useful in peptide labeling experiments, and it has also been used in the generation of SPECT imaging compounds.

[0243] Ring strain and electron deficiency in the oxanorbornadiene increase reactivity towards the cycloaddition rate-limiting step. The retro-Diels Alder reaction occurs quickly afterwards to form the stable 1,2,3 triazole. Limitations of this reaction include poor tolerance for substituents which may change electronics of the oxanorbornadiene and low rates (second order rate constants on the order of 104).

[0244] Another example of click chemistry includes tetrazine ligation. The tetrazine ligation is the reaction of a trans-cyclooctene and an s-tetrazine in an inverse-demand Diels Alder reaction followed by a retro-Diels Alder reaction to eliminate nitrogen gas. The reaction is extremely rapid with a second order rate constant of 2000 M1-s1(in 9: 1 methanol / water) allowing modifications of biomolecules at extremely low concentrations.

[0245] The highly strained trans-cyclooctene is used as a reactive dienophile. The diene is a 3,6-diaryl-s-tetrazine which has been substituted in order to resist immediate reaction with water. The reaction proceeds through an initial cycloaddition followed by a reverse Diels Alder to eliminate N2 and prevent reversibility of the reaction.

[0246] Not only is the reaction tolerant of water, but it has been found that the rate increases in aqueous media. Reactions have also been performed using norbomenes as dienophiles at second order rates on the order of 1 M1• s ' in aqueous media. The reaction has been applied in labeling live cells and polymer coupling.

[0247] Another example of click chemistry includes is [4+1] cycloaddition. This isocyanide click reaction is a [4+1] cycloaddition followed by a retro-Diels Alder elimination of N2.

[0248] The reaction proceeds with an initial [4+1] cycloaddition followed by a reversion to eliminate a thermodynamic sink and prevent reversibility. This product is stable if a tertiary amine or isocyanopropanoate is used. If a secondary or primary isocyanide is used, the produce will form an imine which is quickly hydrolyzed.

[0249] Isocyanide is a favored chemical reporter due to its small size, stability, nontoxicity, and absence in mammalian systems. However, the reaction is slow, with second order rate constants on the order of 102M1- s ' .

[0250] Another example of click chemistry includes quadricyclane ligation. The quadricyclane ligation utilizes a highly strained quadricyclane to undergo [2+2+2] cycloaddition with TI systems.

[0251] Quadricyclane is abiotic, unreactive with biomolecules (due to complete saturation), relatively small, and highly strained (~80 kcal / mol). However, it is highly stable at room temperature and in aqueous conditions at physiological pH. It is selectively able to react with electron-poor TI systems but not simple alkenes, alkynes, or cyclooctynes.

[0252] Bis(dithiobenzil)nickel(II) was chosen as a reaction partner out of a candidate screen based on reactivity. To prevent light-induced reversion to norbornadiene, diethyldithiocarbamate is added to chelate the nickel in the product.

[0253] These reactions are enhanced by aqueous conditions with a second order rate constant of 0.25 M1- s ' . Of particular interest is that it has been proven to be bioorthogonal to both oxime formation and copper-free click chemistry.

[0254] The exemplary click chemistry reactions have high specificity, efficient kinetics, and occur in vivo under physiological conditions. See, e.g., Baskin et al. Proc. Natl. Acad. Set. USA 104(2007): 16793; Oneto et al. Acta biomaterilia (2014); Neves et al. Bioconjugate chemistry 24(2013):934; Koo et al. Angewandte Chemie 51(2012): 11836; and Rossin et al. Angewandte Chemie 49(2010):3375. For a review of a wide variety of click chemistry reactions and their methodologies, see e.g., Nwe K and Brechbiel M W, 2009 Cancer Biotherapy and Radiopharmaceuticals, 24(3): 289-302; Kolb H C et al., 2001 Angew. Chem. Int. Ed. 40: 2004-2021. The entire contents of each of the foregoing references are incorporated herein by reference.

[0255] Exemplary click motif pairs are shown in the table below. Functional groups formed by reaction of click motif pairs are well known in the art.

[0256] Other suitable include the motifs can be found, for example, in Patterson, D.M., et al. “Finding the Right (Bioorthogonal) Chemistry,” ACS Chem. Biol., 2014, 9(3): 592-605; Akgun, B., et al. “Synergic "Click" Boronate / Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells,” J. Am. Chem. Soc., 2017, 139(40): 14285-14291; and Akgun, B. and Hall, D.G. “Fast and Tight Boronate Formation for Click Bioorthogonal Conjugation,” Angew. Chem., Int. Ed. 2016, 55(12): 3909-3913, each of which is hereby incorporated by reference in its entirety

[0257] EXAMPLES

[0258] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non- critical parameters which can be changed or modified to yield essentially the same results.

[0259] Example 1: A Method to Increase the Cellular Permeability of Nucleic Acids Via Specific Molecular Recognition

[0260] Overview

[0261] In this example, we describe the RNA transport function of bifacial Peptide Nucleic Acids (bPNAs). Selective binding of U-rich internal loops (URILs) in RNA by bPNA yields a hybrid complex with increased mammalian cell (HEK-293T) permeability. Notably, identical RNAs lacking a URIL were not transported, nor were URIL-RNAs transported in the absence of bPNA. This methodology enables transport of fully synthetic bPNA-RNA complexes, which elevates the impact of URIL-tagging approaches. URIL-tagging of RNAs enables functionalized, synthetic RNAs to reach cellular targets, opening up a number of therapeutic avenues. In particular, bPNAs modified with E3-ligase ligands can be complexed with synthetic URIL-RNAs that mimic disease-relevant, dysregulated intracellular RNAs and ribonucleoprotein complexes (RNPs). Cellular delivery of the bPNA-RNA complex would allow the URIL-RNA complex to take the place of dysregulated RNA in the targeted RNP, thus driving endogenous degradation of the toxic RNP. This is similar to Proteolysis Targeting Chimeras (PROTACs), but uses RNA in place of a protein ligand to direct degradation to RNPs.

[0262] This strategy for rendering RNAs cell-permeable may be applied to any RNA or oligonucleotide therapeutic, including siRNAs, miRNAs and antisense oligos (ASOs).

[0263] The approach described herein can enable RNP-directed degradation (RNP- PROTACs). It is widely recognized that RNAs govern wide swaths of cellular biology; however, many of the key regulatory processes are funneled through RNPs. Thus, many of the true targets of RNA-driven biology are RNP complexes. The current method uses cell- permeable, synthetic RNA as a ligand to drive RNP -PROTACs. This approach has no competing technology and animates a unique therapeutic platform.

[0264] While RNPs figure prominently in many biological processes, they are particularly salient for neurodegenerative diseases. All studied cases of neurological plaques and prionlike diseases have revealed dysregulation of a protein bearing an RNA binding domain. Further, loss of RNA binding function in prion-like proteins results in loss of neurotoxicity, while protein fibrillization is retained. The most well-studied neurodegenerative disease, amyotrophic lateral sclerosis (ALS), also afflicts the most people. Much of the attention on ALS has focused on two subtypes, C9orf72 and Fus-driven ALS. In both these subtypes, a key RNA player has been identified, the C9 hexanucleotide repeat expansions (G4C2) and the ubiquitous UlsnRNA, which has been shown to bind mutant Fus. Short, synthetic RNA hairpins from these disease-centered RNAs could be equipped with URIL-tags and hybridized with PROTAC -bPNAs, thus enabling degradation of ALS-causative RNPs. Given the lack of effective therapies for ALS and neurodegenerative diseases in general, RNP -PROTACs has the potential to be a high impact therapeutic platform.

[0265] Background and Significance

[0266] Ligand-directed proteolysis: RNA-PROTACs. Proteolysis targeting chimeras (PROTACs) are degradation strategies in which a small-molecule inhibitor is coupled to a ligand for E3-ubiquitin ligase. It has been demonstrated that small-molecule protein binding directs ubiquitination of the target, marking the protein for proteolysis. Functionalization of a bPNA with E3 ligase ligands and triplex hybridization to RNA can direct selective proteolysis of RBPs docked proximal to stem replacement modified sites in an approach we call RNA-PROTACs. Interestingly, IncRNA HOTAIR can direct RBP proteolysis through scaffolding of E3 ligase and substrates Ataxin-1 and Snurportin-1, resulting in ubiqitination and proteolysis of substrates, underscoring the biomimetic aspect of our approach. Unlike PROTACs methods that require conjugation to a transport domain, bPNA has cellpenetrating properties.

[0267] Of particular interest as substrates are prion-like proteins causative in neurodegenerative disorders (eg-ALS, Alzheimer’s disease, Huntington’s disease), which have few therapeutic levers. Prior methods have focused on binding Tau protein 4 to drive ubiquitinylation; however, nearly all proteins that exhibit neurotoxic prion-like properties contain an RRM, and RNA-binding is essential for toxicity. Thus, an RNA-PROTACs approach enabled by bPNA is potentially a general platform for addressing the prion family of diseases. Importantly, the approach described herein allows for precise knockdown of the RNP interactome centered at a specific RNA secondary structural motif. No other method can offer this selective outcome, and thus this strategy could hold a therapeutic advantage.

[0268] Experimental Design

[0269] We have established a robust protocol for installation of intracellular URIL RNAs by transient transfection as well as stable expression via lentiviral transduction. These methods indicate a reliable intracellular URIL targeting with fluorescently labeled bPNA probes. Replacement of the fluorophore module with an E3-ligase ligand (Figures 2A-2B), such as a VHL or pomalidomide ligands, should direct proteasomal degradation to the protein partners in the targeted RNP (Figure 1). This methodology can be extended to a covalently captured URIL RNA that remains linked to the bPNA reagent.

[0270] Cellular Transport of Nucleic Acids

[0271] Our discovery that URIL tagging of synthetic RNAs in vitro with bPNA enables cellular transport of the complex imparts more efficient delivery than other strategies for RNA-directed PROTACs. Fluorescently tagged RNAs (Figures 3 and 4) are only cell permeant if bearing a URIL and bound to bPNA. This was established with RNAs with Alexa Fluor 488 at the 5’ terminus, and further equipped with a URIL. There was no detectable uptake into HEK-293T cells upon treatment with naked RNA, as judged by AF488 (green) fluorescence. When incubated with Cy5-modified bPNA, strong intracellular signals of green and red (Cy5) fluorescence were observed, supportive of transport of the complex. Treatment with Cy5-bPNA alone stained the cells uniformly with red fluorescence, as expected based on the known cell permeant properties of bPNA.

[0272] Summary

[0273] There are examples of RNA-directed degradation of RNA binding proteins in the literature. These may be directed by antisense oligos (ASOs) with phosphorothioate backbones equipped with E3 ligase ligands, delivered gymnotically. Gymnotic transport simply means nucleic acid delivery without a transfection agent, and this has been reported with synthetic backbones such as the PS and fluoroarabino sugar (F-ANA) backbones. While this property may be exploited to delivery PROTACs capable constructs into the cell, gymnotic transfer is both slow and inefficient, with the majority of material endosomally trapped. Additional conceptually related methods use aptamers bearing ligands, which similarly have transport issues. The technology disclosed herein has advantages in the generality of the RNA modification approach as well as the broad applicability to synthetic nucleic acid therapeutics. There are potential therapeutic avenues in the RNA PROTACs arena, as well as in the elevation of known RNA therapeutics through improved cell penetration.

[0274] References

[0275] (1) Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R. J. Protacs: Chimeric Molecules That Target Proteins to the Skpl-Cullin-F Box Complex for Ubiquitination and Degradation. Proc. Natl. Acad. Set. U. S. A. 2001, 98 (15), 8554- 8559.

[0276] (2) Toure, M.; Crews, C. M. Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew. Chem. Int. Ed. 2016 , 55 (6), 1966-1973.

[0277] (3) Yoon, J.-H.; Abdelmohsen, K.; Kim, J.; Yang, X.; Martindale, J. L.; Tominaga- Yamanaka, K.; White, E. J.; Oijalo, A. V.; Rinn, J. L.; Kreft, S. G.; Wilson, G. M.; Gorospe, M. Scaffold Function of Long Non-Coding RNA HOTAIR in Protein Ubiquitination. Nat. Commun. 2013, 4, 2939.

[0278] (4) Chu, T.-T.; Gao, N.; Li, Q.-Q.; Chen, P.-G.; Yang, X.-F.; Chen, Y.-X.; Zhao, Y.-F.; Li, Y.-M. Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin- Proteasome Degradation. Cell Chem Biol 2016, 23 (4), 453-461.

[0279] (5) King, O. D.; Gitler, A. D.; Shorter, J. The Tip of the Iceberg: RNA-Binding Proteins with Prion-like Domains in Neurodegenerative Disease. Brain Res. 2012, 1462, 61-80. (6) Wang, W.; He, S.; Dong, G.; Sheng, C. Nucleic-Acid-Based Targeted Degradation in Drug Discovery. J. Med. Chem. 2022, 65 (15), 10217-10232.

[0280] (7) Ghidini, A.; Clery, A.; Halloy, F.; Allain, F. H. T.; Hall, J. RNA-PROTACs: Degraders of RNA-Binding Proteins. Angew. Chem. Int. Ed Engl. 2021, 60 (6), 3163-3169. https: / / doi.org /

[0281] (8) Crooke, S. T.; Vickers, T. A.; Liang, X.-H. Phosphorothioate Modified Oligonucleotide-Protein Interactions. Nucleic Acids Res. 2020, 48 (10), 5235-5253.

[0282] (9) Souleimanian, N.; Deleavey, G. F.; Soifer, H.; Wang, S.; Tiemann, K.; Damha, M. J.; Stein, C. A. Antisense 2'-Deoxy, 2 '-Fluoroarabino Nucleic Acid (2'F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids. Molecular Therapy - Nucleic Acids 2012, 7, e43.

[0283] (10) Deprey, K.; Batistatou, N.; Kritzer, J. A. A Critical Analysis of Methods Used to Investigate the Cellular Uptake and Subcellular Localization of RNA Therapeutics. Nucleic Acids Res. 2020, 48 (14), 7623-7639.

[0284] Example 2. Synthetic Peptide Carriers Enable siRNA Deliver to Hepatocytes.

[0285] Summary

[0286] Small interfering RNA (siRNA) is one of several ways nucleic acid therapeutics affect gene silencing. In recent years, the U.S. Food and Drug Administration (FDA) has approved several siRNA-based therapeutics. However, there are still significant challenges. One of the most critical hurdles is effective delivery to the cytosol. Various delivery systems have been developed to achieve delivery. Example methods that have been explored fro siRNA delivery include lipid nanoparticles, viral vectors, aptamers, and cellpenetrating peptides. siRNA delivery and gene silencing by RNAi have attracted more interest recently, and the need for enhanced siRNA delivery platforms has propelled investigations to the forefront. See Figure 5. In this Example, we report a method for delivering nucleic acids, including siRNA, based on a hybridization strategy. Our preliminary studies target the expression of ApoB, the major protein component of low- density lipoprotein (LDL) produced in the liver and hepatocyte-derived cells. In this work, we applied the synthetic hybridization approach to siRNA targeting ApoB in a hepatocyte- derived cell line (HepG2). We found that siRNA carrier binding was sufficient to enable functional silencing delivery of uridylate-modified siRNA to HepG2 cells in culture with comparable performance to lipofectamine. Preliminary knockdown studies (RT-qPCR) show low nanomolar IC50 silencing of the endogenous ApoB gene. See Figure 7. Notably, this new synthetic carrier affords a striking improvement in siRNA transport into mammalian cell culture without covalent RNA modification or lipid particle formation. Overall, our initial results demonstrate a new nucleic acid delivery platform with unique advantages, for example, for siRNA therapeutics.

[0287] The initial knockdown studies show ApoB gene silencing with an IC50 in the nanomolar range. Without wishing to be bound by theory, we believe that better knockdown was observed with DNA-modified siRNA (T12 siRNA) due to improved binding of the bPNAs with the thymine.

[0288] Example 3: A Method to Increase the Cellular Permeability of Nucleic Acids Via Conjugation to Transporter Motifs

[0289] Strategies for increasing the cellular permeability of nucleic acids via covalent conjugation of transporter motifs (bPNA and STAs) are illustrated in this Example. Briefly, a transporter motif can be modified at the A-terminus with a chemical handle (Ri) which can be used to modify an oligonucleotide. The transporter motifs used are bifacial peptide nucleic acid (bPNA), which feature a peptide backbone bearing lysine residues that are doubly alkylated on the epsilon nitrogen with alkyl-melamine substituents (Figure 9). In addition, a family of aryl Substituted Tertiary Amines (STAs) can also be conjugated. These motifs also bear dialkylated lysine residues, but the epsilon nitrogen is modified with benzyl derivatives rather than the melamine ring. A range of variants with possible substituents is indicated (Figure 9). The linkage to oligonucleotide (Ri) is also shown (Figure 10). Using phosphoramidite chemistry, the transporter motifs can be incorporated into the terminal (5’) or internal sites in the sequence.

[0290] An example method for linkage based on commercially available materials is a triazole linkage formed by azide-alkyne cycloaddition (click chemistry), as shown (Figure 11). This method using phosphoramidate capping to install a cyclooctyne moiety for strain- driven copper-free click cyclization with an azide terminated peptide. An overall scheme to couple the transporter to the 3’ end of the sense strand of an siRNA duplex with enhanced stabilization chemistry (ESC) is shown.

[0291] Silencing of endogenous genes in hepatocytes. Using the strategy described above, we obtained siRNA-conjugates that were designed to silence ApoB, an endogenous protein expressed in hepatocytes. A small set of conjugates were studied initially by incubation with HepG2 cells over a concentration range of 2 orders of magnitude. We found that a simple conjugate with bPNA (4M) yielded a concentration dependent knockdown of ApoB with an estimated IC50 in the low nanomolar regime while other derivatives yielded significant but more modest effects (Figure 12). Notably, the underivatized siRNA is not known to elicit silencing without additional carriers and silencing in hepatocytes generally requires a livertargeting ligand (triantennerary GalNAc).

[0292] These initial data demonstrate the viability of the bPNA / STA conjugates as RNA carriers. The carrier structure is distinct from those previously reported and importantly shows efficacy with anionic backbones. This is an advantage over other conjugates such as the widely used arginine-based cell-penetrating peptides. The highly basic nature of arginine results in strong electrostatic complexation to anionic RNAs that inhibits function, limiting CPP conjugates of this type to neutral backbone ONTs (PNAs, PMOs); this consequently limits the applications accessible to only splice-switching. Notably, an FDA- approved ONT, SRP-5051, is a PMO conjugate with a CPP from Sarepta Therapeutics that was recently discontinued. There are clearly issues with this approach and the market would be receptive to new approaches that are potentially more generally applicable. If the bPNA / STA conjugate can be used for RNA delivery, then silencing applications (via siRNA or gapmer) are accessible, in addition to splice-switching.

[0293] References

[0294] (1) Cuenoud, B.; Casset, F.; Hiisken, D.; Natt, F.; Wolf, R. M.; Altmann, K.-H.; Martin, P.; Moser, H. E. Dual Recognition of Double-Stranded DNA by 2’ -Aminoethoxy -Modified Oligonucleotides. Angew. Chem. Int. Ed Engl. 1998, 37 (9), 1288-1291.

[0295] (2) Foster, D. J.; Brown, C. R.; Shaikh, S.; Trapp, C.; Schlegel, M. K.; Qian, K.; Sehgal, A.; Rajeev, K. G.; Jadhav, V.; Manoharan, M.; Kuchimanchi, S.; Maier, M. A.; Milstein, S. Advanced siRNA Designs Further Improve in Vivo Performance of GalNAc-siRNA Conjugates. Mol. Ther. 2018, 26 (3), 708-717.

[0296] The compounds, compositions, and methods of the appended claims are not limited in scope by the specific compounds, compositions, and methods described herein, which are intended as illustrations of a few aspects of the claims. Any compounds, compositions, and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compounds, compositions, and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of delivering a nucleic acid to a cell, the method comprising: contacting the nucleic acid with a bifacial Peptide Nucleic Acid (bPNA) comprising a triplex hybrid forming moiety, wherein the bPNA binds to a non-canonical base pairing site in the nucleic acid via triplex hybridization, forming a bPNA-nucleic acid complex; and contacting the cell with the bPNA-nucleic acid complex.

2. The method of claim 1, wherein the nucleic acid comprises RNA.

3. The method of any of claims 1-2, wherein the non-canonical base pairing site comprises a U-rich internal loop (URIL).

4. The method of claim 3, wherein the URIL is a loop that includes at least four non- canonical base pairs, wherein at least 50% of the non-canonical base pairs comprise U-U pairs.

5. The method of any one of claims 3-4, wherein the URIL comprises from 4-8 non- canonical base pairs.

6. The method of any one of claims 1-5, wherein the bPNA is defined by Formula I belowFormula I whereinA is absent, or represents a prosthetic group;X is absent, or represents a first bivalent linking group;L is absent, or represents a second bivalent linking group; n is, individually for each occurrence, an integer selected from 1 and 2; m is an integer selected from 2, 3, and 4;Z represents, individually for each occurrence, a binding motif selected from one of the followingQ1and Q2individually represent -O- or -NRA-;Y is N, -CRB-,R1is, individually for each occurrence, an amino acid sidechain, such as one of the followingR2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; andRBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

7. The method of claim 6, wherein, wherein RArepresents H for each occurrence.

8. The method of any one of claims 6-7, wherein R2represents H for each occurrence.

9. The method of any one of claims 6-8, wherein Q1and Q2represent -O- for each occurrence.

10. The method of any one of claims 6-8, wherein Q1and Q2represent -NH- for each occurrence.

11. The method of any one of claims 6-10, wherein R1is, individually for each occurrence, selected from one of the following12. The method of any one of claims 6-11, wherein R1is, individually for each occurrence, -H or -CH3.

13. The method of any one of claims 6-12, wherein m is 2.

14. The method of any one of claims 6-12, wherein m is 3.

15. The method of any one of claims 6-14, wherein n is 1 in all occurrences.

16. The method of any one of claims 6-14, wherein m is 3 and n is 1 in two occurrences and n is 2 in one occurrence.

17. The method of any one of claims 6-16, wherein X represents a first bivalent linking group.

18. The method of any one of claims 6-17, wherein the first bivalent linking group comprises from 3 to 20 atoms, such as from 3 to 16 atoms or from 3 to 12 atoms.

19. The method of any one of claims 6-18, wherein the first bivalent linking group comprises an alkylene linker or a heteroalkylene linker.

20. The method of any one of claims 6-19, wherein L represents a second bivalent linking group.

21. The method of any one of claims 6-20, wherein the second linking group comprises from 3 to 36 atoms, such as from 3 to 24 atoms or from 3 to 16 atoms.

22. The method of any one of claims 6-21, wherein the second bivalent linking group comprises an alkylene linker or a heteroalkylene linker.

23. The method of any one of claims 6-22, wherein in at least one occurrence, Z represents the binding motif shown belowwhereinQ1and Q2individually represent -O- or -NRA-;Y is N, -CRB-,R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; andRBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

24. The method of any one of claims 6-23, wherein m is 3 and Z represents the binding motif shown below in two occurenceswhereinQ1and Q2individually represent -O- or -NRA-;Y is N, -CRB-,R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;RArepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; andRBrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

25. The method of any one of claims 6-24, wherein the compound is defined byFormula I A belowFormula I A wherein A , L, Y, Q1, Q2, X, n, and m are as defined above26. The method of any one of claims 6-25, wherein the compound is defined byFormula IBFormula IB whereinA, L, R1, m, and n are as defined above; and a is, individually for each occurrence, an integer selected from 3, 4, 5, and 6.

27. The method of any one of claims 6-26, wherein A is absent.

28. The method of any one of claims 6-26, wherein the prosthetic group is not cyanine 5 (Cy5), cyanine 3 (Cy3), or carboxyfluorescein (Cbf).

29. The method of any one of claims 6-26, wherein the prosthetic group is selected from the group consisting of a fluorogenic dye, a protein ligand, a redox-active center, an ROS- generating center, a photoreactive center, a spin label, a group transfer agent, a therapeutic agent, a catalytic center, a click motif, or an NMR active label.

30. The method of claim 29, wherein the prosthetic group is a fluorogenic dye.

31. The method of claim 30, wherein the fluorogenic dye is selected from the group consisting of thiazole derived dyes such as thiazole orange, dimethylindole red and derivatives thereof, symmetric cyanine dyes, asymmetric cyanine dyes, fluoresceins, rhodamines, fluorogenic variants of asymmetric cyanine dyes and fluoresceins such as JF635 and JF646, arsenate dyes such as F1ASH and ReASH, malachite green and derivatives thereof, courmarin dyes, and hydroxybenzylidene dyes.

32. The method of claim 31, wherein the fluorogenic dye is thiazole orange.

33. The method of claim 29, wherein the prothetic group is a protein ligand.

34. The method of claim 33, wherein the protein ligand is selected from the group consisting of a ubiquitin ligase ligand, a ligand for a translational activator, a ligand for a translational inhibitor, a ligand for a transcription activator, a ligand for a transcription inhibitor, a ligand for a nuclease, or a ligand for a cell surface protein.

35. The method of claim 34, wherein the protein ligand is a ubiquitin ligase ligand that binds to an E3 ligase selected from the group consisting of XIAP, VHL, cereblon, and MDM2.

36. The method of any one of claims 1-35, wherein the nucleic acid comprises an oligonucleotide therapeutic.

37. The method of any one of claims 1-36, wherein the nucleic acid comprises an siRNA, an miRNAs, or an antisense oligo (ASO).

38. A method for selectively degrading a target ribonucleoprotein complex in a cell, the method comprising contacting a nucleic acid that mimics a dysregulated intracellular RNA present in the target ribonucleoprotein complex with a bifacial peptide nucleic acid (bPNA) comprising a triplex hybrid forming moiety conjugated to a ubiquitin ligase ligand to form a nucleic acid-bPNA complex, wherein the bifacial peptide nucleic acid binds to a non-canonical base pairing site in the nucleic acid via triplex hybridization; and contacting the cell with the nucleic acid-complex.

39. A method of delivering a nucleic acid to a cell, the method comprising: covalently linking a transporter motif to the nucleic acid to form a transporter motif- nucleic acid conjugate; and contacting the cell with the transporter motif-nucleic acid conjugate; wherein the transporter motif is defined by Formula II belowFormula II whereinR2represents H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;Li represents, individually for each occurrence, a first bivalent linking group of from one to six atoms in length;L2 represents a second bivalent linking group; andE represents, individually for each occurrence, a 5- to 10-membered carbocyclic ring or a 5- to 10-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

40. The method of claim 39, wherein the transporter motif is defined by Formula IIA belowFormula II A wherein E, Li, and R2are as defined above with respect to Formula II,L3 is absent, or represents a third bivalent linking group;X is absent, or represents a fourth bivalent linking group;R1is, individually for each occurrence, an amino acid sidechain, such as one of the followingn is, individually for each occurrence, an integer selected from 1 and 2; and m is an integer selected from 2, 3, and 4.

41. The method of claim 39 or 40, wherein Li is, individually for each occurrence, a bivalent linking group of from one to three atoms in length.

42. The method of any one of claims 39-41, wherein Li is, individually for each occurrence, an alkylene linker or -NH-CH2-CH2-.

43. The method of any one of claims 39-42, wherein E represents, individually for each occurrence, a 6-membered carbocyclic ring or a 6-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

44. The method of any one of claims 39-43, wherein E represents, individually for each occurrence, a 6-membered aryl ring or a 6-membered heteroaryl ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

45. The method of any one of claims 39-44, wherein E represents a phenyl ring or a napthyl ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

46. The method of any one of claims 39-44, wherein E represents a triazine ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

47. The method of any one of claims 39-44, wherein E represents a 6-membered N- containing heterocyclic ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

48. The method of claim 47, wherein the 6-membered N-containing heterocyclic ring comprises a piperidine ring, a piperazine ring, a pyridine ring, a triazine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, an indole ring, a quinoline ring, or a purine ring.

49. The method of any one of claims 39-48, wherein covalently linking the transporter motif to the nucleic acid comprises performing a click chemistry reaction between a transporter motif molecule bearing a first click motif and a nucleic acid bearing a second click motif.

50. The method of any one of claims 39-48, wherein covalently linking the transporter motif to the nucleic acid comprises performing solid phase nucleic acid synthesis using a transporter motif molecule bearing a phosphorami di te moiety.

51. A transporter motif molecule defined by Formula III belowFormula III whereinR2represents H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;Li represents, individually for each occurrence, a first bivalent linking group of from one to six atoms in length;L2 represents a second bivalent linking group;E represents, individually for each occurrence, a 5- to 10-membered carbocyclic ring or a 5- to 10-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group; andG represents a reactive functional group.

52. The transporter motif molecule of claim 51, wherein L2 comprises an oligopeptide linker or a pseudopeptide linker.

53. The transporter motif molecule of claim 51 or 52, wherein the transporter motif is defined by Formula IIIA belowFormula II A wherein E, Li, G, and R2are as defined above with respect to Formula II,L3 is absent, or represents a third bivalent linking group;X is absent, or represents a fourth bivalent linking group;R1is, individually for each occurrence, an amino acid sidechain, such as one of the followingn is, individually for each occurrence, an integer selected from 1 and 2; and m is an integer selected from 2, 3, and 4.

54. The transporter motif molecule of any one of claims 51-53, wherein Li is, individually for each occurrence, a bivalent linking group of from one to three atoms in length.

55. The transporter motif molecule of any one of claims 51-54, wherein Li is, individually for each occurrence, an alkylene linker or -NH-CH2-CH2-.

56. The transporter motif molecule of any one of claims 51-55, wherein E represents, individually for each occurrence, a 6-membered carbocyclic ring or a 6-membered heterocyclic ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, carbonyl, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

57. The transporter motif molecule of any one of claims 51-56, wherein E represents, individually for each occurrence, a 6-membered aryl ring or a 6-membered heteroaryl ring, each optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

58. The transporter motif molecule of any one of claims 51-57, wherein E represents a phenyl ring or a napthyl ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

59. The transporter motif molecule of any one of claims 51-57, wherein E represents a triazine ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

60. The transporter motif molecule of any one of claims 51-57, wherein E represents a 6-membered N-containing heterocyclic ring optionally substituted with from 1 to 4 substituents individually selected from halogen, nitro, cyano, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, an amino group, a C1-C4 alkylamino group, and a C1-C4 dialkylamino group.

61. The transporter motif molecule of claim 60, wherein the 6-membered N-containing heterocyclic ring comprises a piperidine ring, a piperazine ring, a pyridine ring, a triazine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, an indole ring, a quinoline ring, or a purine ring.

62. The transporter motif molecule of any one of claims 51-61, wherein G comprises a click motif.

63. The transporter motif molecule of any one of claims 51-61, wherein G comprises a phosphoramidite moiety.

64. A method for selectively degrading a target ribonucleoprotein complex in a cell, the method comprising contacting a nucleic acid that mimics a dysregulated intracellular RNA present in the target ribonucleoprotein complex with a transporter motif-nucleic acid conjugate comprising the nucleic acid that mimics the dysregulated intracellular RNA covalently bound to a transporter motif and a ubiquitin ligase ligand; and contacting the cell with the transporter motif-nucleic acid conjugate.

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