Bivalent nucleic acid ligands and their uses

Abstract

Gene recognition reagents comprising a divalent nucleobase are provided that bind to both strands of a nucleic acid, such as an expanded repeat sequence associated with an expanded repeat disease. In one example, the expanded repeat disease is a polyQ disease, such as Huntington's disease. Methods of using the reagents are provided, including methods of binding to nucleic acids, such as an expanded repeat sequence associated with an expanded repeat disease, methods of identifying the presence of a nucleic acid comprising an expanded repeat, and methods of treating an expanded repeat disease.

Description

[0001] Statement Regarding Federal Funding

[0002] This invention was made with government support under Grant R21NS098102 awarded by the National Institutes of Health and Grant CHE1039870 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of co-pending U.S. Provisional Application No. 62 / 708,783, filed December 21, 2017, and co-pending U.S. Provisional Application No. 62 / 710,262, filed February 14, 2018, each of which is incorporated herein in its entirety.

[0005] The sequence listing associated with this application is submitted in electronic format via EFS-Web and is hereby incorporated by reference into this specification in its entirety. The name of the text file containing the sequence listing is 6526_1807597_ST25.txt. The size of the text file is 444 bytes and the text file was created on December 21, 2018. Background Art

[0006] 1. Field of the Invention

[0007] Compositions for binding nucleic acids using nucleic acids and nucleic acid oligomer compositions are described herein. Also provided is a method of treating an expanded repeat disease, for example, a PolyQ disease such as Huntington's disease.

[0008] 2. Description of Related Technology

[0009] RNA repeat expansions are prevalent in neuromuscular diseases (or disorders). One such example is Huntington's disease (HD), an autosomal dominant disorder that affects muscle coordination and causes behavioral changes, cognitive decline, and dementia. HD typically presents in middle age, with death occurring 10 to 20 years after onset. It is caused by an expansion of the CAG repeat sequence in the first exon of the huntingtin (htt) gene from a normal range of 6-29 to a pathogenic range of 40-180. The length of the CAG repeat expansion (CAG exp) is inversely correlated with age of onset. Htt encodes a 348 kDa protein that is ubiquitously expressed but most predominantly in neurons of the CNS with multiple physiological roles, including embryonic development and neuroprotection. The CAG repeat sequence is translated into a polyglutamine (polyQ) sequence in the N-terminal region of Htt. Despite a broad understanding of the molecular dysfunction and clinical manifestations, the exact mechanism by which CAGexp causes HD is not fully understood. However, increasing evidence suggests that this is a multivariate disease.

[0010] Loss of protein function may lead to HD, but due to CAG exp Both heterozygous and homozygous patients have similar clinical features, making it unlikely to be the primary cause. Furthermore, individuals have been identified who do not display an abnormal phenotype despite a 50% reduction in normal Htt protein levels due to the deletion of one of the htt alleles. Likewise, mice heterozygous for the httnull mutation do not display clinical features of HD, and homozygotes die in early embryogenesis. These findings highlight the importance of Htt in embryonic development but not in the pathogenesis of HD. Accumulating evidence suggests that a deleterious gain of function is a more plausible cause of the disease. This speculation is supported by the observation that CAG repeats above a similar threshold (30-40 units) in unrelated genes can also cause a variety of neuromuscular diseases besides HD, including DRPLA (gingival-tarsal muscular atrophy), SBMA (spinal and bulbar) muscular atrophy), SCA1 (spinocerebellar ataxia type 1), SCA2 (spinocerebellar ataxia type 2), SCA3 (spinocerebellar ataxia type 3 or Machado-Joseph disease), SCA6 (spinocerebellar ataxia type 6), SCA7 (spinocerebellar ataxia type 7), SCA17 (spinocerebellar ataxia type 17). These diseases, known as polyQ diseases, share many features, including subcortical and cortical atrophy, and nuclear aggregates common to HD.

[0011] Three major cellular toxicity mechanisms have been proposed for HD and other polyQ diseases. The first is polyQ toxicity. PolyQ has a tendency to aggregate and interact with other polyQ-containing proteins to form large amyloid structures. Binding and sequestration of these key proteins will lead to loss of their physiological function, resulting in a dysregulated cascade of molecular and cellular events. The second is a toxic gain of RNA function. Posttranscriptionally, rCAG exp The imperfect hairpin structure is adopted, which sequesters muscleblind-like protein 1 (MBNL1), another RNA splicing regulator, and other key proteins. Although the normal rCAG repeat can also adopt the hairpin motif, it does so in a different sequence context than the expanded version. expAssociation with MBNL1 results in the trapping of the complex in the nucleus as nuclear foci, preventing its export to the cytoplasm to produce Htt protein. The gene transcripts that are mis-spliced ​​due to the loss of MBNL1 are diverse. The third pathogenic mechanism is protein toxicity. It has been shown that in the absence of an ATG start codon, rCAG repeat sequences exceeding a certain length (>42 units) can be translated by repeat-associated non-ATG (RAN) translation, resulting in the production of toxic polyQ and polyalanine (poly A) proteins, as well as polyserine (polyS). The latter two mechanisms are further supported by the following findings: in animal models, the expression of long stretches of untranslated rCAG repeats is harmful, and its behavioral phenotype is similar to HD.

[0012] Collectively, these findings suggest that HD is a multivariate disease caused by toxic increases in RNA and protein function. Thus, one possible strategy for treating HD is to selectively target expanded transcripts, as this would interfere with all three disease pathways. This paper describes bivalent nucleic acid ligands that recognize rCAG repeats and other RNA repeat sequences. Summary of the invention

[0013] In one aspect, a gene recognition reagent is provided. The reagent comprises a nucleic acid or nucleic acid analog backbone, the nucleic acid or nucleic acid analog backbone comprising three or more ribose, deoxyribose or nucleic acid analog backbone residues, a divalent nucleobase sequence connected to the backbone residues, wherein the divalent nucleobase sequence binds to an expanded repeat unit target sequence of a repeat expansion disease on two nucleic acid chains or one or more sequential iterations of the unit target sequence. In another aspect, a composition comprising a gene recognition reagent and a pharmaceutically acceptable carrier is provided.

[0014] In another aspect, a method of binding a nucleic acid comprising an expanded repeat associated with an expanded repeat disease is also provided. The method comprises contacting the nucleic acid comprising the expanded repeat sequence with a gene recognition reagent comprising a nucleic acid or nucleic acid analog backbone, the nucleic acid or nucleic acid analog backbone comprising three or more ribose, deoxyribose, or nucleic acid analog backbone residues, a divalent nucleobase sequence linked to the backbone residues, wherein the sequence of divalent nucleobases binds to a unit target sequence or one or more consecutive iterations of a unit target sequence of an expanded repeat associated with a repeat expansion disease on both nucleic acid strands.

[0015] In another aspect, a method of identifying the presence of a nucleic acid comprising an expanded repeat associated with an expanded repeat disease in a sample obtained from a patient is provided. The method comprises contacting a nucleic acid sample obtained from a patient with a gene recognition reagent comprising a nucleic acid or nucleic acid analog backbone, the nucleic acid or nucleic acid analog backbone comprising three or more ribose, deoxyribose or nucleic acid analog backbone residues, a divalent nucleobase sequence attached to the backbone residues, wherein the sequence of divalent nucleobases binds to one or more consecutive iterations of a unit target sequence or unit repeat sequence of an expanded repeat of an expanded repeat disease on both nucleic acid strands, and determining whether binding and attachment of the gene recognition reagent occurs, which indicates that the sample contains a nucleic acid comprising an expanded repeat associated with an expanded repeat disease, and optionally treating the patient for the expanded repeat disease. The nucleic acid or nucleic acid analog backbone comprises a first end and a second end, and further comprises a first linking group attached to the first end of the backbone, a second linking group attached to the second end, which is non-covalently bound to the first linking group or self-bound.

[0016] In another embodiment, a method of knocking down the expression of a gene containing an expanded repeat associated with an expanded repeat disease in a cell is provided. The method comprises contacting a nucleic acid (e.g., RNA) containing an expanded repeat sequence with a gene recognition reagent comprising a divalent nucleobase sequence containing three or more ribose, deoxyribose, or nucleic acid analog backbone residues attached to the backbone residues, wherein the sequence of divalent nucleobases binds to a unit target sequence or one or more consecutive iterations of a unit target sequence of an expanded repeat of a repeat expansion disease on both nucleic acid strands. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 (A)( Figure 1 , Figure (A)) for targeting rCAG exp - Design of the JB-MPγPNA ligands LG1, LG2, and LG3 in a hairpin structure. K: L-lysine, arrow: N-terminus. (B) Representative binding mode of LG2 in the preferred antiparallel orientation (N-terminus facing the 3'-end of the Watson strand). (C) H-bond interactions of E, I, and F with the corresponding CG, AA, and GC base pairs of RNA. (D) Chemical structure of the MPγPNA backbone to which the J base is covalently attached. (E) Hypothesis of γPNAs and γPNA-DNA duplexes and PNA [as PNA-DNA duplexes] determined by NMR and X-ray.

[0019] Figure 2 . Figures AF provide exemplary structures of nucleic acid analogs.

[0020] Figure 3 Various examples of amino acid side chains are described.

[0021] Figure 4 The structures of exemplary nucleobases are provided.

[0022] Figures 5A-5C Exemplary divalent nucleobase structures are provided, wherein R refers to a nucleotide or nucleotide analog backbone monomer or residue.

[0023] Figure 6 MD simulation results of LG1 binding to RNA duplex containing the sequence r(CAG)4 / r(CAG)4. LG1: NH2-EIF-H, where the γ-side chain is a methyl group. (A) Surface representation of the binding complex with four individual LG1 ligands and RNA duplex containing four CAG repeats before (t = 0) and after (t = 100 ns) simulation. (B) Hydrogen bond interactions of CEG, AJA, and GFC triplets after simulation. (C) Number of H bonds per triplet between the RNA base and the J base of LG1, (D) radius of gyration of the LG1-RNA complex, and (E) root mean square deviation (RMSD) of the LG1-RNA complex relative to the initial structure of the RNA complex.

[0024] Figure 7 The structures of E, I and FJB-MPγPNA monomers are shown.

[0025] Figure 8 and 9 The synthetic schemes of E(15) and F(26) are shown, respectively.

[0026] Fig.10 A scheme for coupling E (15), F (26) and I (27) to the MPyPNA backbone is shown.

[0027] Fig.11 A model RNA target (SEQ ID NO: 1) selected for binding studies described in the Examples is shown.

[0028] Fig.12 LG2 CD titration with R11A. The concentration of R11A was 0.5 μM, while the concentration of LG2 was X: 0, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 8.5, and 11.5 μM. After adding LG2 to R11A, the mixture was incubated at 37°C for 30 min, and then data were collected at 25°C.

[0029] Fig.13CD spectra of LG2 with (A) mismatched R11U, (B) single-stranded WS and CS and (C) double-stranded HP containing a single binding site and (D) CD spectra of LG2P with R11A. CD spectra of RNA (solid line) and [RNA+ligand] mixture (dashed line).

[0030] Fig.14 Effects of ligand orientation (LG2P), mismatched R11U, and single-stranded target (WS+CS). Inset: Fluorescence spectra of the corresponding samples after addition of 2.5 μM pentamidine.

[0031] Fig.15 LG2 NMR titration with R11A. The concentration of R11A was 0.1 mM. Aliquots of LG2 at the indicated molar ratios were added to R11A and incubated at 37°C for 15 min prior to data acquisition. Solvent exchangeable protons are represented by dashed lines and non-exchangeable protons by vertical solid lines.

[0032] Fig.16 Native chemical ligation is schematically shown, as described in the Examples.

[0033] Fig.17 Reaction progress of parent compound M after addition of 4MP at 37°C. (A) Overview of the reaction sequence. Initial formation of M# and M## intermediates, followed by conversion to reactive LG2N* intermediates, and finally to the cyclic product cLG2N. (B) MALDI-TOF MS spectra of M after addition of 4MP and incubation at 37°C for 0, 15, and 60 min. Samples were prepared in 0.1X PBS buffer.

[0034] Fig.18 .MALDI-TOF spectra of (A) [LG2N+R11A], (B) [LG2N+R11U] and (C) LG2N, incubated with 2ME at 37°C for 16 hours. Prior to MS analysis, the samples were reconstituted with 4 mM guanidine chloride and heat denatured at 95°C for 5 minutes. The concentrations of LG2N, R11A, R11U and 2ME were 22, 1, 1 and 500 μM, respectively. In positive mode, no RNA molecules were observed in the MALDI-TOF spectra. The Y axes in (B) and (C) are the same as in (A); they are omitted to save space.

[0035] Fig.19Competitive binding assay. The concentrations of R24A, R127A and R96U were 100, 18 and 22 nM, each containing 1.1 μM binding sites; LG2N in lanes 2-6 and 8 were 11, 22, 44, 88, 88 and 88 μM; 4MP and TCEP were 500 and 100 μM, respectively (arranged in that order). The ratio of binding sites to ligands is as shown. Samples were prepared in 0.1X PBS buffer and incubated at 37°C for 24 h, then separated on 2% agarose gel and stained with SYBR-Gold.

[0036] Fig. 20 Gel shift assay at physiologically simulated ionic strength. Fig. 9 The samples were prepared in the same manner as shown in . The concentrations of R24A, R127A and R96U were 100, 18 and 22 nM, each containing 1.1 μM binding sites; LG2N in lanes 2-6 and 8 were 11, 22, 44, 88, 88 and 88 μM, respectively; 4MP and TCEP were 500 and 100 μM, respectively (arranged in that order). The ratio of binding sites to ligands is shown. The samples were incubated at 37°C for 24 hours and then separated on 2% agarose gel and stained with SYBR-Gold.

[0037] Fig.21 . A general method for synthesizing the ligands described herein is shown. DETAILED DESCRIPTION OF THE INVENTION

[0039] Unless otherwise expressly stated, the use of numerical values ​​within the various ranges specified in this application is expressed as approximate values, as if the minimum and maximum values ​​within the range were both preceded by the word "about". In this way, slight variations above and below the range can be used to obtain substantially the same results as the values ​​within the range. In addition, unless otherwise stated, the disclosure of the range is intended to include a continuous range of every value between the minimum and maximum values. As used herein, "a" and "an" refer to one or more.

[0040] As used herein, the term "comprising" is open ended and may be synonymous with "including," "comprising," or "characterized by." The term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The term "consisting of" excludes any elements, steps, or ingredients not specified in the claim. As used herein, embodiments "comprising" one or more of the stated elements or steps also include, but are not limited to, embodiments "consisting essentially of" and "consisting of" these elements or steps.

[0041] Provided herein are compositions and methods for binding to a target sequence in a nucleic acid, for example, for binding to a repeat expansion associated with a disease involving repeat expansions in a nucleic acid sequence. Figure 1 is a schematic diagram illustrating the binding of non-limiting examples of recognition agents described herein (gene recognition agents, ligands) to CAG repeats in RNA hairpin structures seen in HD and other PolyQ diseases. Terminal self-ligating groups or π-stacked aromatic groups, such as the exemplary self-ligating groups described in the Examples below, and as in U.S. Patent Application Publication No. 2016 / 0083433, which is incorporated herein by reference. Self-ligating groups, pi-stacking groups, and similar groups on a nucleic acid chain or between two nucleic acid chains (if containing divalent) are collectively referred to herein as "concatenating groups". A variety of recognition agents are bound (hybridized) to a template nucleic acid through Watson-Crick or Watson-Crick-like cooperative base pairs. In cells, the template nucleic acid is an RNA or DNA molecule, although in vitro, the template nucleic acid can be any RNA or DNA, as well as modified nucleic acids or nucleic acid analogs. Recognition agents that are in close enough proximity (eg, bound to adjacent sequences on the template nucleic acid) will self-assemble or π-π stack, thereby linking to form substantially longer oligomers or polymers. Further details are provided below.

[0042] As used herein, a "patient" is an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, such as a monkey and a chimpanzee), a non-primate (e.g., a cow, a pig, a camel, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose).

[0043] As used herein, the term "treat" or "therapy" refers to a beneficial or desired result, such as improvement of one or more symptoms of a disease, and the term "treat" or "therapy" also includes, but is not limited to, alleviation or improvement of one or more symptoms of a repeat expansion disease, such as a PolyQ repeat expansion disease, such as Huntington's disease. "Treatment" can also mean prolonging survival in the absence of treatment.

[0044] In the context of disease markers or symptoms, "reduction" refers to a clinically relevant and / or statistically significant reduction in the level. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, to a level within the normal range acceptable to individuals without the disease, or below the assay level detected. In some aspects, the reduction is reduced to a level within the normal range accepted for individuals without the disease, which can also be referred to as normalization of the level. In some aspects, reduction is normalization of the level of signs or symptoms of the disease, a reduction in the difference between the tested level of the disease sign and the normal level of the disease sign (e.g., when the value of the subject must be reduced to reach the normal value, a higher normal level is reached, and when the value of the subject must be increased to the normal value, a lower normal level is reached). In some aspects, the method includes clinically relevant inhibition of mRNA expression of a PolyQ repeat expansion disease such as Huntington's disease, for example, as demonstrated by clinically relevant results after treating the subject with a recognition agent described in the present invention.

[0045] As used herein, a "therapeutically effective amount" is intended to include an amount of an identification agent as described herein that, when administered to a subject with a disease, is sufficient to achieve treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on: the identification agent (agent), the mode of administration, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of previous treatment or concomitant treatment (if any), and other individual characteristics of the subject to be treated. In the case of the gene identification agents described herein, an exemplary dosage range is 0.1 pg to 1 mg per dose.

[0046] "Therapeutically effective amount" also includes an amount of an agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The identified agents employed in the methods described herein can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0047] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, talc, magnesium, calcium or zinc stearate or stearic acid) or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium sulfate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; (10) glycols such as propylene glycol; (11) polyols such as propylene glycol; Alcohols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL and (22) other non-toxic compatible substances used in pharmaceutical preparations.

[0048] As used herein, the term "cell" refers to any type of cell from any animal, such as, but not limited to, rats, mice, monkeys, and humans. For example, but not limited to, the cell can be a progenitor cell, such as a stem cell, or a differentiated cell, such as an endothelial cell, a smooth muscle cell.

[0049] "Expression" or "gene expression" refers to the overall information flow from a gene (without limitation, a functional genetic unit such as RNA or protein for producing a gene product in a cell, or other expression system encoded on a nucleic acid, which comprises: a transcriptional promoter and other cis-acting elements, such as response elements and / or enhancers; an expression sequence that usually encodes a protein (open reading frame or ORF) or a functional / structural RNA, and a polyadenylation sequence to produce a gene product (usually a protein, optionally with post-translational modifications or a functional / structural RNA). "Expression of a gene under the transcriptional control of a specified sequence" or "under the control of a specified sequence" refers to a sequence that contains a sequence operably linked (usually functionally linked in cis) to the specified sequence. The gene expression of the gene of the sequence. The specified sequence can be all or part of a transcription element (but not limited to a promoter, an enhancer and a response element), and can regulate and / or affect the transcription of the gene in whole or in part. "Gene for expressing the gene product" is a gene that can express the gene product when placed in a suitable environment, for example, when transformed, transfected, transduced, etc. into a cell and subjected to cellular expression, and under suitable expression conditions. In the case of a constitutive promoter, "suitable conditions" refers to generally only needing to introduce the gene into a host cell. In the case of an inducible promoter, "suitable conditions" refers to applying a certain amount of a corresponding inducer to an expression system (such as a cell) that effectively causes gene expression.

[0050] As used herein, the term "knock down" refers to that the expression of one or more genes in an organism is generally significantly reduced relative to a functional gene, for example relative to a therapeutically effective degree. Gene knockdown also includes complete gene silencing. As used herein, "gene silencing" refers to substantially completely preventing the expression of a gene. Knockdown and gene silencing may occur at the transcription stage or the translation stage. By knocking down or silencing one or more genes at the translation stage, RNA (e.g., mRNA) in the targeting cell using the recognition reagent can modify gene expression.

[0051] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid analogs include, for example but not limited to: 2'-O-methyl substituted RNA, locked nucleic acid, unlocked nucleic acid, triazole-linked DNA, peptide nucleic acid, morpholino oligomers, dideoxynucleotide oligomers, diol nucleic acid, threose nucleic acid acid and combinations thereof, including optional ribonucleotides or deoxyribonucleotide residues. In this article, with respect to nucleic acids and nucleic acid analogs, "nucleic acid" and "oligonucleotide" are short single-stranded structures composed of epitope nucleotides, which can be used interchangeably. Oligonucleotides can be referred to by the length of the chain (i.e., the number of nucleotides) by the nomenclature "polymer (-mer)". For example, an oligonucleotide with 22 nucleotides will be referred to as a 22 polymer.

[0052] "Nucleic acid analogs" are compositions comprising a sequence of nucleobases arranged on a substrate such as a polymer backbone, and can bind to DNA and / or RNA by Watson-Crick hybridization or Watson-Crick-like hydrogen bond base pairing. Non-limiting examples of common nucleic acid analogs include peptide nucleic acids, such as γPNA, morpholino nucleic acids, phosphorothioates, locked nucleic acids (2'-O-4'-C-methylene bridges, including oxy, thio or amino versions thereof), unlocked nucleic acids (C2'-C3' bonds are cleaved), 2'-O-methyl substituted RNA, threose nucleic acids, ethylene glycol nucleic acids, etc.

[0053] Conformationally pre-organized nucleic acid analogs are nucleic acid analogs that have a backbone (pre-organized backbone) that forms only right-handed or left-handed helices, depending on the structure of the nucleic acid backbone. As shown herein, an example of a conformationally pre-organized nucleic acid analog is γPNA, which has a chiral center on the γ carbon and, depending on and due to the chirality of the group on the γ carbon, only forms a right-handed or left-handed helix. Similarly, locked nucleic acids contain a ribose with a bridge between the 2' oxygen and the 4' carbon, which "locks" the ribose into a 3'-endo (north) conformation.

[0054] In the context of the present disclosure, "nucleotide" refers to a monomer comprising at least one nucleobase and a backbone element (main chain portion), which is ribose or deoxyribose in nucleic acids such as RNA or DNA. "Nucleotide" also generally includes a reactive group that allows polymerization under specific conditions. In natural DNA and RNA, these reactive groups are 5' phosphate and 3' hydroxyl. In order to chemically synthesize nucleic acids and analogs thereof, bases and backbone monomers may include modified groups, such as blocked amines known in the art. "Nucleotide residue" refers to a single nucleotide incorporated into an oligonucleotide or polynucleotide. Similarly, "nucleobase residue" refers to a nucleobase incorporated into a nucleotide or nucleic acid or its analog. "Gene recognition reagent" generally refers to a nucleic acid or nucleic acid analog comprising a nucleobase sequence, which can hybridize to a nucleic acid analog sequence on a complementary nucleic acid or nucleic acid by cooperative base pairing, such as Watson-Crick base pairing or Watson-Crick-like base pairing.

[0055] In more detail, the nucleotides used for RNA, DNA or nucleic acid analogs have a structure AB, wherein A is a main chain monomer portion, and B is a nucleobase as described herein. The main chain monomer can be any suitable nucleic acid main chain monomer, such as ribose triphosphate or deoxyribose triphosphate, or a monomer of a nucleic acid analog, such as a peptide nucleic acid (PNA), such as γPNA (γPNA). In one example, the main chain monomer is a ribose monophosphate, diphosphate or triphosphate ribose or a deoxyribose monophosphate, diphosphate or triphosphate ribose, such as 5' monophosphate, diphosphate or triphosphate of ribose or deoxyribose. The main chain monomer includes both structural "residue" components, such as ribose in RNA, and any active groups modified when the monomers are connected together, such as 5' triphosphate and 3' hydroxyl of ribonucleotides, which are polymerized into RNA when modified, leaving a phosphodiester bond. Similarly for PNA, the C-terminal carboxyl and N-terminal amine active groups of the N-(2-aminoethyl)glycine main chain monomer condense during polymerization to leave a peptide (amide) bond. On the other hand, the active group is a phosphoramidite group that can be used for the synthesis of phosphoramidite oligomers, as widely known in the art. The nucleotides also optionally contain one or more protecting groups known in the art, such as 4,4'-dimethoxytrityl (DMT), and as described herein. Many other methods for preparing synthetic gene recognition reagents are known and depend on the specific chemical process of the backbone structure and base addition process. Determining which active groups are utilized in the connection of nucleotide monomers and which groups are protected in the bases, as well as the steps required for preparing oligomers are within the capabilities of ordinary technicians in the field of chemistry and the synthesis of nucleic acid analog oligomers.

[0056] Non-limiting examples of common nucleic acid analogs include peptide nucleic acids, such as γPNA, phosphorothioates (e.g., Figure 2 (A)), locked nucleic acid (2'-O-4'-C-methylene bridge, including oxygen, sulfur or its amino version, e.g., Figure 2 (B)), unlocking the nucleic acid (the C2'-C3' bond is cleaved, e.g. Figure 2 (C)), 2'-O-methyl substituted RNA, morpholino nucleic acids (e.g., Figure 2 (D)), threose nucleic acid (e.g., Figure 2 (E)), glycol nucleic acid (e.g., Figure 2 (F), showing R and S forms) etc. Figure 2 (AF) show the monomer structures of various examples of nucleic acid analogs. Figure 2(AF) Each shows two monomer residues incorporated into a longer chain, as indicated by the wavy lines. The incorporated monomers are referred to herein as "residues," and the portion of the nucleic acid or nucleic acid analog other than the nucleobase is referred to as the "backbone" of the nucleic acid or nucleic acid analog. For example, for RNA, an exemplary nucleobase is adenine, the corresponding monomer is adenosine triphosphate, and the introduced residue is an adenosine monophosphate residue. For RNA, the "backbone" consists of ribose subunits linked by phosphates, so that the backbone monomers are ribose triphosphates before incorporation and ribose monophosphate residues after incorporation. Like γPNA, locked nucleic acids ( Figure 2 (B)) are conformationally preorganized.

[0057] A "moiety" is a portion of a molecule and includes a class of "residues" which are the portion of a compound or monomer that remains in a larger molecule, such as a polymer chain, after the compound or monomer has been incorporated into the larger molecule, such as a nucleotide into a nucleic acid or an amino acid into a polypeptide or protein.

[0058] The term "polymer composition" is a composition comprising one or more polymers. As a class, "polymers" include, but are not limited to, homopolymers, heteropolymers, copolymers, block polymers, block copolymers, and may be natural and synthetic. Homopolymers contain one type of structural unit or monomer, while copolymers contain more than one type of monomer. An "oligomer" is a polymer containing a small number of monomers, e.g., 3 to 100 monomer residues. Similarly, the term "polymer" includes oligomers. The terms "nucleic acid" and "nucleic acid analogs" include nucleic acids and nucleic acid polymers and oligomers.

[0059] A polymer "comprises" or is "derived from" a monomer if the monomer is incorporated into the polymer. Thus, the polymer comprises an incorporated monomer that is different from the monomer prior to its introduction into the polymer because at least some linker groups are introduced into the polymer backbone or some groups are removed during the polymerization process. If the bond is present in the polymer, the polymer is said to contain a particular type of bond. The incorporated monomer is a "residue". Common monomers of nucleic acids or nucleic acid analogs are called nucleotides.

[0060] In the context of chemical components such as molecules, compounds, components, groups, moieties, ions, etc., "non-reactive" means that the component does not react to any substantial extent with other chemical components in its intended use. Non-reactive components are selected so as not to interfere or to interfere negligibly with the intended use of the component, moiety or group as a recognition agent. In the context of linker moieties described herein, they are non-reactive because they do not interfere with the binding of the recognition agent to the target template and do not interfere with the attachment of the recognition agent on the target template.

[0061] As used herein, "alkyl" refers to a straight chain, branched chain or cyclic hydrocarbon group, including, for example, 1 to about 20 carbon atoms, such as but not limited to C 1-3 , C 1-6 , C 1-10 Groups, such as but not limited to straight chain, branched alkyl, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. "Substituted alkyl" refers to an alkyl substituted at one or more, such as 1, 2, 3, 4, 5 or even 6 positions, and the substituent is attached to any available atom to produce a stable compound, as described herein. "Optionally substituted alkyl" refers to an alkyl or substituted alkyl. "Halogen", "halide" and "halo" refer to -F, -Cl, -Br and / or -I. "Alkylene" and "substituted alkylene" refer to divalent alkyl and divalent substituted alkyl, respectively, including but not limited to methylene, ethylene, trimethylene, tetramethylene, pentamethylene, heptamethylene, octamethylene, nonamethylene or decamethylene. "Optionally substituted alkylene" refers to alkylene or substituted alkylene.

[0062] "Alkenyl" or "alkenyl" refers to a straight chain, branched or cyclic hydrocarbon group, including, for example, 2 to about 20 carbon atoms, such as but not limited to C 1-3 , C 1-6 , C 1-10 A group having one or more, for example 1, 2, 3, 4 or 5 carbon-carbon double bonds. "Substituted alkene" refers to an alkene substituted at one or more, for example 1, 2, 3, 4 or 5 positions, with the substituents attached to any available atom to produce a stable compound, as described herein. "Optionally substituted alkene" refers to an alkene or a substituted alkene. Similarly, "alkenylene" refers to a divalent alkene. Examples of alkenylene include, but are not limited to, vinylene (-CH=CH-) and all stereoisomeric and conformational isomeric forms thereof. "Substituted alkenylene" refers to a divalent substituted alkene. "Optionally substituted alkenylene" refers to an alkenylene or a substituted alkenylene.

[0063] "Alkyne" or "alkynyl" refers to a straight or branched unsaturated hydrocarbon having a specified number of carbon atoms and at least one triple bond. Examples of (C2-C8)alkynyl include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-heptyn, 2-heptyn, 3-heptyn, 1-octyne, 2-octyne, 3-octyne and 4-octyne. Alkynyl can be unsubstituted or optionally substituted with one or more substituents described below. The term "alkynylene" refers to a divalent alkyne. Examples of alkynylene include, but are not limited to, ethynylene, propynylene. "Substituted alkynylene" refers to a divalent substituted alkynyl.

[0064] The term "alkoxy" refers to an -O-alkyl group having a specified number of carbon atoms. For example, (C1-C6)alkoxy includes -O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), -O-isopropyl (isopropoxy), -O-butyl (butoxy), -O-sec-butyl (sec-butoxy), -O-tert-butyl (tert-butoxy), -O-pentyl (pentoxy), -O-isopentyl (isopentyloxy), -O-neopentyl (neopentyloxy), -O-hexyl (hexyloxy), -O-isohexyl (isohexyloxy) and -O-neohexyl (neohexyloxy). "Hydroxyalkyl" refers to a (C1-C6)alkyl group in which one or more of the hydrogen atoms of the alkyl group are replaced by an -OH group. 10 )alkyl. Examples of hydroxyalkyl include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2CH2OH and branched forms thereof. The term "ether" or "oxyether" refers to an alkyl group in which one or more of the carbon atoms of the alkyl group is replaced by an -O- group. The term ether includes -CH2-(OCH2-CH2)qOP1 compounds in which P1 is a protecting group, -H or (C1-C 10 Exemplary ethers include polyethylene glycol, diethyl ether, methyl hexyl ether, and the like.

[0065] "Heteroatom" refers to N, O, P and S. Compounds containing N or S atoms can be optionally oxidized to the corresponding N-oxide, sulfoxide or sulfone compounds. "Heterosubstituted" refers to organic compounds described in any embodiment herein, wherein one or more carbon atoms are replaced by N, O, P or S.

[0066] "Aryl" alone or in combination refers to an aromatic ring system, such as phenyl or naphthyl; "aryl" also includes an aromatic ring system optionally fused to a cycloalkyl ring. "Substituted aryl" is an aryl group that is independently substituted with one or more substituents attached at any available atom to produce a stable compound, wherein the substituents are as described herein. "Optionally substituted aryl" refers to an aryl or substituted aryl group. "Arylene" means a divalent aryl group, and "substituted arylene" refers to a divalent substituted aryl group. "Optionally substituted arylene" refers to an arylene or substituted arylene group. As used herein, the term "polycyclic aryl" and related terms, such as "polycyclic aromatic group" refer to a group consisting of at least two fused aromatic compounds. "Heteroaryl" or "heterosubstituted aryl" refers to an aryl group substituted with one or more heteroatoms such as N, O, P and / or S.

[0067] "Cycloalkyl" refers to a monocyclic, bicyclic, tricyclic or polycyclic 3 to 14 membered ring system, which is saturated, unsaturated or aromatic. The cycloalkyl may be attached via any atom. Fused rings are also contemplated for cycloalkyl, wherein the cycloalkyl is fused to an aryl or heteroaryl ring. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Cycloalkyl may be unsubstituted or optionally substituted with one or more substituents as described below. "Cycloalkylene" refers to a divalent cycloalkyl. The term "optionally substituted cycloalkylene" refers to a cycloalkylene substituted with 1, 2 or 3 substituents, attached to any available atom to produce a stable compound, wherein the substituents are as described herein.

[0068] "Carboxyl" or "carboxylic" refers to a group having the specified number of carbon atoms (if specified) and terminating in a -C(O)OH group, thus having a -RC(O)OH structure, where R is a divalent organic group including a straight chain, branched chain or cyclic hydrocarbon. Non-limiting examples of these include: C 1-8 Carboxyl groups, such as glycolic acid, propionic acid, 2-methylpropionic acid, butyric acid, 2,2-dimethylpropionic acid, valeric acid, etc. "Amine" or "amino" refers to a group having the specified number of carbon atoms and terminating in a -NH2 group, thus having a -R-NH2 structure, wherein R is an unsubstituted or unsubstituted divalent organic group, such as a linear, branched or cyclic hydrocarbon, and optionally contains one or more heteroatoms.

[0069] Combinations of the foregoing terms refer to any suitable combination of the foregoing, for example, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroaryl Alkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl. For example, "arylalkylene" refers to a divalent alkylene group in which one or more hydrogen atoms in the alkylene group are replaced by aryl groups such as (C3-C8)aryl. Examples of (C3-C8)aryl(C1-C6)alkylene groups include, but are not limited to, 1-phenylbutene, phenyl-2-butene, 1-phenyl-2-methylpropene, phenylmethylene, phenylpropene and naphthylethylene. The term "(C3-C8)cycloalkyl-(C1-C6)alkylene" refers to a divalent alkylene group in which one or more hydrogen atoms in the C1-C6 alkylene group are replaced by a (C3-C8)cycloalkyl group. Examples of (C3-C8)cycloalkyl-(C1-C6)alkylene groups include, but are not limited to, 1-cyclopropenylbutene, cyclopropenyl-2-butene, cyclopentyl-1-phenyl-2-methylpropene, cyclobutanemethylene, and cyclohexylpropene.

[0070] An "amino acid" has the structure H2N-C(R)-C(O)OH, where R is a side chain, such as an amino acid side chain. An "amino acid residue" refers to the remainder of an amino acid when incorporated into an amino acid chain, such as when incorporated into a recognition agent disclosed herein, for example, having the structure -NH-C(R)-C(O)-, H2N-C(R)-C(O)- (when at the N-terminus of a polypeptide), or -NH-C(R)-C(O)OH (when at the C-terminus of a polypeptide). An "amino acid side chain" is a side chain of an amino acid, including proteinogenic or non-proteinogenic amino acids. An amino acid has the following structure:

[0071]

[0072] , wherein R is an amino acid side chain. Non-limiting examples of amino acid side chains are shown in Figure 3 In. Glycine (H2N-CH2-C(O)OH) has no side chain.

[0073] "Peptide nucleic acid" refers to a nucleic acid analog or a DNA or RNA mimetic in which the sugar phosphodiester backbone of DNA or RNA is replaced by N-(2-aminoethyl)glycine units. Gamma PNA (γPNA) is an oligomer or polymer of γ-modified N-(2-aminoethyl)glycine monomers having the following structure:

[0074] wherein at least one of R1 or R2 attached to the γ carbon is not hydrogen, making the γ carbon a chiral center. When R1 and R2 are hydrogen (N-(2-aminoethyl)-glycine backbone) or are the same, there is no such chirality about the γ carbon. The combined PNA or γPNA monomer,

[0075]

[0076] The term "residues" of a PNA or γPNA herein refers to the remaining structure after incorporation into an oligomer, wherein each residue has an R group that is the same or different from its base (nucleobase), such as adenine, guanine, cytosine, thymine and uracil bases, or other bases, such as the monovalent and divalent bases described herein, such that the order of nucleobases on the PNA is its "sequence", such as DNA or RNA. The nucleobase sequence in a nucleic acid or nucleic acid analog oligomer or polymer (e.g., a PNA or γPNA oligomer or polymer) binds to a complementary sequence of adenine, guanine, cytosine, thymine and / or uracil residues in a nucleic acid or nucleic acid analog by nucleobase pairing in a Watson-Crick or Watson-Crick-like manner, essentially the same as double-stranded DNA or RNA.

[0077] "Guanidine" or "guanidinium" groups can be added to recognition agents to increase solubility and / or bioavailability. Since PNAs are produced in a similar manner to synthetic peptides, a simple method for adding guanidine groups is to add one or more terminal arginine (Arg) residues to the N-terminus and / or C-terminus of the PNA, such as γPNA, recognition agents. Similarly, arginine side groups, or a guanidine-containing moiety, e.g. Wherein n is, for example but not limited to, in the range of 1-5, or a salt of the above group, which can be attached to the main chain of the recognition reagent described herein. The guanidine-containing group is a group containing a guanidine portion and can have less than 100 atoms, less than 50 atoms, such as less than 30 atoms. In one aspect, the guanidine-containing group has the following structure: Wherein L is a linker according to any aspect described herein, such as a non-reactive aliphatic hydrocarbon linker, such as a methylene, ethylene, trimethylene, tetramethylene or pentamethylene linker. In some aspects, the guanidine-containing group has the following structure: Wherein n is 1-5, for example, the guanidine group can be arginine.

[0078] "Nucleobases" include the primary nucleobases: adenine, guanine, thymine, cytosine and uracil, and modified purine and pyrimidine bases such as, but not limited to, hypoxanthine, xanthene, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine. Figure 4 and 5A -5C also depicts non-limiting examples of nucleobases, including monovalent nucleobases (e.g., adenine, cytosine, guanine, thymine, or uracil, which bind to one strand of a nucleic acid or nucleic acid analog) and divalent nucleobases (e.g., JB1-JB16 described herein), which simultaneously bind to complementary nucleobases on both DNA strands, and "clamp" nucleobases, such as "G-clamps," which bind to complementary nucleobases with enhanced strength. Additional purine, purine-like, pyrimidine, and pyrimidine-like nucleobases are known in the art, for example, as disclosed in U.S. Pat. Nos. 8,053,212, 8,389,703, and 8,653,254. For Figure 4 The bivalent nucleobases JB1-JB16 shown in A, Table A shows the specificity of different nucleobases. It is worth noting that the JB1-JB4 series binds complementary bases (CG, GC, AT and TA), while JB5-JB16 bind mismatches and can therefore be used to bind two base matches and / or mismatches. Bivalent nucleobases are described in more detail in U.S. Patent Application Publication No. 20160083434A1 and International Patent Publication No. WO 2018 / 058091, both of which are incorporated herein by reference.

[0079] Table A: Divalent Nucleobases

[0080]

[0081] *Diaminopurine, an adenine analog.

[0082] As used herein, "JB# nucleobase", such as "JB4 nucleobase", refers to all bases with the name JB4, representing C / G (combined G / C), including JB4, JB4b, JB4c, JB4d and JB4e. Exemplary γPNA structures that are not end-modified with aromatic groups as described herein, but can be as described herein, have been disclosed in International Publication No. WO 2012 / 138955 and are incorporated herein by reference. As shown herein, miniPEGγPNA includes one or more groups comprising a polyethylene glycol (PEG, polyoxyethylene) portion.

[0083] Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to each other to form interchain base pairs. Base pairs are formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can be base-paired (hybridized) in a Watson-Crick manner (e.g., A to T, A to U, C to G) or in any other manner that allows the formation of duplexes. When RNA is used instead of DNA, uracil instead of thymine is a nucleobase complementary to adenosine. If two sequences comprising complementary sequences form duplexes under the following conditions, they can hybridize: under specified conditions, such as in water, saline (e.g., saline or 0.9% w / v saline) or phosphate buffer, or under other stringent conditions, such as but not limited to, 0.1XSSC (saline sodium citrate) is diluted to 10X SSC, wherein 1X SSC is 0.15M NaCl and 0.015M sodium citrate in water. The hybridization of complementary sequences is determined by, for example, salt concentration and temperature, and the melting temperature (Tm) is reduced, then mismatches increase and stringency increases. Sequences that perfectly match are considered "fully complementary," although one sequence (e.g., a target sequence in an mRNA) may be longer than the other, as is the case with the small recognition agents described herein and the related longer targets to which they are attached, such as mRNAs containing repeat expansions. The two complementary nucleic acid strands bind in an antiparallel orientation, with one strand binding in a 5' to 3' direction and the other in a 3' to 5' direction. PNAs allow for both parallel and antiparallel orientations, although for gamma PNAs, antiparallel binding is preferred.

[0084] According to one aspect of the present invention, a gene recognition reagent is provided. In various aspects, the recognition reagent is self-concatenating, which means that it contains a linking group, i.e., an end group that is covalently linked or non-covalently linked, for example, by pi stacking, when hybridizing to an adjacent sequence on a target nucleic acid. The recognition reagent contains three or more, for example, 3-10 or 3-8 consecutive nucleic acid or nucleic acid analog monomer residues, whose end groups are, for example, self-concatenating or pi stacking, and in order to target a double-stranded RNA hairpin sequence, contains a nucleobase sequence that hybridizes (binds) to a repeat expansion sequence associated with a repeat expansion disease. In the case of polyQ disease, the rCAG expThe double-stranded sequence of the hairpin formed by the sequence is the target sequence, and the reagent has a divalent nucleobase sequence selected from the following: EIF or (EIF)n, wherein n is two or more integers, such as 2, 3, 4 or 5, such as EIFEIF; IFE or (IFE)n, wherein n is two or more integers, such as 2, 3, 4 or 5, such as IFEIFE; or FEI, or (FEI)n, wherein n is two or more integers, such as 2, 3, 4 or 5, such as FIEFEI, wherein E is a divalent nucleobase that binds C / G, such as JB3 or JB3b (Watson chain / Crick chain), F is a divalent nucleobase that binds G / C, such as JB4, JB4b, JB4c, JB4d or JB4e, and I is a divalent nucleobase that binds A / A, such as JB6 or JB6b, and is thus able to bind to the two hybridized chains of the hairpin formed by the CAG repeat sequence, such as Figure 1 shown.

[0085] The recognition reagents described herein combine the characteristics of small molecules, e.g., low molecular weight, ease of large-scale production, low production cost, cell permeability and desired pharmacokinetics, and sequence-specific recognition of oligonucleotides by Watson-Crick base pairing. The connection of oligomer recognition reagents has been demonstrated to be useful for both self-connection and π-stacking connection methods and is described with reference to the examples, which are intended to be illustrative and not limiting in all respects. Thus, the present invention is capable of many variations in detailed implementation, which can be derived from the description contained herein by one of ordinary skill in the art.

[0086] An example of the use of the recognition agents described herein is in the treatment of genetic diseases with small sequence repeat expansions, such as those listed in Table B.

[0087] Table B – Genetic Disorders Associated with Expanded Repeats

[0088]

[0089]

[0090] Based on Table B, exemplary sequences of divalent nucleobases of recognition agents that will target the gene product are provided in Table C, including sequences in the 5' to 3' direction relative to the sense strand. It should be understood that, depending on their sequence, not all repetitive sequences will form a hairpin structure under normal conditions, but can be induced into a three-stranded "hairpin" structure by the listed recognition agents. Moreover, the sequences listed in Table C are exemplary only, and other sequences are expected to form triplex structures depending on the alignment of the folded sequences in the natural hairpin structure or the hairpin structure induced by the recognition agent. Similarly, the repetitive nature of the sequence requires that for three base repeats, each sequence can use three different frameshifts (see Table C, (GAA)n is the third column), and for four base repeats, four different frameshifts can be used. It is useful. Therefore, for three base sequences, the permutations and combinations of double-stranded binding alignments and the ability to move the frame of the recognition agent within each alignment (for example, Figure 1 The EIF, IFE and FEI of the rCAGexp sequence alignment shown in the table result in 9 permutations and combinations of the unit recognition reagent, which can be repeated in the recognition reagent. For example, for the sequence (GAA)n, under normal conditions, it is not expected that the folding alignment forms a hairpin double strand, but it is expected that three different sequences can form a triplex structure. In Table C, the only repeating sequence showing all permutations and combinations is the sequence of (GAA)n, and for other repetitions, only exemplary sequences are provided. It is worth noting that due to the repeated structure, any sequence provided in Table C can be repeated, for example from 2-5X, such as G / AA / AA / G, also refers to G / AA / AA / GG / AA / AA / G and 13-6-14 (JB13 series-JB6 series-JB14 series) also includes 13-6-14-13-6-14. In Table C, nucleobase sequences are first identified by the target nucleobase to which they bind (e.g., JB4 binds G / C, and are also identified by a "JB" reference number, e.g., "4" refers to "JB4," including JB4, JB4b, JB4c, JB4d, and JB4e.

[0091] Table C - Examples of Identification Reagents

[0092]

[0093]

[0094]

[0095] In Table C, the unit recognition reagent target sequences (bound bases) list each base pair of the two strands bound by a single divalent nucleobase of the recognition reagent as "B1 / B2", where B1 is the first base strand and B2 is the base of the second strand bound by the divalent nucleobase of the recognition reagent. Thus, for the sequence (CAG)n, the sequence of the unit target sequence is C / GA / AG / C, referring to the antiparallel alignment of the sequence 5'-CAG-3', while for the sequence (CCG)n, the sequence of the second alignment of the target sequence is C / GG / CC / C, referring to the antiparallel alignment of 5'-CGC-3' to 5'-CCG-3', and the sequence C / GC / CG / C refers to the antiparallel alignment of the sequence 5'-CCG-3'.

[0096] In the case of recognition agent binding, a "unit target sequence" is the shortest repetitive sequence found in: a repetitive nucleobase sequence of a target nucleotide sequence, or a repetitive double-stranded nucleobase sequence including mismatches in a double-stranded sequence, or double strands of the same or different nucleic acid molecules, which are aligned in a triplex structure when bound by a bivalent recognition agent, as described herein.

[0097] In one aspect, a recognition reagent is provided, comprising: a peptide nucleic acid or γ-peptide nucleic acid backbone having a first end and a second end, prepared from 3 or more, e.g., 3 to 10 or 3, 4, 5, 6, 7, 8, 9 or 10, or 3-8 PNA or γPNA backbone residues; a sequence of nucleobases attached or linked to a plurality of nucleic acid or nucleic acid analog backbone residues in the sequence; a -SH, -OH or -SS-Lg portion at the first end of the backbone, and a -C(O)-S-Lg or C(O)-O-Lg portion (a leaving group Lg, linked to the backbone by an ester or thioester bond) at the second end of the backbone, wherein the sequence of the nucleobases is complementary to a unit target sequence or two or more consecutive iterations of a unit target sequence (two or more direct repeats of a specified sequence, without a gap) within a target sequence in a nucleic acid, e.g., when a plurality of recognition modules bind to adjacent sequences of the target sequence, they engage with each other. In one aspect, Lg is biocompatible and / or non-toxic. Non-limiting examples of Lg include substituted or unsubstituted (C1-C8)alkyl, substituted or unsubstituted (C3-C8)aryl, (C3-C8)aryl (C1-C6)alkylene, (C1-C8)carboxyl, optionally substituted with an amino acid side chain, or containing a guanidine group, or

[0098]

[0099] , wherein o is 1-20, each instance of R6 is independently an amino acid side chain, and R7 is -OH or -NH2.

[0100] In another aspect, a recognition reagent is provided, comprising: a nucleic acid or nucleic acid analog backbone having a first end and a second end prepared from three or more, e.g., 3 or more molecules, e.g., prepared from 3 to 10 or 3, 4, 5, 6, 7, 8, 9 or 10, or 3-8 nucleic acid or nucleic acid analog backbone residues, which are optionally conformationally pre-organized; a nucleobase sequence attached or linked to a plurality of nucleic acid or nucleic acid analog backbone residues in sequence; a first aryl moiety linked to the first end of the nucleic acid or nucleic acid backbone; and a second aryl moiety, which is optionally the same as the first aryl moiety, linked to the second end of the nucleic acid or nucleic acid backbone. The aryl moieties are independently 2 to 5 ring-fused polycyclic aromatic moieties, such as substituted or unsubstituted aryl or heteroaryl moieties having 2 to 5 fused rings, such as, but not limited to, unsubstituted or substituted pentene, indene, naphthalene, azulene, heptene, biphenyl, as-indenene, s-indenene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, acephenanthrene, aceanthrene, triphenylene, pyrene, Naphthonaphthalene / tetracene, septobalene, chrysene or perylene, optionally substituted with one or more heteroatoms such as O, N and / or S, for example, xanthene, riboflavin (vitamin B2), mangiferin or mangiferin, and may be identical or different, and identical in multiple aspects, as long as each of them is able to stack with the aromatic moiety of an adjacent recognition agent when the recognition agent is hybridized to an adjacent sequence of the target nucleic acid. The recognition agent is capable of binding to both strands of a hairpin formed by an expanded repeat sequence, the expanded repeat sequence is, for example, associated with an expanded repeat disease listed in Table B, such as a PolyQ disease. On the one hand, in order to bind to a CAG repeat sequence, as described above, having the sequence EIF or (EIF) n, wherein n is an integer of two or more, such as 2, 3, 4 or 5, such as EIFEIF; IFE or (IFE) n, wherein n is an integer of two or more, such as 2, 3, 4 or 5, such as IFEIFE; or FEI, or (FEI) n, wherein n is an integer of two or more, such as 2, 3, 4 or 5, such as FIEIFEI.

[0101] In one aspect, the recognition reagent is self-contained and has the following structure:

[0102]

[0103] wherein X is S or O; n is an integer between 1 and 6, including 1 and 6; m is an integer between 0 and 4, including 0 and 4. R1 and R2 are each independently: H; guanidine-containing, for example

[0104]

[0105] Where n = 1, 2, 3, 4 or 5; amino acid side chains, for example:

[0106]

[0107] Straight or branched (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene, optionally substituted with ethylene glycol units containing 1 to 50 ethylene glycol moieties; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(SCH2-CH2) q -SP1; -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; r and s are each independently an integer from 1 to 50, inclusive;

[0108] R3 is H or a leaving group and is biocompatible and / or non-toxic in one aspect, such as substituted or unsubstituted (C1-C8)alkyl, substituted or unsubstituted (C3-C8)aryl, (C3-C8)aryl (C1-C6)alkylene, (C1-C8)carboxyl, optionally substituted with an amino acid side chain, or guanidine-containing

[0109]

[0110] , wherein o is 1-20, each instance of R6 is independently an amino acid side chain, and R7 is -OH or -NH2; R4 is (C1-C 10 ) divalent hydrocarbon or (C1-C 10) divalent hydrocarbon, such as -O-, -OH, -C(O)-, -NH-, -NH2, -C(O)NH-; R5 is -OH, -SH or a disulfide bond protecting group; and each instance of R is independently a nucleobase, which produces a nucleobase sequence complementary to the target sequence of the nucleobase in the target nucleic acid, so that each of the multiple recognition modules binds to the target sequence of the nucleobase on the template nucleic acid. On the one hand, R5 has a structure of -SH, -OH or -SS-R8, wherein R8 is one or more amino acid residues, amino acid side chains, linear, branched or hetero-substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene. In one aspect, R1 or R2 is an amino acid side chain or a guanidine-containing group, such as

[0111]

[0112] wherein n=1, 2, 3, 4 or 5. In one aspect, R1 and R2 are different, R1 is H and R2 is not H, or R2 is H and R1 is not H. For binding to natural nucleic acids, such as RNA or DNA, R1 can be H and R2 is not H, thereby forming a "right-handed" L-γPNA. A "left-handed" D-γPNA, where, for example, R2 is H and R1 is not H, does not bind to natural nucleic acids. In one aspect, the recognition agent is substituted with one or more guanidine-containing groups, such as

[0113]

[0114] Wherein n=1, 2, 3, 4 or 5.

[0115] In one aspect, R1 or R2 is (C1-C6)alkyl substituted with: -(OCH2-CH2) q OP1; -(OCH2-CH2) q -NHP1; -(SCH2-CH2) q -SP1; -(OCH2-CH2) r -OH; -(OCH2-CH2) r -NH2; -(OCH2-CH2) r -NHC(NH)NH2; or -(OCH2-CH2) r -SS[CH2CH2] sNHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50.

[0116] The recognition reagent with an aromatic terminal group has the ability to be connected as a result of non-covalent bonding, such as pi stacking, of the aromatic group of the adjacent hybrid recognition reagent. Therefore, in another aspect, a recognition reagent (recognition module) is provided, the structure of which is:

[0117]

[0118] , wherein R is independently a nucleobase, and each instance of R may be the same or different;

[0119] n is an integer from 1 to 6, for example 1, 2, 3, 4, 5 or 6;

[0120] Each B is independently a ribose 5' phosphate residue, a deoxyribose 5-phosphate residue or a nucleic acid analog backbone residue, and in one aspect, is a backbone residue of a conformationally preorganized nucleic acid analog such as γPNA or LNA;

[0121] L is independently a linker, e.g., a non-reactive linker or a non-reactive, non-place-occupying linker, and each instance of L can be the same or different; and

[0122] Each instance of Ar is independently a 2 to 5 ring fused polycyclic aromatic moiety, such as a substituted or unsubstituted aryl or heteroaryl moiety having 2 to 5 fused rings, such as, but not limited to, unsubstituted or substituted pentene, indene, naphthalene, azulene, heptene, biphenyl, as-indenene, s-indenene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acetophenanthren, acetothren, triphenylene, pyrene, Naphthonaphthalene / tetracene, pleiadene, picene or perylene, optionally substituted with one or more heteroatoms such as O, N and / or S, such as xanthene, riboflavin (vitamin B2), mangiferin or mangiferin, and may be the same or different, and are the same in aspects. In some aspects, when the recognition agent hybridizes to the adjacent sequence of the target nucleic acid, each Ar is stacked with the Ar group of the adjacent recognition agent.

[0123] The part in the compound, such as an aromatic moiety or a core base, is covalently connected to the recognition reagent backbone, and is therefore referred to as being "connected" to the backbone. Depending on the chemical nature used to prepare the compound, the connection can be direct, or directly connected by a "joint", which is a part that covalently connects two other parts or groups. On the one hand, the terminal aromatic (aryl) group is connected to the recognition reagent through a joint. The joint is a non-reactive part of the backbone that connects the aromatic group to the recognition reagent, and in some aspects includes 1-10 carbon atoms (C1-C 10 ), which is optionally substituted by heteroatoms such as N, S or O, or a non-reactive bond, such as an amide bond (peptide bond) formed by reacting an amine with a carboxyl group. 10 Examples of alkylene groups are straight or branched alkylene (divalent) moieties, optionally containing cyclic moieties, such as methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene or decamethylene moieties (i.e., -CH2-[CH2]n-, where n = 1 to 9), optionally containing amide bonds. Linkers are non-occupying in that they do not sterically hinder or otherwise interfere to a significant degree with the binding of the recognition agent to the target template, and do not interfere with the attachment of the recognition agent to the target template. Linkers are the remainder resulting from the attachment of an aromatic group to the backbone of the recognition agent, for example, or in a non-limiting example,

[0124] It is produced by linking an acetic acid-substituted aryl compound (A), such as pyrene-1-acetic acid, to a Dab (n=1), Orn (n=2) or Lys (n=3) residue.

[0125] In other aspects, a linker or linker group is an organic moiety that connects two parts of a compound, e.g., covalently connects two parts of a compound, such as, but not limited to, the connection of an aromatic group to the backbone of a recognition agent, the connection of a nucleobase to the backbone of a nucleic acid or nucleic acid analog, and / or the connection of a guanidine group to a recognition agent in the context of the present invention. The linker typically comprises a direct bond or an atom such as oxygen or sulfur, a unit such as C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenylalkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroaryl The invention also provides a alkyl radical comprising a alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group, an alkyl group In one aspect, the linker comprises about 5 to 25 atoms, e.g., 5-20, 5-10, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19 or 20 atoms, or a total of 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms and heteroatoms, e.g., O, P, N or S atoms.

[0126] For bonding to PNA (e.g., γPNA), a convenient and useful linker is one that reacts an amine with a carboxyl group to form an amide bond, for example, using well-known peptide synthesis chemistry to add an amino acid to the recognition agent, wherein the amino acid can be pre-modified with a chemical moiety (e.g., an aryl moiety or a guanidinium group), as shown in the following examples, and adding an aryl-modified amino acid to connect the pyrenyl moiety to the recognition agent, and using arginine to provide the guanidinium group. Connection to non-peptide nucleic acid analogs can be achieved using any suitable connection chemistry that is well known, such as by using carbodiimide chemistry, such as EDC (EDAC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) to connect an amine-modified Ar group to the recognition agent, for example, via a terminal phosphate.

[0127] When attaching the aromatic group to the backbone of the recognition agent, the linker is of an appropriate size or length to place the aromatic group in a π-π stacking position during attachment of the recognition agent to the target nucleic acid sequence, as described herein.

[0128] In another aspect, the recognition agent has the following structure:

[0129]

[0130] in,

[0131] Each instance of R is independently a nucleobase, and each instance of R may be the same or different.

[0132] n is an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6;

[0133] R1 and R2 are each independently: H, containing a guanidine group, such as

[0134]

[0135] Where n=1, 2, 3, 4 or 5 are amino acid side chains, for example:

[0136]

[0137] Unsubstituted or substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(OCH2-CH2-0) q -SP1; -CH2-(SCH2-CH2) q -SP1, -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] sNHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50; and in one aspect, R1 and R2 are different, R1 is H and R2 is not H, or R2 is H and R1 is not H;

[0138] R 10 or R 11 One of the 12 , R 13 or R 14 wherein one of the following is -L-R3, wherein each instance of R3 is independently a 2 to 5 ring fused polycyclic aromatic moiety, such as a substituted or unsubstituted aryl or heteroaryl moiety having 2 to 5 fused rings, such as, but not limited to, unsubstituted or substituted pentene, indene, naphthalene, azulene, heptene, biphenyl, as-indenene, s-indenene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acetophenanthrenene, acetothrenene, triphenylene, pyrene, naphthacene / tetracene, pleiadene, picene or perylene, optionally substituted with one or more heteroatoms such as O, N and / or S, such as xanthene, riboflavin (vitamin B2), mangiferin or mangiferin, and can be the same or different, and are the same in multiple aspects, and in some examples, each stacks with the R3 group of an adjacent recognition agent when the recognition agent is hybridized to an adjacent sequence of the target nucleic acid, and wherein L is a linker, such as a non-reactive linker or a non-reactive, non-place-occupying linker, and each instance of L can be the same or different, and can comprise an amino acid residue, or a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, an arylalkyl, an arylalkenyl, an arylalkynyl, a heteroarylalkyl, a heteroarylalkenyl, a heteroarylalkynyl, a heterocyclylalkyl, a hetero Cycylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkenylalkynyl, alkynyl, arylalkenylalkynylalkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheteroheterocyclylalkyl, alkylheteroheterocyclylalkenyl , alkylheteroalkylalkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more carbon atoms, such as methylene or methine (CH=), are optionally replaced by heteroatoms such as O, S or N, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, and R 10 , R 11 , R 12 , R 13 and R 14 The rest of the residues are independently: H; one or more consecutive amino acid residues; guanidine-containing; amino acid side chain; straight or branched (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene, optionally substituted with ethylene glycol units containing 1 to 50 ethylene glycol moieties; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(SCH2-CH2) q -SP1; -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2)r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50.

[0139] In one aspect, R4 and R7 are -L-R3, and in another aspect, R4 and R7 are -L-R3, and R 11 and R 14 In one aspect, the linker comprises about 5 to 25 atoms, such as 5-20, 5-10, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms, or a total of 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C and heteroatoms, such as O, P, N or S atoms. In another aspect, R1, R2, R 10 , R 11 , R 12 , R 13 , or R 14 One or more of them is (C1-C6)alkyl substituted by: -(OCH2-CH2) q OP1; -(OCH2-CH2) q -NHP1; -(SCH2-CH2) q -SP1; -(OCH2-CH2) r -OH; -(OCH2-CH2) r -NH2; -(OCH2-CH2) r -NHC(NH)NH2; or -(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50.

[0140] In another aspect, the recognition reagent comprises a PNA backbone and thus has the following structure:

[0141]

[0142] in,

[0143] n is an integer from 1 to 8, including 1, 2, 3, 4, 5, 6, 7 or 8;

[0144] m is an integer from 1 to 5, such as 1 to 3, including 1, 2, 3, 4 or 5;

[0145] R2 is: containing guanidine group, for example

[0146] Where n = 1, 2, 3, 4 or 5; amino acid side chains, for example:

[0147]

[0148] Unsubstituted or substituted (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C1-C8)hydroxyalkyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene, or (C3-C8)cycloalkyl(C1-C6)alkylene; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(OCH2-CH2-0) q -SP1; -CH2-(SCH2-CH2) q -SP1, -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; r and s are each independently an integer from 1 to 50, inclusive;

[0149] R3 is an unsubstituted fused-ring polycyclic aromatic moiety, such as pentene, indene, naphthalene, azulene, heptene, biphenyl, as-indenene, s-indenene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acetophenanthren, acetothrenene, triphenylene, pyrene, naphthacene / tetracene, pleiadene, picene or perylene; and

[0150] R 11 , R 13 and R 14 Each is independently H, containing a guanidine group, such as

[0151] wherein n=1, 2, 3, 4 or 5, an amino acid side chain, or one or more consecutive amino acid residues, such as one or more Arg residues. In one aspect, R3 is pyrene.

[0152] In another aspect, R2 is -CH2-(OCH2-CH2)r-OH, wherein r is an integer from 1 to 50, such as 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in one example is 2. In another aspect, one or more of R2, R5, R7 or R8 is (C1-C6) alkyl substituted with: -(OCH2-CH2) q OP1; -(OCH2-CH2) q -NHP1; -(SCH2-CH2) q -SP1; -(OCH2-CH2) r -OH; -(OCH2-CH2) r -NH2; -(OCH2-CH2) r -NHC(NH)NH2; or -(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50.

[0153] Although previous PNA-based recognition agents were shown to lack chirality, in one example, the gamma carbon (to which R1 and R2 are attached) is in the (R) orientation, where R2 is H and R1 is not H, or the gamma carbon atom is in the (S) orientation, where R1 is H and R2 is not H. Furthermore, in one example, when present, in R10 , R 11 , R 12 , R 13 and / or R 14 In another example, when present, at R 10 , R 11 , R 12 , R 13 and / or R 14 In position , one or more or all of the chiral amino acid residues may be D-amino acids.

[0154] Also provided are pharmaceutically acceptable salts of any of the foregoing.

[0155] Pharmaceutically acceptable salts of any compound described herein may also be used in the methods described herein. Pharmaceutically acceptable salt forms of the compounds described herein may be prepared by conventional methods known in the pharmaceutical art, and include veterinary acceptable salts. For example, but not limited to, in the case where the compound contains a carboxylic acid group, a suitable salt thereof may be formed by reacting the compound with a suitable base to provide a corresponding base addition salt. Non-limiting examples include: alkali metal hydroxides, such as potassium hydroxide, sodium hydroxide, and lithium hydroxide; alkaline earth metal hydroxides, such as barium hydroxide and calcium hydroxide; alkali metal alkoxides, such as potassium ethoxide and sodium propoxide; and various organic bases, such as piperidine, diethanolamine, and N-methylglutamine.

[0156] Non-limiting examples of pharmaceutically acceptable basic salts include: aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium, and zinc salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, chloroprocaine, choline, N,N'-dibenzylethylenediamine (phenylmethionine), dicyclohexylamine, diethanolamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, benzidine, iso-propylamine, lidocaine, lysine, meglumine, N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine and tris-(hydroxymethyl)-methylamine (trimethylamine).

[0157] Non-limiting examples of pharmaceutically acceptable acid salts include acetate, adipate, alginate, arginine, aspartate, benzoate, benzenesulfonate (benzenesulfonate), bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, citrate, cyclopentanepropionate, digluconate, dihydrogen phosphate, dinitrobenzoate, dodecyl sulfate, ethanesulfonate, fumarate, galactonate, galacturonate, glucoheptanoate, gluconate, Glutamate, glycerophosphate, hemisuccinate, hemisulphate, pimelic acid, dihydrogen hydrobromide, dihydrogen hydroadipate, dihydrogen adipate, dihydrogen isethionate, isobutyrate, lactate, lactate, malate, maleate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogen phosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, palmitate, pectinate, persulfate, propyl phenylacetate, propyl acetate phosphate, phosphonate and phthalate.

[0158] Various salt forms are also considered to be pharmaceutically acceptable salts. Common, non-limiting examples of various salt forms include: bitartrate, diacetate, difumarate, dimeglumine, diphosphate, disodium and trihydrochloride.

[0159] As such, "pharmaceutically acceptable salts" as used herein are intended to mean active ingredients (drugs) comprising a salt form of any compound described herein. The salt form preferably imparts improved and / or desired pharmacokinetic / pharmacodynamic properties to the compounds described herein.

[0160] The compositions described herein can be administered by any effective route. Examples of delivery routes include, but are not limited to: topical, such as transdermal, inhalation, enema, ocular, otic, and intranasal delivery; enteral, such as oral, gastric feeding tube, and rectal; and parenteral, such as intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoresis, transmucosal, epidural, and intravitreal injection, with oral, intravenous, intramuscular, and transdermal methods being preferred in many cases. Suitable dosage forms may include single-dose or multi-dose vials or other containers, such as medical syringes, containing compositions containing active ingredients for treating repeat expansion diseases, as described herein.

[0161] The dosage regimen can be adjusted to provide the best desired response (e.g., a therapeutic or preventive response). For example, a single bolus can be administered, several separate doses can be administered over time, or the composition can be administered continuously or in a pulsed manner, wherein the dose or partial dose is administered at regular intervals, such as every 10, 15, 20, 30, 45, 60, 90 or 120 minutes, every 2 to 12 hours per day, or every other day, etc., proportionally reduced or increased as required by the treatment situation. In some cases, it may be particularly advantageous to prepare the composition in a dosage unit form, such as a parenteral or inhaled composition, for ease of administration and uniform dosage. The use specifications of the dosage unit form depend directly on (a) the unique characteristics of the active compound and the specific therapeutic or preventive effect to be achieved, and (b) the inherent limitations of the technical field of preparing active compounds such as those for treatment of individual sensitivity.

[0162] Useful dosage forms include: intravenous, intramuscular or intraperitoneal solutions, oral tablets or liquids, topical ointments or creams and transdermal devices (e.g., patches). In one aspect, the compound is a sterile solution comprising an active ingredient (drug or compound) and a solvent such as water, saline, lactated Ringer's solution or phosphate buffered saline (PBS). The solution may contain other excipients such as polyethylene glycol, emulsifiers, salts and buffers.

[0163] The preparation of the therapeutic / pharmaceutical compositions is carried out according to accepted pharmaceutical methods, for example as described below: Lei Ming The science and practice of pharmacy (21st ed., ed.), Paul Beringer et al., Lippincott, Williams & Wilkins, Baltimore, MD Easton, Pa. (2005) (see, e.g., Chapters 37, 39, 41, 42, and 45 for exemplary formulations of powders, liquids, parenteral, intravenous, and oral solids and methods of making them).

[0164] The dosage regimen can be adjusted to provide the best desired response (e.g., a therapeutic or preventive response). For example, a single bolus can be administered, several separate doses can be administered over time, or the composition can be administered continuously or in a pulsed manner, wherein the dose or partial dose is administered at regular intervals, such as every 10, 15, 20, 30, 45, 60, 90 or 120 minutes, every 2 to 12 hours per day, or every other day, etc., proportionally reduced or increased as required by the treatment situation. In some cases, it may be particularly advantageous to prepare the composition in a dosage unit form, such as a parenteral or inhaled composition, for ease of administration and uniform dosage. The use specifications of the dosage unit form depend directly on (a) the unique characteristics of the active compound and the specific therapeutic or preventive effect to be achieved, and (b) the inherent limitations of the technical field of preparing active compounds such as those for treatment of individual sensitivity.

[0165] Useful dosage forms include: intravenous, intramuscular or intraperitoneal solutions, oral tablets or liquids, topical ointments or creams and transdermal devices (e.g., patches). In one aspect, the compound is a sterile solution comprising an active ingredient (drug or compound) and a solvent such as water, saline, lactated Ringer's solution or phosphate buffered saline (PBS). The solution may contain other excipients such as polyethylene glycol, emulsifiers, salts and buffers.

[0166] In addition to a gene recognition agent, aspects of the invention provide methods for binding a nucleic acid comprising an expanded repeat associated with an expanded repeat disease, comprising contacting the nucleic acid comprising the expanded repeat with a gene recognition agent as described in any aspect herein. In one aspect, the method is performed in vitro or ex vivo. In another aspect, the method is performed in vivo by administering to a patient a suitable pharmaceutical formulation comprising the gene recognition agent in an amount effective to knock down the expression of a gene having an expanded repeat or to treat the patient.

[0167] In another aspect, a method for identifying the presence of a nucleic acid comprising an expanded repeat associated with an expanded repeat disease, e.g., (CAG)n, in a sample obtained from a patient is provided. The method comprises contacting a nucleic acid sample obtained from a patient with a gene recognition reagent according to any aspect described herein. Next, binding and ligation of the gene recognition reagent occurs in the presence of a target sequence in the nucleic acid in the sample, such that the binding and ligation indicates the presence of the target sequence in the nucleic acid in the sample. Any effective method may be used to detect and / or quantify the binding and ligation of the gene recognition reagent. The presence of a nucleic acid comprising an expanded repeat sequence in the target sequence is associated with an expanded repeat disease, at which point the patient's expanded repeat disease may be treated, for example, by administering to the patient a pharmaceutical dosage form comprising a gene recognition reagent as described herein.

[0168] Thus, a method of treating a patient suffering from a disease associated with an expanded repeat of a nucleotide sequence is provided. Examples of diseases are those listed in Table B, or PolyQ diseases, such as Huntington's disease, DRRPA (Dental Arch-tarsal Muscular Atrophy), SBMA (Spinal and Bulbous Muscular Atrophy), SCA1 (Spinocerebellar Ataxia Type 1), SCA2 (Spinocerebellar Ataxia Type 2), SCA3 (Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease), SCA6 (Spinocerebellar Ataxia Type 6), SCA7 (Spinocerebellar Ataxia Type 7), and SCA17 (Spinocerebellar Ataxia Type 7). The disease is treated by administering to the patient a composition or pharmaceutical dosage form comprising a gene recognition agent as described herein, wherein the gene recognition agent is selected to bind to a characteristic repeat sequence of a particular disease, such as (CAG)n for a polyQ disease such as Huntington's disease. The composition of the pharmaceutical dosage form is administered to the patient in an amount and dosage regimen effective to treat the disease. Thus, also provided is the use of a composition comprising a gene recognition agent as described herein for treating a disease associated with an expanded repeat sequence of a nucleotide sequence, such as a PolyQ disease, such as Huntington's disease, DRPLA (Dental-Breast-Louve Muscular Atrophy), SBMA (Spinal and Bulbous Muscular Atrophy), SCA1 (Spinocerebellar Ataxia Type 1), SCA2 (Spinocerebellar Ataxia Type 2), SCA3 (Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease), SCA6 (Spinocerebellar Ataxia Type 6), SCA7 Spinocerebellar Ataxia Type 7) and SCA17 (Spinocerebellar Ataxia Type 17).

[0169] In some embodiments, the present disclosure provides a compound comprising: a) a series of peptide nucleic acid (PNA) units, wherein the series of units includes i) a first unit; ii) a last unit; and iii) at least one intermediate unit between the first unit and the last unit, wherein each unit in the series of units includes: a) a main chain portion, wherein: 1) the main chain portion of the first unit is covalently bonded to the main chain portion of another unit; 2) the main chain portion of the last unit is covalently bonded to the main chain portion of another unit; and 3) the main chain portion of each intermediate unit is covalently linked to the main chain portions of the other two units; and B) a divalent nucleobase is covalently bonded to the main chain portion; b) a first aromatic portion covalently linked to the first unit; and c) a last aromatic portion covalently linked to the last unit.

[0170] In some embodiments, the present disclosure provides a compound comprising: a) a series of PNA units, wherein the series units include i) a first unit; ii) a last unit; and iii) at least one intermediate unit between the first unit and the last unit, wherein each unit in the series units includes: a) a main chain portion, wherein: 1) the main chain portion of the first unit is covalently bonded to the main chain portion of another unit; 2) the main chain portion of the last unit is covalently bonded to the main chain portion of another unit; 3) the main chain portion of each intermediate unit is covalently bonded to the main chain portions of the other two units; and b) a divalent nucleobase is covalently bonded to the main chain portion; b) two aromatic moieties, one covalently linked to the first unit and the other covalently linked to the last unit.

[0171] In some embodiments, the present disclosure provides a compound, wherein the first aryl moiety is covalently linked to the first unit via a first linker moiety, and the last aryl moiety is covalently linked to the last unit via a last linker moiety. In some embodiments, the present disclosure provides a compound, wherein the first linker moiety and the last linker moiety each independently comprise a guanidine group. In some embodiments, the present disclosure provides a compound, wherein the first linker moiety and the last linker moiety each independently comprise an amino acid residue. In some embodiments, the present disclosure provides a compound, wherein the first linker moiety and the last linker moiety each independently comprise three guanidine-containing amino acid residues.

[0172] In some embodiments, the present disclosure provides a kind of compound, wherein the first joint part and the last joint part each independently comprise three continuous arginine residues. In some embodiments, the present disclosure provides a compound in which the series unit is 3 to 8 units. In some embodiments, the present disclosure provides a compound in which the series unit is γ-PNA. In some embodiments, the present disclosure provides a kind of compound, wherein the first aryl part and the last aryl part each independently comprise a 2 to 5 ring fused polycyclic aromatic part, such as polyaromatic hydrocarbons such as pyrene. In some embodiments, the present disclosure provides a compound in which the first aryl part and the last aryl part are identical. In some embodiments, the present disclosure provides a compound further comprising an ethylene glycol unit, such as a diethylene glycol unit.

[0173] In some embodiments, the present disclosure provides a compound, wherein the compound presents nucleobases in an order complementary to a target nucleic acid sequence. In some embodiments, the present disclosure provides a compound wherein the target nucleic acid sequence is associated with a repeat expansion disease. In some embodiments, the present disclosure provides a compound wherein the repeat expansion disease is myotonic dystrophy type 1 (DM1) or myotonic dystrophy type 2 (DM2). In some embodiments, the present disclosure provides a compound wherein the first aryl moiety of one compound and the last aryl moiety of the other compound are stacked when the two compounds are hybridized to a nucleic acid.

[0174] In some embodiments, the present disclosure provides compounds of the formula:

[0175] H- L Arg-CAGCAG- L Arg-NH2(P1);

[0176] H- L Arg- L Dab(Pyr)-CAGCAG- L Orn(Pyr)- L Arg-NH2(P2);

[0177] H- L Arg- L Orn(Pyr)-CAGCAG- L Orn(Pyr)- L Arg-NH2(P3);

[0178] H- L Arg- L Lys(Pyr)-CAGCAG- L Lys(Pyr)- L Arg-NH2(P4);

[0179] H- L Arg- L Lys(Pyr)-CATCAG- L Lys(Pyr)- L Arg-NH2(P5); or

[0180] H- L Arg- L Lys(Pyr)-CTGCTG- L Lys(Pyr)- LArg-NH2(P6), wherein Orn is ornithine, Dab is diaminobutyric acid, and Pyr is a carboxyl-functionalized aromatic compound, such as pyrene, for example pyrene-1-carboxylic acid, pyrene-2-carboxylic acid, pyrene-4-carboxylic acid, pyrene-1-acetic acid, pyrene-2-acetic acid or pyrene-4-acetic acid.

[0181] In some embodiments, the disclosure provides a method for binding a nucleic acid, the method comprising contacting the nucleic acid with a compound of the disclosure, wherein the compound binds to the nucleic acid upon contact. In some embodiments, the disclosure provides a method for knocking down mRNA expression in a cell, the method comprising contacting the cell with a compound of the disclosure, wherein the compound knocks down the expression of the mRNA in the cell when binding to a DNA sequence corresponding to the mRNA in the cell.

[0182] In some embodiments, the present disclosure provides a gene recognition reagent, which includes: a nucleic acid or nucleic acid analog backbone having a first end and a second end, and having three to eight ribose-5-phosphate, deoxyribose 5-phosphate or nucleic acid analog backbone residues; the same or different divalent nucleobases connected to multiple 5-phosphate ribose, 5-phosphate deoxyribose or nucleic acid analog backbone residues; a first aryl moiety connected to the first end of the nucleic acid or nucleic acid analog backbone via a linker; a second aryl moiety and optionally a second aryl moiety identical to the first aryl moiety connected to the second end of the nucleic acid or nucleic acid analog backbone via a linker.

[0183] In some embodiments, the present invention provides a detection method comprising a) hybridizing a first probe nucleic acid to a first repeating portion of a target nucleic acid, wherein the first probe nucleic acid includes a first end connected to a first emitter portion and a second end connected to a second emitter portion; and b) hybridizing a second probe nucleic acid to a second repeating portion of the target nucleic acid, wherein the second probe nucleic acid includes a first end connected to a third emitter portion and a second end connected to a fourth emitter portion; wherein i) the first and second repeating portions of the target nucleic acid are associated with a repeat expansion disease; ii) binding of the first probe nucleic acid and the second probe nucleic acid to the target indicates that the first or second emitter portion is close to the third or fourth emitter portion; and iii) the presence of the first or second emitter portion close to the third or fourth emitter portion results in a change in the emission wavelength of the close-range emitter portion.

[0184] In some embodiments, the detection method also includes detecting the change of the emission wavelength of the close-range emission part. In some embodiments, the first probe nucleic acid is hybridized with the first repeating part to increase the affinity of the second probe nucleic acid and the second repeating part. In some embodiments, the first or second emitter part is close to the presence of the third or fourth emitter part resulting in the pi-pi stacking interaction between the first or second emitter part and the third or fourth emitter part. In some embodiments, the target nucleic acid is directly obtained from a biological sample.

[0185] Example 1

[0186] NMR, X-ray, and biochemical studies have shown that the rCAG-hairpin structure is relatively dynamic compared to canonical RNA duplexes, with the internal AA bulges exhibiting large motion amplitudes. This molecular scaffold contains internal AA mismatches between every two canonical GC / CG base pairs ( Figure 1 (A) ), similar to a "pothole" in the road, provides a unique and feasible receptor-like binding site for exogenous ligands. Relatively short nucleic acid ligands targeting the rCAG-hairpin structure were developed ( Figure 1 (B)). Using Janus bases (J bases or JBs), E, ​​I, and F ( Figure 1 (C)), which are able to form divalent H-bond interactions with: the nucleobases in both strands of the RNA duplex, the conformationally preorganized MPyPNA backbone ( Figure 1 (D and E)). The J base described here is part of a larger set of 16 double-sided nucleic acid recognition elements designed to bind to all 16 possible RNA base pair combinations ( Figures 5A-5C ). They differ from other J-bases, including the "Janus-wedges," in that they are all identical in shape, size, and chemical function, so they can be combined in a modular fashion to recognize any combination of RNA base pairs.

[0187] Molecular dynamics (MD) simulations. To evaluate the feasibility of the bivalent recognition design concept, we performed MD simulations of the ligand LG1 bound to an RNA duplex containing four rCAG repeats. The C-terminal lysine residue was omitted and the MP side chain was replaced by a methyl group (MeγPNA) to simplify the computational model. The CEO, AIA, and GFC triplets were constructed and optimized via a 6-31 basis set and grafted onto their respective RNA and MeγPNA backbones. The structure of the bound RNA-LG1-RNA complex was generated using the NAB module of Ambertools. The final structure was solvated by water molecules and ions, energy minimized, and simulated for 100 ns. The resulting complex remained fairly stable throughout the simulation, with the four individual LG 1 ligands fitting tightly between the two RNA strands ( Figure 6 (A)). The number of H-bonds remains constant throughout the simulation, five for each of the CEO and GFC triads and four for AIA ( Figure 6 (B)). However, attempts to simulate the binding of fewer than four LG1 ligands were unsuccessful. Due to the frayed terminal base pairs ( Figure 6 (D and E)), the complex was disassembled and some distorted structures were formed.

[0188] Synthesis of chemical building blocks and ligands. We synthesized J-bases E and F, and the corresponding JB-MPγPNA monomers 1 to 3 ( Figure 7 ). J-base I is not yet ready because it can be purchased from the market. E and F are respectively prepared according to Scheme 1 ( Figure 8 ) and Option 2 ( Fig. 9 ) were synthesized. The Boc protection / deprotection sequence in compounds 5 to 9 was found to be preferred for improving the chemical yield of the NBS reaction and suppressing side reactions in the subsequent condensation and cyclization steps. It was found that in order to allow the Stille coupling to proceed smoothly, it was preferred to further protect 12. This was accomplished by Boc-anhydride performed at elevated temperatures. Despite the apparent bulk, we had no problems in coupling E to the MPγPNA backbone or assembling the corresponding monomers on MBHA resin. The core structure of F, 4-amino-2-(methylthio)pyridine-5-carbonitrile (17), was prepared according to a published protocol. Subsequent conversion of the cyano group to the amidine followed by Boc protection gave 21. However, attempts to fully protect the exocyclic amine with a large excess of Boc-anhydride under various conditions were unsuccessful as this resulted in the formation of multiple spots on TLC with different numbers of Boc-groups that were difficult to separate by column chromatography. To address this challenge, we implemented Boc protection in two steps. 22 was subjected to oxidation and hydrolysis followed by alkylation and hydrogenolysis to give F. Once prepared, E (15), F (26) and I (27) were coupled to the MPγPNA backbone ( Fig.10 , Scheme 3). Removal of the Alloc protecting group produced the desired monomers 1 to 3. Ligands LG1, LG2, and LG3, as well as LG2P in the opposite (parallel) orientation to LG2P, were prepared on MBHA resin using a PAL-linker and HBTU as a coupling agent. LG2P was included as a negative control because the parent LG2 ligand showed the most potential to bind to the rCAG repeat sequence. A lysine residue was incorporated at the C-terminus to improve aqueous solubility. After the last monomer coupling was completed, the ligand was cleaved from the resin, precipitated with ether, purified by RP-HPLC, and confirmed by MALDI-TOF mass spectrometry (Table D).

[0189] Table D: Molecular weights of LG1, LG2, LG3, LG2P and parent compound M

[0190]

[0191]

[0192] *Contains Na,

[0193]

[0194] Target selection and sample preparation. A series of model RNA targets were selected for binding studies ( Fig.11 The R11X series contains 24 rCXG repeat sequences ( Fig.11 (A)), after adopting the second hairpin structure, it can provide 11 binding sites for the ligand ( Fig.11 (B)). R11A contains a perfect match (AA), while R11U and R11C contain respective UU and CC mismatches for ligand binding. R11G (X=G) has not been identified because it has been shown to form G-quadruplexes and hairpin structures, which may confound the interpretation of experimental results. Single-stranded RNA targets (WS (Watson strand) and CS (Crick strand)) with LG2 binding sites underlined ( Fig.11 (C)), and a double-stranded hairpin HP containing a single ligand binding site ( Fig.11 (D) ) were also examined. Samples were prepared by incubating pre-annealed RNA with the respective ligands at 37°C in 0.1X PBS buffer (1 mM NaPi, 13.7 mM NaCl, 0.27 mM KCl; pH 7.4). This particular buffer was chosen based on our initial screen, where we found that R11A adopted a stable hairpin structure and that the ligands were able to bind.

[0195] Spectroscopic characterization of ligand binding. A combination of UV-Vis and circular dichroism (CD) was used to determine the binding properties of the ligands. UV-Vis measurements showed that all three ligands were able to bind R11A. Evidence for their interaction could be gleaned from the hypochromic and bathochromic shifts in the [R11A+ligand] absorption at 252 and 330 nm. The UV absorbance in the 275-375 nm range corresponds to π-π* transitions of the E group. This finding was confirmed by the CD data, which showed a clear increase and bathochromic shift in the signal at ∼270 nm. Of the series, LG2 exhibited the greatest difference in CD amplitude. CD titration of LG2 with R11A reached a saturation point of 11:1, consistent with the predicted number of ligand binding sites for R11A ( Fig.12 ). We assume that R11A adopts a uniform hairpin structure because it is thermally annealed before ligand addition; however, other combinations of intra- and intermolecular folding are possible. In contrast, LG2 is ligated with the mismatched R11U ( Fig.13(A)) and R11C (data not shown), single-chain WS and CS ( Fig.13 (B)), double-stranded HP containing a single binding site ( Fig.13 (C)) or the mismatched orientation of LG2P and R11A ( Fig.13 (D) No significant differences were observed in the CD signals after incubation with R11U and R11C. Since the results of R11U and R11C mismatches were nearly identical in all respects, from this point on, all discussions of base mismatches focus on R11U. Taken together, these results suggest that the interaction between ligand and RNA occurs in a sequence- and orientation-specific manner, with a preference for duplexes over single-stranded RNA targets and for targets with multiple contiguous binding sites over isolated single sites.

[0196] Ligand binding was confirmed by fluorescence measurements. To further confirm the findings from UV-Vis and CD, we measured the fluorescence signals of the ligands with and without RNA targets after excitation at 330 nm (λmax of the E base). We observed a significant fluorescence quenching of LG3 compared to LG1 and LG2. This drastic reduction in the fluorescence signal can be attributed to photoinduced electron transfer quenching, which is expected to be most effective for LG3 because the fluorophore (E) is stacked between the other two J bases. This interpretation is consistent with the observation that the fluorescence signal of LG3 was fully recovered upon heating (data not shown). For all three ligands, the emission signal was further reduced upon addition of R11A, but most significantly for LG2, which was reduced by 60% compared to 35% and 33% for LG1 and LG3, respectively. We observed similar fluorescence quenching patterns after incubation of LG2 with R11U, [WS+CS], HP, and LG2P with R11A ( Fig.14 ), although less drastic compared to LG2 with R11A. Similar observations were made for related classes of bicyclic pyrimidine analogs, indicating that the fluorescence yield of these fused-ring fluorophores is highly sensitive to the local environment. To test the hypothesis that nonspecific binding of ligand to RNA could lead to fluorescence quenching, we incubated the respective RNA targets with pentamidine before adding LG2 and vice versa. The data show that the fluorescence signal of LG2 with R11U and [WS+CS] and LG2P with R11A was fully restored, but LG2 with R11A was still inhibited ( Fig.14 , inset). This result is consistent with LG2 binding to R11A in a different mode from that of pentamidine, presumably via a defined divalent H-bond interaction ( Figure 1 B and C).

[0197] NMR titration. To gain insight into the binding mode of LG2, we performed a series of multinuclear and multidimensional NMR experiments on LG2, R11A and their combination. Samples were prepared in the same 0.1X PBS buffer as before, containing a 9:1 volume ratio of H2O:D2O. NOESY data were collected at mixing times of 300, 200 and 100 milliseconds to obtain proton-proton distances, while COZY experiments were performed to map the proton spin system of each residue. The hydrogen bonding interaction between LG2 and R11A is expected to result in line broadening and downfield chemical shift of the imino proton signals of the canonical GC / CG pair due to the opening of the base pair, and the appearance of a new set of imino proton signals is due to the formation of H bonds between the ligand and the RNA. Consistent with the CD data, 1 H-NMR experiments showed that R11A adopts a stable hairpin structure in 0.1X PBS buffer, as evidenced by a clear imino proton signal at 12.35 ppm. This chemical shift assignment was further supported by variable temperature measurements, showing a gradual decay of the peak intensity with increasing temperature, while the peak intensity of the non-exchangeable protons remained fairly constant. Partial assignment of the nucleobase protons was performed by NOESY experiments. As expected, upon addition of LG2, the imino proton signal of R11A gradually broadened and shifted downfield ( Fig.15 ). Moreover, the chemical shifts of C4-NH and G2-NH were significantly affected, indicating their interaction with the ligand. Despite much effort, we were unable to assign and monitor the chemical shift of A6-NH when titrating LG2 against R11A due to the degeneracy of the repeat sequence. Nevertheless, this result suggests that the GC / CG base pair opening is mediated by the ligand. However, we did not observe any new imino proton signals in the range of 10-20 ppm, as expected for H-bond formation between the ligand and RNA.

[0198] Template-directed native chemical ligation (NCL). As observed with LG2 and R11A, weak and transient interactions of ligand with RNA are unlikely to result in meaningful biological responses. To further improve the binding affinity of LG2, we synthesized a second LG2 derivative, LG2N ( Fig.16 (A)) containing a C-terminal thioester and N-terminal cystine. This ligand was prepared using a second-generation N-acylurea linker. The bifunctional probe design exploits the conformational pre-organization of MPγPNA to prevent the two functional groups from spontaneously reacting with each other upon disulfide bond reduction. The cystine group is used to provide greater control in probe processing. Previous studies have shown that ligands of the same length containing all natural nucleobases have a reduced (acyclic) half-life of approximately 1 hour at physiological temperature (37°C). We expect LG2N* to have a similar half-life. Fig.16(B) Depicts the reaction pathway of LG2N after disulfide bond reduction, which is the expected chemical state of LG2N in the reducing intracellular environment. In the presence of RNA target, the ligand is expected to form transient divalent H-bond interactions with the nucleobases of the RNA target adjacent to each other due to intermolecular base stacking (step 2). After the disulfide bond is broken, the ligand will undergo template-directed NCL to form a ligation product, thereby binding the RNA template more tightly (step 3). In the absence of RNA target, the ligand will self-inactivate by forming a ring product through an intramolecular NCL reaction (step 4).

[0199] To confirm the prediction that LG2N would remain stable for a limited period of time after disulfide bond reduction before undergoing cyclization, we monitored the reaction progress of the parent compound M, which was in situ derivatized with LG2N* ( Fig.17 (A)). We chose MALDI-TOF over HPLC and other analytical techniques to quantify the reaction products due to the relatively short time frame for intramolecular NCL, which can be completed in less than 5 min with the former. Attempts were made to quench the reaction with an electrophile prior to HPLC analysis. However, this effort was unsuccessful due to the rapid intramolecular reaction of the ligand. 4-Mercaptophenol (4MP) and 2-mercaptoethanol (2ME) were investigated as possible reducing agents. Both produced similar kinetic profiles for the parent compound M; however, the mass-to-charge ratio (m / z) of the intermediate produced by the former overlapped with that of the hydrolyzed parent compound and was therefore difficult to distinguish. For this reason, we chose 4MP for reaction monitoring and gel shift analysis by MALDI-TOF, while 2ME was employed in subsequent melting experiments due to its transparency in the nucleobase absorption region. Our data indicate that upon addition of 4MP to the parent compound M, two reactive species corresponding to the transesterification (M#) and C-terminal thioester (M##) are initially formed ( Fig.17 (A)). They last <15 min and then transform into the reactive LG2N* intermediate and finally into the cyclic product (cLG2N) ( Fig.17 (B)). LG2N* is stable at physiological temperature. It is present as the major product at the 1-h reduction time point, accounting for approximately 75% of the total products in the mixture, with the remaining 25% being primarily cLG2N. The half-life of LG2N* is approximately 3 h, which is approximately three times the natural lifetime. We attribute the chemical stability of LG2N* to the expanded aromatic ring size of the E base, which provides better base stacking interactions, resulting in less conformational flexibility of the ligand.

[0200] Next, we performed UV melting experiments to determine the effect of template-directed NCL on the thermal stability of RNA. Samples were prepared in 0.1X PBS buffer with a 2:1 ratio of the number of LG2N to R11A binding sites and incubated at 37°C for 16 h before UV melting analysis. As expected, without a reducing agent, LG2N had minimal effect on the melting transition (Tm) of R11A, with the highest ΔTm being ~+1°C. Similar findings were seen for LG2, which contains neither a C-terminal thioester nor an N-terminal cysteine. However, in the presence of 2ME, the results were similar whether the incubation of [LG2N+R11A] was performed at ambient temperature or at 37°C. In both cases, the Tm of R11A was significantly increased, ranging from 59-68°C. The derivative of the melting curves showed a broad sigmoidal distribution, indicating the presence of a series of cascade products that are formed and bound to the RNA template. This finding is expected to be applicable to template-directed synthesis. Despite similar Tm, the profiles of the two melting curves are quite different, with one incubated at elevated temperature showing an inverted absorbance in the range of 25–50°C. This melting behavior is characteristic of hydrophobic / aromatic interactions, a phenomenon that has been previously observed in thermophilic foldamers and other aromatic systems such as perylene and pyrene. We attribute the intensity inversion profile to J-group interactions of the cascade product, which are presumably formed in greater amounts at 37°C than at ambient temperature. Upon heating, hydrophobic interactions become more pronounced at a certain point (approximately 50°C), beyond which repulsion occurs. The enhanced thermal stability of R11A is consistent with the formation of the NCL product and its binding to the RNA template.

[0201] To confirm that the Tm enhancement of R11A was due to template-directed cascade and binding of the resulting product to the RNA template, we performed MALDI-TOF analysis on UV-melted samples before heating. The formation of the cascade product was evident from the appearance of new peaks with gradually increasing m / z values ​​with a step size of ∼1319 Daltons, corresponding to the mass of the ligand ( Fig.18 (A)). However, in the absence of target, only the mismatched R11U ( Fig.18 (B)) or a single LG2N ( Fig.18 (C)) No products of these connections were observed. Likewise, there was no evidence that the connected products were formed by single-stranded [WS+CS] or double-stranded HP (data not shown). This result is consistent with the occurrence of template-directed synthesis mediated by the R11A hairpin structure.

[0202] To determine whether LG2N can bind rCAG expTo distinguish between normal repeats, we performed competition binding assays using two RNA targets containing r(CAG)n repeats in the normal (R24A, n=24, identical to R11A) and pathogenic (R127A, n=127) range, as well as a mismatched r(CUG) 96 Control (using R96U). After adopting the respective hairpin motifs, these RNA transcripts will provide 11, 62 and 47 binding sites for LG2N. Two sets of samples were prepared, one in 0.1X PBS and the other in a physiologically relevant buffer (10mM NaPi, 150mM KCl, 2mM MgCl2; pH 7.4). In both groups, equimolar mixtures of R24A (100nM chain concentration, 1.1μM binding sites) and R127 A (18nM chain concentration, 1.1μM binding sites) were incubated with various concentrations of LG2N as well as 4MP, and TCEP at 37°C for 24 hours. TCEP was included to ensure that disulfide bonds were completely reduced. The reaction mixtures were analyzed by agarose gel and stained with SYBR-Gold for visualization. Fig.19 Inspection of the Figure 2 indicated that LG2N preferentially binds to R127A over R24A, as evidenced by the faster disappearance of the top two bands in lanes 2 to 5 than the bottom band. This binding event only occurred in the presence of a reducing agent and with a perfectly matched RNA target, as no evidence of binding was observed in the absence of 4MP and TCEP (compare lane 6 to lane 1) and with the mismatched R96U (compare lane 8 to lane 7). We did not observe the formation of any shifted bands, as would be expected from complexing of the ligand with the RNA, indicating that SYBRGold is unable to intercalate into the RNA-ligand complex. This finding is not surprising, given that ligand binding is known to cause dramatic changes in RNA conformation ( Figure 6 In contrast, we did not observe any signs of ligand binding at physiologically relevant ionic strengths ( Fig. 20 ), indicating that RNA hairpins are unable to mediate template-directed ligand oligomerization under these conditions.

[0203] Many neuromuscular diseases, more than 20 in total, including HD, myotonic dystrophy type 1 (DM1) and type 2 (DM2), spinocerebellar ataxias (SCAs), fragile X syndrome (FXS), Friedreich's ataxia (FRDA), and subsets of amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), are caused by unstable repeat expansions. Expansions in the coding regions of genes can lead to changes in protein function, whereas expansions occurring in noncoding regions can cause disease without interfering with the protein sequence through toxic increases in RNA function and / or unintentional production of deleterious polypeptides through RNA translation. Many of these genetic diseases are inherited in an autosomal dominant manner, requiring only an expansion (or mutation) in one allele to cause disease. Therefore, one approach to interfering with such disease pathways is to target either the affected allele or the corresponding gene transcript. Between the two, the latter offers greater accessibility for exogenous molecules and greater recognition specificity for bivalent ligands such as JB-MPyPNAs, due to their tendency to adopt an imperfect hairpin structure.

[0204] We focused our efforts on rCAG repeats due to their extensive genetic penetration in HD as well as other neurodegenerative diseases including but not limited to MJD, SBMA, DRPLA, and SCA. Molecules that selectively bind to mutant htt transcripts and interfere with the above disease pathways may be useful in treating not only HD but also other related genetic diseases. The nucleic acid ligands described herein differ from conventional antisense agents in several ways. First, they bind to the CAG-RNA hairpin motif via divalent hydrogen bonding interactions. Second, they are relatively small in size, one triplet repeat unit in length. Therefore, they are easier to synthesize, structurally modify, and scale up via solution chemistry as opposed to solid phase chemistry. Third, due to the molecular weight of the small molecule outer rim, such “molecular” systems generally have more favorable pharmacokinetic properties than typical antisense agents. Fourth, J-based recognition is more sequence-specific and selective. More specifically, because a single-base mismatch that would normally occur on one face of a natural nucleobase occurs on both faces of the J base; and it is more selective, preferring double-stranded targets over single-stranded RNA targets because the binding free energies of the two end products are very different. However, unlike small molecules or riboswitches, recognition of J bases follows a well-defined set of rules, namely through bivalent Watson-Crick H-bond interactions, rather than a wider variety of combinations of attractions. In addition, JB-ligand design is modular. In this way, ligands can be prepared and modified to bind to any RNA repeat sequence. In the initial design stage, weak and transient interactions between JB-ligands and normal-length rCAG repeats at low salt concentrations were ideal, as this would allow further improvement of recognition selectivity by tuning the binding affinity of the ligand and by adjusting the ionic strength of the buffer.

[0205] Materials and methods

[0206] UV melting analysis. All UV melting samples were prepared by mixing the ligand with the RNA target at the specified concentration in 0.1XPBS buffer and annealing by incubation at 90°C for 5 minutes, followed by gradual cooling to room temperature. UV melting curves were collected using an Agilent Cary UV-Vis 300 spectrometer equipped with a thermoelectrically controlled multi-sample cell holder. UV melting spectra were collected by monitoring the UV absorption at 260 nm from 25 to 95°C in the heating run and from 95 to 25°C in the cooling run, both at a rate of 1°C per minute. The cooling and heating curves were almost identical, indicating that the hybridization process was reversible. The recorded spectra were smoothed using a 20-point neighbor average algorithm. The first derivative of the melting curve was used to determine the melting temperature of the complex.

[0207] CD analysis. Samples were prepared in 0.1X PBS buffer. All spectra represent the average of at least fifteen scans collected between 200 and 375 nm at a rate of 100 nm / min in a 1 cm pathlength cuvette at 25°C. The CD spectrum of the buffer solution was subtracted from the sample spectrum and then smoothed by a five-point neighbor average algorithm. Steady-state fluorescence measurements. All steady-state fluorescence samples were prepared by mixing the ligand with the specified concentration of RNA in 0.1lX PBS buffer and annealing by incubation at 90°C for 5 minutes and then gradually cooling to 37°C. Prior to measurement, the samples were incubated at 37°C for 1 hour. Steady-state fluorescence data were collected at 25°C by a Cary Eclipse fluorescence spectrometer. (εex=330nm, εem=340-600nm) with a slit size.

[0208] Competitive binding assay R24A was purchased from IDT. R127A [r(CAG) 127 ] and R96U[r(CUG) 96 ]. R24A, R126A, and R96U were prepared in 0.1X PBS buffer and annealed by heating to 90°C for 5 minutes and then gradually cooling to room temperature. Ligands and RNA were mixed at the indicated concentrations and incubated at 37°C for 24 hours. The samples were then loaded onto a 2% agarose gel containing 1X Tris-borate buffer and separated by electrophoresis at 100 V for 25 minutes. The gel was stained with SYBR-Gold and visualized by UV-Transilluminator.

[0209] MD simulations, ligand synthesis and monomer synthesis: The procedures are provided in the attached Supplementary Information and are hereby incorporated into this paper.

[0210] General Technology

[0211] Unless otherwise stated, all starting materials and chemicals were purchased from commercial suppliers and used without further purification. Unless otherwise stated, all reactions were carried out under anhydrous conditions with dry solvents under nitrogen atmosphere. Removal of solvents in vacuo refers to distillation using a rotary evaporator connected to a high vacuum pump. The solid, syrup or liquid products obtained were dried under high vacuum. Analytical thin layer chromatography was performed on pre-coated silica gel plates (60F-254, thickness 0.25 mm); visualization of compounds was by UV light or staining with ninhydrin or iodine, or heating as a developer. NMR spectra were recorded at 300 or 500 MHz with CDCl3, DMSO and D2O as solvents and TMS as internal standard. The following abbreviations are used to explain multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, ddt = doublet of doublet of triplet, dt = doublet of triplet td = triplet of doublet, ABq = AB 四重峰 , dd = doublet of doublet, tt = triplet of triplet, br = wide, wtc. High resolution mass spectrometry (HRMS) was performed using ESI-ESI-TOF mass analyzer and DART analyzer. Low resolution mass spectrometry (LRMS) was performed using ESI-ion trap-MS mass analyzer. Unless otherwise stated, a C18 (15cm x2.1mm, 5μm particles) chromatographic column was used, and linear gradient elution (method A) was performed from 95%H2O (0.01%TFA) to 95%MeCN (0.01%TFA) in 40 minutes, and oligomers were purified on RP-HPLC at a flow rate of 1.0mL / min. MALDI spectra were measured using a TOF / TOF spectrometer. Absorption curves were recorded on a UV-Vis spectrophotometer. Circular dichroism (CD) spectra were recorded on a spectropolarimeter. Fluorescence (emission) was recorded on a fluorescence spectrophotometer.

[0212] MD simulation

[0213] The C-terminal lysine residue was omitted from γPNA, and the MP side chain was substituted with a methyl group (MeγPNA, published X-ray crystal structure, PDB-ID 3PA0). The CEG, AIA, and GFC triplets were constructed using chimera1 and optimized with the HF / 6-31G* basis built in Gaussain. The helical structure of the bound RNA-HD1-RNA complex was created from the triplets using the NAB module of Ambertools, and the modified MeγPNA backbone from the crystal structure was grafted onto the helical structure (see Figure 3). Figure 2A, initial structure). Four such RNA-HD1-RNA complexes were generated, in which the RNA was a duplex containing four rCAG repeats and the number of HD1 ligands bound to the RNA varied from one to four. n -RNA complexes with TIP3P 4 Water molecules are solvated and ions are added to neutralize the charge. The energy of the system is minimized and then the RNA and HD1 ligand atoms are cleaved. The samples were heated to 300 K under harmonic suppression of . The suppression was then released stepwise through a series of six short simulations, and finally an unsuppressed NPT simulation was run at 300 K and 1 bar pressure for 100 ns using the Nose-Hoover thermostat and Parinello-Rahman barostat, respectively. Electrostatic interactions were treated using the particle mesh Ewald method, and all simulations were performed using the GROMACS Amber99-parmbsc0 force field with χOL3 modification for RNA, and the force field parameters for the HD1 ligand were created using the generic amber force field.

[0214] Resin loading [(MP)(PAL)]

[0215] (1) Loading MP. 1 g MBHA resin (1 mmol / g, Peptide International, RMB-2100-PI) was soaked in DCM in a reaction vessel for 1 hour, and then washed with DCM (3X) and 5% DIEA in DCM (3X), see Fig.21 . After confirming that the amine groups have been neutralized (blue) by Kaiser-test, the following solutions are added in sequence to a 15 mL standard tube: 3.5 mL NMP, 450 μL solution A, 460 μL solution B, and 550 μL solution C. The mixture is vortexed for 10 seconds, allowed to stand for 3 minutes, and then added to the resin in the reaction vessel. The reaction is allowed to proceed for 1 hour with gentle shaking. The reaction mixture is discarded under positive air pressure and the resin is washed with DMF (3X), DCM (3X), 5% DIEA in DCM (1X), and DCM (3X). The resin is capped by adding the capping solution to the reaction vessel and gently stirring the vessel for 45 minutes (2X). After discarding the last capping solution, the resin is washed with DCM (3X) and the capping is confirmed to be complete by Kaiser-test (light yellow).

[0216] Solution A: 43 mg Fmoc-MP in 500 μL NMP (0.200 M)

[0217] Solution B: 87 μL DIEA (0.500 M) in 913 μL pyridine

[0218] Solution C: 55 mg HATU in 750 μL NMP (0.201 M)

[0219] Capping solution (Ac2O / NMP / pyridine: 112 / 2): 2 mL Ac2O, 4 mL NMP and 4 mL pyridine

[0220] (2) Load PAL and Lys (more than 1 g of resin). The Fmoc protecting group was removed by treating the resin with a 20% piperidine solution in DMF (2X) for 7 minutes each, followed by washing with DMF (3X) and DCM (3X). After confirming that Fmoc had been successfully removed by the Kaiser-test (blue), a coupling solution was prepared by mixing the following materials: 3 mL of 0.2 M monomer solution, 1.5 mL of 0.52 M DIEA solution, and 1.5 mL of 0.39 M HBTU solution. The mixture was activated for 3 minutes and then added to the resin. The reaction was allowed to proceed for 30 minutes under gentle stirring of the reaction vessel. The completion of the reaction was confirmed by the Kaiser test (light yellow). The resin was washed with DMF (3X), 5% DIEA solution (1X), and DCM (1X), and the unreacted amines were then capped by adding the capping solution to the resin and the reaction vessel was gently stirred for 30 minutes. After removal of the capping solution, the resin was washed with 20% piperidine (2X), DMF (2X) and DCM (2X).

[0221] Monomer solution: 303 mg Fmoc-PAL (0.200 M) in 3 mL NMP 281 mg Fmoc-Lys(Boc)-OH (0.200 M) in 3 mL NMP

[0222] DIEA solution: 364 μL DIEA (0.52 M) in 3.6638 mL DMF

[0223] HBTU solution: 740 mg HBTU (0.39 M) in 5 mL DMF

[0224] Capping solution: (Ac2O / NMP / pyridine: 1 / 25 / 25 by volume)

[0225] Fmoc-deprotection The Fmoc protecting group was removed by treating the resin with a solution of 20% piperidine (0.5 mL, 50 mg resin) in DMF (2X) for 7 min each, followed by washing with DMF (3X) and DCM (3X). Deprotection of Fmoc was confirmed by Kaiser-test (blue).

[0226] After confirming the successful removal of Fmoc (blue) by the Kaiser test for monomer coupling, the resin was washed with DMF (4X) and DCM (5X). The coupling solution was prepared by mixing the following materials: 150 μL of 0.2M monomer solution, 75 μL of 0.52M DIEA solution, and 75 μL of 0.39M HBTU solution. The mixture was activated for 10 minutes. The resin was washed with pyridine (1X) before the monomer solution was added to the resin. The reaction was allowed to proceed for 2-4 hours under gentle stirring in the reaction vessel. The reaction was confirmed to be complete by the Kaiser test (light yellow). The resin was washed with DMF (3X) and DCM (3X).

[0227] Capping Unreacted amines were capped with freshly prepared capping solution. Capping solution (1:25:25; acetic anhydride:NMP:Py) was added to the resin and the reaction vessel was stirred for 4 minutes. The resin was washed with DMF (4X) and DCM (5X).

[0228] After the last Fmoc deprotection step, the resin was washed with DMF (5X) and DCM (8X). The resin was dried under vacuum for 15 minutes. To the dried resin, freshly prepared 95% TFA: 5% m-cresol (0.6 mL for 50 mg of resin) was added and the reaction vessel was kept in standby mode. After 1 hour at room temperature, the TFA solution was collected in a centrifuge tube. Again, the cleavage solution (0.5 mL for 50 mg of resin) was added to the resin and the reaction vessel was allowed to stand for 30 minutes. The TFA solution was combined with the previously collected solution.

[0229] Precipitation To the collected TFA solution, add cold dry ether (-60°C, 14 mL) and shake. Precipitation occurs within 30 minutes. The precipitated oligomers are collected by centrifugation and washed with cold ether (2x).

[0230] Purification The crude oligomer was dissolved in 0.5 mL of 95% water, 5% acetonitrile and 0.1% TFA. The resulting solution was purified by reverse phase analytical column.

[0231] Example 2 - Monomer Synthesis

[0232]

[0233] Monomer E (1): Tetrakis(triphenylphosphine)palladium (28.22 mg, 24.42 μmol) was added to a mixture of phenylsilane (0.12 mL, 0.98 mmol) and allyl monomer 29a (0.60 g, 0.49 mmol) in anhydrous dichloromethane (10 mL) at room temperature. Complete conversion of the starting material to the corresponding acid was observed in 15 h by TLC (eluent: EA:Hex(SO:SO); Rf of 29a was 0.6; product Rf = 0.0 (dragging)). Silica gel was added to the reaction mixture at room temperature and the solvent was removed on a rotary evaporator. The crude silica gel absorbed product was purified by flash silica gel chromatography to give the desired product. Yield: 0.44 g, 78%. 1 H NMR (S0 0.13 MHz, DMSO-d6, rotamer): δ 1.08 / 1.09 (s / s, 9H), 1.25-1.28 (m, 18H), 1.37-1.38 (m, 18H), 1.41 / 1.6S (s / s, 9H), 3.12-3.64 (m, 13H), 3.77-3.90 (m, 2H), 3.90-4.00 (m, 2H), 3.99-4.4.34 (m, 4H), 7.21-7.49 (m, 5H), 7.54-7.79 (m, 2H), 7.89 (d, J = 7.2 Hz, 2H), 8.55 / 8.64 (s / s, 1H); 13 C NMR (125.77 MHz, DMSO-d6, rotamer): δ 27.2-27.6 (18C, t Bu), 35.8, 46.7, 54.9, 59.7, 60.6 / 60.6(2C), 65.6 / 65.6, 69.6 / 69.6, 69.8 / 69. 9, 70.4 / 70.4, 72.2 / 72.2, 82.1(3C), 82.2, 83.2, 83.4, 86.2, 108.1, 120.1(2C), 125.2, 127.0(4C), 127.6(4C), 140.7(2C), 143.8 / 143.8, 147.9, 149.2 / 149.3, 149.3, 149.5, 149.7, 155.0 / 155.9, 156.3 / 156.5, 162.3, 166.6 / 166.6; HRMS: [C 61 H 85 N7O 17 +H] m / z calculated: 1188.6080, found: 1188.6071.

[0234]

[0235] Monomer I (2): The compound was prepared by the above process of compound 1, using compound 29b (3.10 g, 4.39 mmol) as the starting material. TLC (eluent: MeOH:DCM (5.95); Rr of 29b = 0.2; R of 2 f =0.0). Yield: 2.22 g, 76%. 1 H NMR (300.13MHz, DMSO-d6): δ1.09 (bs, 9H), 3.11-3.52 (m, 15H), 3.87-3.96 (m, 2H), 4.24 (dd, J=13.4, 5.4Hz, 4H), 7. 19-7.44 (m, SH), 7.69 (d, J=7.5Hz, 2H), 7.89 (d, J=7.4Hz, 2H), 10.71-10.75 (m, 1H), 11.06 (bs, 1H), 12.46 (bs, 1H); 13 C NMR (126 MHz, DMSO-d6, rotamers): δ 27.8 (3C), 47.2 / 47.2, 61.1 (3C), 65.6 / 66.0, 70.1 (2C), 70.3 / 70.4, 70.9 (3C), 72.7, 107.8, 120.6-128.1 (10C), 139.7 / 140.1, 141.2 (2C), 144.3, 151.8, 156.3, 164.6, 170.9; HRMS: [C 34 H 42 N4O 10 m / z calculated for [(+H): 667.2979, found: 667.2988.

[0236]

[0237] Monomer F (3): This compound was prepared by the above process for compound 1, using compound 29c (2.50 g, 2.00 mmol) as the starting material. TLC (eluent: EA: Hex (50:50); Rf of 29c = 0.6; Rf of 3 = 0.0 (dragging)). Yield: 1.94 g, 80%. 1H NMR (300.13 MHz, DMSO-d6, rotamer): δ 1.08 (s, 9H), 1.35 (s, 9H), 1.36 (s, 9H), 1.41 / 1.42 (s / s, 18H), 3.20-3.62 (m, 20H), 3.77-4.15 (m, 4H), 4.18-4.34 (m, 4H), 4.82–5.35 (m, 2H), 7.17-7.46 (m, 5H), 7.70 (d, J=7.0 Hz, 2H), 7.88 (d, J=7.5 Hz, 2H), 8.93 / 8.95 (s / s, 1H); 13 C NMR (125.77 MHz, DMSO-d6, rotamer): δ 27.3-27.5 (18C, t Bu), 46.7 / 46.7, 60.6 (2C), 60.7 (2C), 69.6 (2C), 69.7, 69.9, 70.4 (3C), 72.2, 82.1, 82.1, 82.9, 82.9, 83.3, 83.4, 108.0, 110.8, 118 .4, 120.1(2C), 123.1, 125.2 / 125.3, 127.0(4C), 127.6(4C), 140.7(2C), 143.8, 146.9, 149.5, 156.6, 157.1, 162.4 / 162.4.HRMS: [C 60 H 85 N7O 19 m / z calculated for [(+H): 1208.5980, found: 1208.5959.

[0238]

[0239] (4-chloro-pyridin-2-yl)-carbamic acid tert-butyl ester (5): To a stirred solution of 2-amino-4-chloropyridine (1) (50.00 g, 0.39 mol) in 556 mL of anhydrous THF in an ice bath was added Et3N (81.37 mL, 0.58 mol) dropwise in an inert atmosphere over 10 min. After stirring at the same temperature for another 10 min, a solution of Boc2O (84.88 g, 0.39 mol) in THF (100 mL) was added dropwise over 30 min, followed by a catalytic amount of DMAP (4.75 g, 0.04 mol). After stirring at room temperature overnight, the reaction mixture was diluted with ethyl acetate (200 mL). The combined organic layers were filtered through a sinter funnel, and the filtrate was washed with saturated NaHCO3 solution and dried over Na2SO4. The solvent was removed on a rotary evaporator to give the pure product. Yield: 67.59 g, 76%. 1 H NMR (300.13MHz, CDCl3): δ1.54 (s, 9H), 6.95 (dd, J=5.5, 1.8Hz, 1H), 8.12 (d, J=1.9Hz, 1H), 8.24 (d, J=5.5Hz, 1H), 9.96 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ28.5 (3C), 81.5, 112.9, 118.7, 146.2, 148.3, 152.6, 153.8; ESI-MS: [C 10 H 13 m / z calculated for ClN2O2+H]: 229.1, found: 229.2.

[0240]

[0241] Ethyl 2-((tert-butoxycarbonyl)amino)-4-chloronicotinate) (6): To a stirred solution of tert-butyl (4-chloro-pyridin-2-yl)carbamate (30.50 g, 0.13 mol) in 534 mL of anhydrous THF in a dry ice acetone bath (-78°C) was added n-BuLi (24.86 mL, 0.27 mol, 11 M) dropwise over 1 h in an inert atmosphere. After stirring at -78°C for an additional 30 min, ClCO2Et (13.93 mL, 0.15 mol) was added dropwise over the course of 10 min. The progress of the reaction was checked by TLC. After complete consumption of the starting material, the reaction mixture was slowly quenched with 1% HCl (200 mL). The resulting reaction mixture was transferred to a separatory funnel containing 10% HCl (50 mL) and EA (200 mL). The crude product was extracted from the aqueous layer with EA, and the combined organic layers were dried over Na2SO4. The resulting solvent was removed on a rotary evaporator, and the resulting crude product was purified by column chromatography to obtain ethyl 2-((tert-butoxycarbonyl)amino)-4-chloronicotinate as a pale yellow solid. Yield: 29.68 g, 74%. 1H NMR (500.13 MHz, CDCl3): δ 1.40 (t, J = 7.2 Hz, 3H), 1.50 (s, 9H), 4.41 (q, J 7.2 Hz, 2H), 7.08 (d, J = 5.2 Hz, 1H), 8.31 (d, J = 5.2 Hz, 1H), 8.62 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ14.1, 28.3 (3C), 62.3, 81.8, 120.8, 144.7, 149.9, 150.0, 151.3, 151.8, 164.9; HRMS: [C 13 H 17 m / z calculated for ClN2O4+Na]: 323.0774, found: 323.0777.

[0242]

[0243] Ethyl 2-amino-4-chloronicotinate (7): Ethyl 1,2-((tert-butyloxycarbonyl)amino)-4-chloronicotinate (29.00 g, 0.10 mol) was added to a mixture of concentrated HCl (52.60 mL, 0.58 mol) and dichloromethane (64 mL) at room temperature (slow addition). After 2 hours at room temperature, the completion of the starting material was monitored by TLC, and the reaction mixture was then cooled to 0°C and neutralized with 2N NaOH solution. The reaction mixture was transferred to a separatory funnel containing 100 mL of dichloromethane, and the compound was extracted from the aqueous layer into the organic layer. The combined dichloromethane layers were dried over anhydrous Na2SO4 and concentrated. The crude material was purified by silica gel column chromatography to give pure ethyl 2-amino-4-chloronicotinate as a white solid. Yield: 18.38 g, 95%. 1 H NMR (500.13MHz, CDCl3): δ1.41 (t, J=7.1Hz, 3H), 4.42 (q, J=7.1Hz, 2H), 6.13 (bs, 2H), 6.68 (d, J=5.3Hz, 1H), 7.97 (d, J=5.4Hz, 1H); 13 C NMR (125.77 MHz, CDCCl3): δ 14.2, 61.8, 108.3, 115.9, 145.6, 151.2, 159.9, 166.4; HRMS: m / z calculated for [C8H9ClN2O2+H]: 201.0431, found: 201.0422.

[0244]

[0245] Ethyl 2-amino-4-chloro-5-bromonicotinate (8): To a solution of 7 (18.00 g, 0.09 mol) in anhydrous DMF was added NBS (18.36 g, 0.10 mol) in portions at room temperature. Analysis of the resulting solution by TLC showed that 8 was completely consumed after 4 hours. The resulting mixture was quenched with water (100 mL) and extracted with ethyl acetate (EA, 2×100 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate (Na 2SO 4), filtered and concentrated. The resulting residue was purified by silica gel column chromatography (100-200 mesh) to obtain only ethyl 2-amino-4-chloro-5-bromonicotinate (8) as a light yellow solid. Yield: 22.07 g, 88%. 1H NMR (500.13MHz, CDCl3): δ1.41 (t, J=7.1Hz, 3H), 4.43 (q, J=7.1Hz, 2H), 5.86 (s, 2H), 8.24 (s, 1H); 13 C NMR (125.77 MHz, CDCl3): δ 14.2, 62.3, 109.7, 110.4, 144.4, 152.8, 158.0, 165.9. HRMS: m / z calculated for [C8H8BrClN2O2+H]: 278.9536, found: 278.9541.

[0246]

[0247] Ethyl 2-((tert-butoxycarbonyl)amino)-4-chloro-5-bromonicotinate) (9): To an ice-cold solution of 8 (20.00 g, 71.55 mmol) in anhydrous dichloromethane (CH2Cl2) was added diisopropylethylamine (DIEA, 27.42 mL, 157.41 mmol) followed by triphosgene (7.64 g, 25.76 mmol). After 1 hour at 0°C, tert-butyl alcohol (33.96 mL, 357.76 mmol) was added over the course of 5 minutes and the ice bath was removed. The progress of the reaction was monitored by TLC and after 24 hours at room temperature, the starting material had been completely consumed. The resulting mixture was quenched with water and extracted with dichloromethane (CH2Cl2, 2×100 mL). The collected organic layer was washed with brine, saturated sodium bicarbonate solution (NaHCO3), dried over anhydrous sodium sulfate, filtered and concentrated. The crude material obtained was purified by silica gel column chromatography (100-200 mesh) to give ethyl 2-(((tert-butoxycarbonyl)amino)amino-4-chloro-5-bromonicotinate (9) as a white solid. Yield: 13.58 g, 50%. 1 H NMR (500.13MHz, CDCl3): δ1.41 (t, J=7.2Hz, 3H), 1.50 (s, 9H), 4.42 (q, J 7.2Hz, 2H), 8.37 (s, 1H), 8.57 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ14.1, 28.3 (3C), 62.6, 82.2, 113.9, 116.3, 119.9, 144.0, 149.5, 151.7, 164.2; HRMS: [C 13 H 16m / z calculated for BrClN2O4+H]: 379.0060, found: 379.0063.

[0248]

[0249] 5-Bromo-2-((tert-butoxycarbonyl)amino)-4-chloronicotinic acid) (10): 41.41 mL of 1 M sodium hydroxide (118.53 mmol) was added to a mixture of 9 (10.00 g, 26.34 mmol) in ethanol:THF (3:1, 76 mL) at 0 °C over 15 min. After complete consumption of the starting material as monitored by TLC, the reaction mixture was concentrated. The obtained crude material was transferred to a separatory funnel containing water (200 mL) and extracted with EA (1 x 100 mL), and the organic layer was discarded. 10% HCl (200 mL) and EA (100 mL) were carefully added to the aqueous layer (pH ~ 3-5). The compound was extracted in EA layer, dried over anhydrous sodium sulfate and concentrated to give pure 5-bromo-2-((tert-butoxycarbonyl)amino)-4-chloronicotinic acid (10) as a light yellow solid. Yield: 6.02 g, 65%. 1 H NMR (500.13MHz, DMSO-d6): δ1.43 (s, 9H), 8.69 (s, 1H), 9.88 (s, 1H); 13 C NMR (125.77MHz, DMSO-d6): δ27.9 (3C), 80.1, 116.3, 124.5, 141.2, 148.8, 150.8, 152.6, 164.1; HRMS: calculatedm / z for [C 11 H 12 m / z calculated for BrClN2O4+H]: 350.9747, found: 350.9712.

[0250]

[0251] (11) It was difficult to purify this compound by column chromatography. Therefore, we proceeded to the next step without any purification or analysis. HRMS: [C 17 H 23 m / z calculated for BrClN5O5+H]: 492.0649, found: 492.0655.

[0252]

[0253] Di-tert-butyl(8-bromo-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidine-2,5-diyl)dicarbamate) (12): To an ice-cold solution of 10 (5.00 g, 11.07 mmol) in anhydrous dimethylformamide (DMF, 28 mL) was added N-methylmorpholine (NMM, 1.64 mL, 14.94 mmol) followed by ethyl chloroformate (1.63 mL, 13.84 mmol). After 1 hour at 0°C, Boc-guanidine (2.38 g, 14.94 mmol) was added in one portion and the ice bath was removed. The progress of the reaction was monitored by TLC; the reaction was followed by HPLC. After 24 hours at room temperature, the starting material was completely consumed. The resulting mixture was concentrated to dryness to give Boc-guanidine adduct as a crude mixture, to which water was added and stirred at room temperature for 10 minutes. The solid obtained was filtered through filter paper and dried overnight under vacuum. NaH (60%, 1.77 g, 44.28 mmol) was carefully added to an ice-cold solution of the resulting solid in anhydrous DMF. After 24 hours, the DMF was removed in vacuo and water was carefully added. The resulting solid was collected by filtration and dried in vacuo. The crude product obtained was purified by silica gel column chromatography (100-200 mesh) to give 12 as a yellow solid. Yield: 3.64 g, 72% (in two steps). 1 H NMR (500.13MHz, DMSO-d6): δ1.48 (s, 9H), 1.50 (s, 9H), 8.54 (s, 1H), 10.85 (s, 1H), 11.58 (s, 1H), 11.78 (s, 1H); 13 C NMR (125.77MHz, DMSO-d6): δ27.7(3C), 27.8(3C), 80.3, 83.2, 103.2, 109.2, 149.0, 150.4, 152.0, 152.6, 153.7, 154.2, 161.7; HRMS: [C 17 H 22 m / z calculated for BrN5O5+H]: 456.0883, found: 456.0890.

[0254]

[0255] Di-tert-butyl(8-bromo-4-(tert-butoxy)pyrido[4,3-d]pyrimidine-2,5-diyl)bis((tert-butoxycarbonyl)-carbamate))(13): To a solution of 12 (3.10 g, 6.79 mmol) in anhydrous THF was added DIEA (3.50 mL, 20.38 mmol) and DMAP (0.17 g, 1.36 mmol) followed by Boc2O (5.93 g, 27.18 mmol) at room temperature. After 4 h at room temperature, Boc2O (2.97 g, 13.59 mmol) was added and the reaction mixture was heated to 50°C. The consumption of the starting material was monitored by TLC, and after 24 hours, the reaction mixture was diluted with a saturated solution of NaHCO3 and EA. The organic layer was separated from the aqueous layer and dried over sodium sulfate. The solvent was removed in vacuo, and the obtained crude material was purified by silica gel column chromatography to obtain pure 13 as a white solid. Yield: 3.15 g, 65%. 1 H NMR (500.13MHz, DMSO-d6): δ1.27 (s, 18H), 1.45 (s, 18H), 1.65 (s, 9H), 9.06 (s, 1H); 13 C NMR (125.77MHz, DMSO-d6): δ27.2(6C), 27.4(6C), 27.5(3C), 82.7(2C), 83.8(2C), 87 .6, 109.7, 118.2, 148.7, 148.8(2C), 149.6(2C), 150.8, 155.0, 156.4, 166.5; HRMS: [C 31 H 46 m / z calculated for BrN5O9+H]: 712.2557, found: 712.2540.

[0256]

[0257] Di-tert-butyl(8-allyl-4-(tert-butoxy)pyridor-4,3-dlpyrimidine-2,5-diyl)bis((tert-butoxycarbonyl)carbamate))(14): To a stirred and degassed solution of 13 in anhydrous toluene (21 mL) (3.00 g, 4.21 mmol) was added allyltributyltin (2.79 g, 8.42 mmol) followed by tetrakistriphenylphosphinepalladium(0) (0.49 g, 0.42 mmol) at room temperature. The resulting solution was flushed with argon to form an inert atmosphere and the solution was refluxed at 125°C for 15 hours. The resulting mixture was cooled to room temperature, silica gel was added, the solvent was removed in vacuo, and the slurry was loaded on silica gel column chromatography to afford 14 as a light yellow solid. Yield: 1.70 g, 60%. 1 H NMR (500.13MHz, CDCl3): δ1.32 (s, 18H), 1.47 (s, 18H), 1.71 (s, 9H), 3.78 (dq, J=6. 6, 1.3Hz, 2H), 4.94-5.15 (m, 2H), 6.09 (ddt, J=16.7, 10.1, 6.4Hz, 1H), 8.49 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ27.8(6C), 27.9(6C), 28.2(3C), 32.4, 82.4(2C), 83.3(2C), 86.3, 10 8.8, 116.5, 131.4, 136.0, 148.1(2C), 149.8(2C), 150.3(2C), 155.6, 156.8, 167.1; HRMS: for[C 34 H 51 N5O9+H] m / z calculated value: 674.3765, found value: 674.3746.

[0258]

[0259] 2-(2,5-bis(bis(tert-butoxycarbonyl)amino)-4-(tert-butoxy)pyrido[4,3-d]pyrimidin-8-yl)acetic acid) (15, E): To an ice-cold stirred solution of 14 (1.00 g, 1.48 mmol) in CH3CN:CCl4:H2O (23:23:34 mL) was added Na1O4 (2.54 g, 11.87 mmol) followed by ruthenium(III) chloride hydrate (0.07 g, 0.30 mmol). After 2 h at 0°C, the reaction mixture was filtered through a bed of celite and concentrated in vacuo to 3 / 4 volume at 35°C. To the mixture was added water (20 mL) and EA (20 mL) at 25°C. The organic layer was separated from the aqueous layer and dried over sodium sulfate. The resulting solvent was removed under vacuum and the obtained crude material was purified by silica gel column chromatography to give 15(E) as a light brown solid. Yield: 0.51 g 50%. 1 H NMR (300.13MHz, CDCl3): δ1.34 (s, 18H), 1.56 (s, 18H), 1.71 (s, 9H), 4.03 (s, 2H), 8.62 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ28.0(6C), 28.1(6C), 28.3(3C), 37.1, 83.2(2C), 85.1(2C), 87. 7, 109.0, 124.5, 149.6, 149.9(2C), 150.1, 155.9(2C), 156.5(2C), 167.4, 170.4; HRMS: [C 33 H 49 N5O 11 m / z calculated for [(+H): 692.3507, found: 692.3491.

[0260]

[0261] 4-amino-2-(methylthio)pyrimidine-5-carbonitrile (17): A mixture of 2-(ethoxymethylene)malononitrile (100.00 g, 0.82 mol), S-methylisothiourea hemisulfate (73.81 g, 0.82 mol) and triethylamine (370.92 mL, 2.66 mol) in absolute ethanol (819 mL) was stirred at room temperature. After 2 days, the solvent was removed in vacuo and the resulting residue was washed with water to give the desired compound as a white solid. Yield: 122.48 g, 90%. 1 H NMR (500.13MHz, DMSO-d6): δ2.45 (s, 3H), 7.87 (s, 2H), 8.43 (s, 1H); 13 C NMR (125.77 MHz, DMSO-d6): δ 13.4, 85.3, 115.6, 160.5, 161.4, 174.6; ESI-MS: m / z calculated for [C6H6N4S+H]: 167.0, found: 167.1.

[0262]

[0263] 4-amino-N'-hydroxy-2-(methylthio)pyrimidine-5-carboximidamide (18): A mixture of 17 (50.00 g, 0.30 mol), hydroxylamine hydrochloride (24.04 g, 0.35 mol) and triethylamine (83.92 mL, 0.60 mol) in methanol (602 mL) was refluxed at 75°C. After 2 days, the reaction mixture was cooled to room temperature and the precipitated solid was collected by filtration and washed with methanol and hexane to give 18 as a light yellow solid. Yield: 46.15 g, 77%. 1 H NMR (500.13MHz, DMSO-d6): δ2.43 (s, 3H), 5.96 (s, 2H), 7.59 (s, 1H), 8.10 (s, 1H), 8.35 (s, 1H), 9.87 (s, 1H); 13 C NMR (125.77 MHz, DMSO-d6): δ 13.2, 103.6, 149.9, 152.8, 159.6, 169.3; ESI-MS: m / z calculated for [C6H9N5OS+H]: 200.1, found: 200.0.

[0264]

[0265] (E)-N'-acetoxy-4-amino-2-(methylthio)pyrimidine-5-carboximidamide (19): To a solution of 18 (31.00 g, 0.16 mol) in glacial acetic acid was added acetic anhydride (51.48 mL, 0.55 mol) at room temperature. Analysis of the resulting solution by TLC showed that 18 had been completely consumed after 15 hours. The acetic acid was removed under vacuum and saturated sodium bicarbonate solution and EA (200 mL each) were added to the crude material (pH-8-9). The aqueous layer was extracted twice with EA and the organic layer was dried over sodium sulfate. The EA was removed under vacuum to give pure 19 as a white solid. Yield: 28.02 g, 90%. 1 H NMR (500.13MHz, DMSO-d6): δ2.15 (s, 3H), 2.45 (s, 3H), 6.89 (s, 2H), 7.78 (s, 1H), 7.96 (s, 1H), 8.40 (s, 1H); 13 C NMR (125.77MHz, DMSO-d6): δ13.3, 19.5, 102.4, 154.5, 154.6, 159.8, 167.9, 171.3; HRMS: [C8H 11 m / z calculated for [N5O2S+H]: 242.0712, found: 242.0718.

[0266]

[0267] 4-amino-2-(methylthio)pyrimidine-5-carboximidamide (20): A mixture of 19 (25.00 g, 0.10 mol), formic acid (97.73 mL, 2.59 mol) and palladium / carbon (10%, 5.51 g, 5.18 mmol) in methanol (130 mL) was refluxed at 80°C. After 15 hours, the reaction mixture was cooled to room temperature and the solvent was removed in vacuo to give the formate salt of the desired product. Yield of formate salt of the product: 29.96 g, 80%. (For NMR analysis: methanol (10 mL) and TEA (5 mL, pH-9-10) were added to the obtained crude product (1 g) and stirred for 15 min. The obtained solid was collected by filtration and washed with hexane.) 1H NMR (500.13 MHz, DMSO-d6): δ2.43 (s, 3H), 5.96 (s, 2H), 7.59 (s, 1H), 8.11 (s, 1H), 8.36 (s, 1H), 9.87 (s, 1H); 13C NMR (125.77 MHz, DMSO-d6): δ13.3, 103.6, 149.9, 152.9, 159.6, 169.4; HRMS: m / z calculated for [C6H9N5S+H]: 184.0657, found: 184.0664.

[0268]

[0269] Tert-Butyl((4-amino-2-(methylthio)pyrimidin-5-yl)(imino)methyl)carbamate) (21): To an ice-cold stirred solution of 20 as formate salt (22.00 g, 68.47 mmol) in THF:water (100:125 mL) was added sodium bicarbonate (34.51 mL, 0.41 mol) portionwise. When the pH of the resulting solution reached 8-9, phenylacetic anhydride (17.19 g, 78.74 mmol) in 25 ml THF was slowly added. Analysis of the resulting solution by TLC indicated complete consumption of 20 after 24 h. The reaction mixture was transferred to a separatory funnel containing 100 mL water and 200 mL EA. The aqueous layer was extracted with EA (2X) and the combined EA layers were dried over sodium sulfate. EA was removed under vacuum and the mixture was purified by silica gel column chromatography to give pure 21 as a white solid. Yield: 15.71 g, 81%. 1 H NMR (300.13MHz, CDCl3): δ1.51 (s, 9H), 2.50 (s, 3H), 7.61 (s, 2H), 8.42 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ13.9, 28.1(3C), 79.6, 104.6, 155.2(2C), 161.1(2C), 173.9; HRMS: [C 11 H 17 m / z calculated for [N5O2S+H]: 284.1181, found: 284.1189.

[0270]

[0271] Tert-butyl(Z)-((4-(bis(tert-butoxycarbonyl)amino)-2-(methylthio)pyrimidin-5-yl)((tert-butoxycarbonyl)imino)methyl)(tert-butoxycarbonyl)carbamate) (22) To an ice-cold stirred solution of 21 (15.00 g, 52.94 mmol) in THF (106 mL) was added DIPEA (46.11 mL, 264.69 mmol) and DMAP (1.29 g, 10.59 mmol) in portions followed by Boc-anhydride (57.77 g, 264.69 mmol) in 25 mL Solution in THF (slowly added). Monitor the reaction process by TLC, and the result shows that 21 has been completely consumed after 24 hours. The reaction mixture is transferred to a separatory funnel comprising 200mL water and 100mL EA. The mixture is extracted with EA (2X), and the combined EA layer is dried over sodium sulfate. EA is removed in vacuo, and the crude mixture is purified by silica gel column chromatography to obtain pure 22, which is a white solid. Yield: 28.96g, 80%. 1 13C NMR (125.77MHz, CDCl3): δ14.6, 28.0(6C), 28.1(6C), 28.1(3C), 83.2, 83.7(2C), 84 .9(2C), 119.2, 148.3, 150.1, 157.3(2C), 158.8, 159.5(2C), 160.4, 176.1; HRMS: [C 31 H 50 N5O 10 m / z calculated for [S+H]: 684.3278, found: 684.3275.

[0272]

[0273] Compound (23): To an ice-cold stirred solution of 22 (15.00 g, 21.94 mmol) in DCM (55 mL) was added mCPBA (10.60 g, 61.42 mmol) in portions. The progress of the reaction was analyzed by TLC, which showed that 22 was completely consumed after 3 hours. The reaction mixture was transferred to a separatory funnel containing 200 mL of a saturated solution of sodium bicarbonate and 100 mL of DCM. The mixture was extracted with DCM (2X) and the combined DCM layers were dried over sodium sulfate. The DCM was removed in vacuo and the crude mixture was used in the next step without further purification.

[0274]

[0275] Tert-butyl(Z)-((4-(bis(tert-butoxycarbonyl)amino)-2-oxo-1,2-dihydro-pyrimidin-5-yl)((tert-butoxycarbonyl)imino)methyl)(tert-butoxycarbonyl)carbamate)(24): To an ice-cold stirred solution of crude 23 (15.00 g) in THF (55 mL) was slowly added 2N NaOH (54.84 mL, 109.68 mmol). The starting material consumption was monitored by TLC. After 30 min, the reaction mixture was transferred to a separatory funnel containing 200 mL of 1% HCl and 100 mL of EA. The aqueous layer was extracted with EA (2X) and the combined EA layers were dried over sodium sulfate. The EA was removed in vacuo and the crude material was purified by silica gel column chromatography. Yield: 10.75 g, 75% in two steps. 1 H NMR (300.13MHz, DMSO-d6): δ1.37 (s, 18H), 1.39 (s, 18H), 1.41 (s, 9H), 8.65 (s, 1H), 12.80 (s, 1H); 13 C NMR (125.77MHz, DMSO-d6): δ27.4(15C), 82.0, 82.9(2C), 83.5(2C), 108.0, 145.2, 147.5(2C), 149.1(2C), 152.8, 155.0, 157.0, 162.9; HRMS: [C 30 H 47 N5O 11 m / z calcd for [(4-(4-hydroxyethyl)propene]: 654.3350, found: 654.3359.

[0276]

[0277] Benzyl(Z)-2-(4-(bis(tert-butoxycarbonyl)amino)-2-oxo-5-(N,N,N'-tris(tert-butoxycarbonyl)carbamimidoyl)pyrimidin-1(2H)-yl)acetate (25): To a mixture of compound 24 (10.00 g, 15.30 mmol) and K2CO3 (4.23 g, 30.59 mmol) in anhydrous DMF (75 mL) was added benzyl acetate-2-bromoacetate (2.42 mL, 15.30 mmol) dropwise over 10 min. The progress of the reaction was monitored by TLC and was found to be complete after 4 h. The reaction mixture was filtered through a sinter funnel and DMF was evaporated under vacuum. Water was added to the resulting crude material and stirred for 5 minutes. The precipitate obtained was collected by filtration and washed with water and hexane to obtain pure compound 25 as a white solid. (Note: If the compound is not precipitated well, an extraction method can be used to obtain the compound. If necessary, a short bed chromatographic column can be used to obtain the pure compound). Yield: 10.92 g, 89%. 1 H NMR (500.13MHz, CDCl3): δ1.46 (s, 18H), 1.48 (s, 18H), 1.50 (s, 9H), 4.75 (s, 2H), 5.22 (s, 2H), 7.27-7.47 (m, 5H), 7.95 (s, 1H); 13 C NMR (125.77MHz, CDCl3): δ27.8(6C), 27.9(6C), 28.0(3C), 51.0, 68.2, 83.0, 83.7(2C), 85.2(2C), 110.6, 128.6(3C), 128.7(2C), 128.8, 134.5, 148.2, 149.5(2C), 152.0(2C), 154.4, 157.1, 163.9, 166.1; HRMS: [C 39 H 55 N5O 13 m / z calculated for [(+H): 802.3875, found: 802.3882.

[0278]

[0279] (Z)-2-(4-(bis(tert-butoxycarbonyl)amino)-2-oxo-5-(N,N,N'-tris(tert-butoxycarbonyl)carbamimidoyl)pyrimidi-1(2H)-yl)acetic acid) (26, F): Palladium on carbon (10% loading, 0.66 g, 0.62 mmol) was carefully added to a solution of 25 (5.00 g, 6.24 mmol) in EA (50 mL) under an argon balloon. After 5 min, the argon balloon was replaced with a hydrogen balloon and the reaction mixture was stirred under the same atmosphere for another 1 h. Complete consumption of the starting material was assessed by TLC. The resulting reaction mixture was carefully filtered through a celite bed and the filtrate was evaporated on a rotary evaporator to give pure compound 26 as a white solid. Yield: 3.99 g, 90%. 1 H NMR (500.13MHz, DMSO-d6): δ1.36 (s, 18H), 1.41 (s, 18H), 1.42 (s, 9H), 4.85 (s, 2H), 9.10 (s, 1H), 13.45 (s, 1H); 13 C NMR(125.77MHz, DMSO-d6):0 27.4(6C), 27.4(6C), 27.5(3C), 50.9, 82.2, 83.0(2C), 83.3(2C), 108.3, 145.1, 146.9(2C), 149.4(2C), 154.1, 156.1, 157.0, 162.5, 168.4; HRMS:

[0280] [C 32 H 49 N5O 13 m / z calculated for [(+H): 712.3405, found: 712.3386.

[0281]

[0282] 2-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)acetic acid (27): This compound was purchased from a commercially available source.

[0283]

[0284] Allyl(R)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)propyl)glycinate (28): To a cooled solution of N-Fmoc(O-MP(tert-butyl)-L-serinol (5.00 g, 10.93 mmol) in saturated DCM:water (28 mL) was added Dess-Martin reagent (9.96 g, 23.49). The reaction mixture was stirred at the same temperature for another 30 min and at room temperature for 1 h to complete the conversion of the alcohol to the aldehyde. The reaction mixture was diluted with ether, saturated sodium bicarbonate solution, 10% sodium thiosulfate, and 4% ethyl acetate. The mixture was stirred for 5 min. The entire reaction mixture was transferred to a separatory funnel and the organic compound was extracted with ether (2x). The ether layer was dried over sodium sulfate and removed with a rotary evaporator to obtain a crude aldehyde. The aldehyde was used for the next step without further purification. Glycine allyl acid PTSA salt (6.28 g, 21.86 mmol) and DIEA (5.90 mL, 33.89 mmol) were added to an anhydrous DCM (44 mL) solution of the aldehyde and reacted for 1 hour. A portion of NaB (OAc) 3 H (5.79 g, 27.33 mmol) was added to the reaction mixture. After stirring at room temperature overnight, the reaction mixture was diluted with DCM (100 mL) and a saturated solution of sodium bicarbonate. The reaction mixture was transferred to a separatory funnel and the layer was separated from the aqueous layer. The combined organic layer was dried over sodium sulfate and removed on a rotary evaporator, and the crude mixture was purified by flash silica gel column chromatography. Yield: 4.85 g, 85%. 1 HNMR (500.13MHz, CDCl3): δ1.19 (s, 9H), 2.77-2.92 (m, 2H), 3.49-3.69 (m, 13H), 3. 88 (bs, 1H), 4.23 (t, J=6.9Hz, 1H), 4.39 (d, J=7.5Hz, 2H), 4.64 (dt, J=5.9, 1.4Hz, 2H ), 5.23-5.36 (m, 2H), 5.73 (d, J=8.4Hz, 1H), 5.92 (ddt, J=17.3, 10.4, 5.8Hz, 1H), 7. 32(td, J=7.5, 1.2Hz, 2H), 7.36-7.43(m, 2H), 7.60-7.68(m, 2H), 7.74-7.79(m, 2H); 13C NMR (125.77MHz, CDCl3): δ27.5 (3C), 47.3, 50.3, 50.5, 50.6, 50.7, 61.2, 65.5, 66.7, 70.5, 70.8, 71.2, 71.4, 73.1, 1 18.7, 119.9, 120.0, 120.0, 125.2, 127.0, 127.6, 127.7, 131.8, 141.3, 144.0, 144.0, 156.4, 171.8, 174.3; ESI-MS: [C 31 H 42 N2O71+H] m / z calculated value: 555.3, found value: 555.3.

[0285]

[0286] Allyl monomer (29a): To a mixture of 15(E) (0.50 g, 0.72 mmol) and DIEA (163.67 μL, 0.94 mmol) in anhydrous DMF (6 mL) was added a portion of HBTU (0.32 g, 0.83 mmol) at room temperature. After 10 min at room temperature, a solution of backbone 28 (0.60 g, 1.08 mmol) in DMF (4 mL) was added to the above reaction mixture. The progress of the reaction was monitored by TLC (eluent: EA:Hex (50:50); R of the backbone f =0.2; R of the product f =0.6; R of E f =0.1). After the starting material was completely consumed, DMF was removed in vacuo at 45°C. 1% HCl (20 ml) and EA (20 ml) were added to the obtained crude material, and the organic layer (2x) was separated from the aqueous layer using a separatory funnel. The combined organic layers were dried, removed, and the obtained crude product was purified by flash silica gel column chromatography. Yield: 0.62 g, 70%. 1 H NMR (500.13 MHz, DMSO-d6, rotamer): δ 1.08 / 1.09 (s, 9H), 1.21 1.33 (m, 18H), 1.36 / 1.38 (s, 18H), 1.41 / 1.65 (s, 9H), 3.36-3.70 (m, 10H), 3.82-4.27 (m, 8H), 4.32 (bs, 2H), 4.41-4.71 (m, 2H), 5.1 3-5.41 (m, 2H), 5.81-6.01 (m, 1H), 7.30-7.57 (m, 5H), 7.71 / 7.73 (d, J=9.7Hz, 2H), 7.89 / 7.99 (d, J=7.5Hz, 2H), 8.56 / 8.60 (s, 1H); 13C NMR (125.77 MHz, DMSO-d6, rotamers): δ 27.2-27.6 (18C), 46.7, 59.7, 60.6 / 60.6, 64.7, 65.3, 65.5, 69.6, 69.6, 69.8, 70.0, 70.4, 70.4, 72.2, 81.8-86.3 (6C), 108.1, 117.7, 118.2, 120.1 (2C), 125.1, 127.0 (2C), 127.3, 127.6 (2C), 127.8 / 127.9, 132.1, 132.3, 140.7, 143.8, 147 .9, 149.3, 149.5, 149.6, 155.0 / 155.1, 155.9, 156.5, 166.6, 168.8, 169.2, 170.2; HRMS: [C 64 H 89 N7O 17 m / z calculated for [(+H): 1228.6393, found: 1228.6398.

[0287]

[0288] Allyl monomer (29b): This compound was prepared using the above procedure for compound 29a using compound 27 (1.00 g, 5.88 mmol) and backbone 28 (4.08 g, 7.35 mmol) as starting materials. TLC (eluent: 5% MeOH:DCM (5:95); R of the backbone f =0.6; R of the product f =0.2; R of 27 f＝ 0.0). Yield: 3.5 g, 84%. 1 H NMR (500.13 MHz, DMSO-d6, rotamer): δ 1.10 / 1.10 (s, 9H), 3.30-3.59 (m, 14H), 3.74-4.13 (m, 2H), 4.17-4.42 (m, 4H), 4.60 (dd, J=33.4, 4.8 Hz, 2H), 5.00-5.44 (m, 2H), 5.82-6.01 (m, 1H), 7.10-7.55 (m, 6H), 7.69 (d, J=7.4 Hz, 2H), 7.89 (d, J=7.5 Hz, 2H), 10.75 (bs, 1H), 11.08 (s, 1H); 13C NMR (125.77 MHz, DMSO-d6, rotamer) δ 27.2 (3C), 46.7, 60.6 (2C), 64.7, 65.2, 65.5, 69.6, 69.8, 69.9, 70.4 (2C), 72.2, 107.2, 117.7, 118.1, 120.1 (2C), 125.2, 127.0 (2C), 127.3, 127.6 (2C), 132.2 / 132.3, 139.2, 139.7, 140.7, 143.8 / 143.8, 151.2 / 151.3, 155.8, 164.1 / 164.1, 168.9, 169.4, 170.6; HRMS: [C 37 H 46 N4O 10 m / z calculated for [(4-(4-hydroxyethyl)propene]: 707.3292, found: 707.3290.

[0289]

[0290] Allyl monomer (29c): This compound was prepared by the above procedure for compound 29a using compound 26 (3.50 g, 4.92 mmol) and backbone 28 (3.41 g, 6.15 mmol) as starting materials. TLC (eluent: EA: hexane (40:60); R of the backbone f =0.1; R of the product f =0.7; R of 26 f =0.1). Yield: 4.80 g, 78%. 1 HNMR (300MHz, DMSO-d6): δ1.09 (bs, 9H), 1.34 / 13.6 (s, 18H), 1.41 / 1.42 (s, 27H), 3.35-3.59 (m, 14H), 4.07-4.49 (m, 4H), 4.61 (dd, J=37.6, 5.4Hz, 3 H), 4.97-5.42 (m, 3H), 5.77-6.04 (m, 1H), 7.32 (t, J=7.5Hz, 2H), 7.50-7.5 7 (m, 3H), 7.89 (d, J = 7.5Hz, 2H), 7.99 (d, J = 8.4Hz, 2H), 8.94 / 8.96 (s, 1H); 13C NMR (125.77 MHz, DMSO-d6, rotamers): δ 27.2-27.5 (18C), 46.7, 60.2 / 60.7 (2C), 64.7, 65.5, 69.5, 69.6, 69.8, 69.9, 70.4 / 70.4 (2C), 72.2, 82.2-83.4 (6C), 108.1, 117.7, 1 20.1(2C), 124.4, 125.1, 127.0(2C), 127.4, 127.6(2C), 127.8, 132.2(2C), 140.7(2 C), 143.8(2C), 145.1, 146.9(2C), 149.5(2C), 157.0, 162.5, 166.5, 168.4; HRMS: [C 63 H 89 N7O 19 m / z calculated for [(+H): 1248.6291, found: 1248.6256.

[0291] The following numbered items describe non-limiting aspects of the invention:

[0292] Item 1. A gene recognition reagent comprising a nucleic acid or nucleic acid analog backbone and comprising 3 or more ribose, deoxyribose or nucleic acid analog backbone residues, a divalent nucleobase sequence connected to the backbone residues, wherein the sequence of the divalent nucleobase binds to a unit target sequence of an expanded repeat of a repeat expansion disease on two nucleic acid chains, or one or more consecutive iterations of the unit target sequence.

[0293] Item 2. The gene recognition reagent of Item 1, wherein the nucleic acid or nucleic acid analog main chain comprises a first end and a second end, and further comprises a first connecting group connected to the first end of the main chain, and a second connecting group connected to the second end, which is non-covalently bonded or self-bonded to the first connecting group.

[0294] Item 3. The gene recognition reagent described in Item 1 or 2, wherein when the two chains are arranged in an antiparallel direction, the sequence of the divalent nucleobase binds to a unit target sequence of an expanded repeat of a repeat expansion disease on each of the two chains or two or more consecutive iterations of the unit target sequence.

[0295] Item 4. A gene recognition reagent according to any one of Items 1 to 3, wherein the unit target sequence is a continuous iteration (GAA) on the two chains. n , (CGG) n , (CCG) n , (CAG) n , (CTG) n , (CCTG) n , (ATTCT)n , or (GGGGCC) n Or a sequence complementary to any of the aforementioned sequences.

[0296] Item 5. The gene recognition reagent of any one of Items 1 to 4, wherein the unit target sequence is C / GA / AG / C.

[0297] Item 6. A gene recognition reagent according to any one of Items 1 to 5, which has a nucleobase sequence in the order:

[0298] JB3 nucleobase, JB6 nucleobase, and JB4 nucleobase;

[0299] JB6 nucleobase, JB4 nucleobase, and JB3 nucleobase;

[0300] or JB4 nucleobase, JB3 nucleobase and JB6 nucleobase;

[0301] , or two or more consecutive iterations of the foregoing.

[0302] Item 7. The gene recognition reagent of Item 6, which has a nucleobase sequence in the order of:

[0303] JB3 or JB3b; JB6 or JB6b; and JB4, JB4b, JB4c, JB4d or JB4e;

[0304] JB6 or JB6b; JB4, JB4b, JB4c, JB4d or JB4e; and JB3 or JB3b;

[0305] JB4, JB4b, JB4c, JB4d or JB4e; JB3 or JB3b; and JB6 or JB6b,

[0306] or two or more consecutive iterations of the foregoing.

[0307] Item 8. A gene recognition reagent according to any one of Items 1 to 5, which has a nucleobase sequence in the order:

[0308] JB3, JB6 and JB4;

[0309] JB6, JB4 and JB3; or

[0310] JB4, JB3 and JB6,

[0311] or two or more consecutive iterations of the foregoing.

[0312] Item 9. The gene recognition reagent of any one of Items 1 to 3, which has a nucleobase sequence of the unit recognition reagent sequence listed in Table C.

[0313] Item 10. The gene recognition reagent according to any one of Items 1 to 9, wherein the two chains are the same nucleic acid molecule.

[0314] Item 11. The gene recognition reagent according to any one of Items 1 to 10, wherein both strands are RNA.

[0315] Item 12. The gene recognition reagent according to any one of Items 1 to 11, which consists of a sequence of 3 to 8 nucleobases, such as 3, 4, 5, 6, 7 or 8 nucleobases.

[0316] Item 13. The gene recognition reagent according to any one of Items 1 to 12, wherein the main chain residues are nucleic acid analog main chain residues.

[0317] Item 14. The gene recognition reagent of Item 13, wherein the main chain residues are conformationally pre-organized.

[0318] Item 15. The gene recognition reagent of Item 14, wherein the main chain residues are locked nucleic acid residues.

[0319] Item 16. The gene recognition reagent of Item 14, wherein the main chain residues are peptide nucleic acid (PNA) residues.

[0320] Item 17. The gene recognition reagent of Item 14, wherein the main chain residues are γPNA residues.

[0321] Item 18. The gene recognition reagent according to any one of Items 1 to 14, which has the following structure:

[0322]

[0323] wherein X is S or O; n is an integer between 1 and 6, including 1 and 6; m is an integer between 0 and 4, including 0 and 4; R1 and R2 are each independently: H; a guanidine-containing group such as

[0324]

[0325] Where n = 1, 2, 3, 4 or 5; amino acid side chains, such as

[0326]

[0327] Straight or branched (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene, optionally substituted with ethylene glycol units containing 1 to 50 ethylene glycol moieties; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2)q -NHP1; -CH2-(SCH2-CH2) q -SP1; -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; and r and s are each independently an integer from 1 to 50;

[0328] R3 is H or a leaving group, such as a linear or branched (C1-C8)alkyl, a substituted or unsubstituted (C3-C8)aryl, a (C3-C8)aryl (C1-C6)alkylene, a (C1-C8)carboxyl, which is optionally substituted with an amino acid side chain or a guanidine-containing group, or wherein o is 1-20, each instance of R6 is independently an amino acid side chain, and R7 is -OH or -NH2;

[0329] R4 is (C1-C 10 ) divalent hydrocarbon or (C1-C 10 ) divalent hydrocarbons, such as -O-, -OH, -C(O)-, -NH-, -NH2, -C(O)NH-;

[0330] R5 is -OH, -SH or a disulfide bond protecting group; and

[0331] each instance of R is independently a nucleobase that produces a nucleobase sequence that is complementary to a unit target sequence of nucleobases in one or more target nucleic acids, such that each of the plurality of recognition modules binds to a target sequence of nucleobases on the template nucleic acid and is linked to one another,

[0332] or a pharmaceutically acceptable salt thereof.

[0333] Item 19. The gene recognition reagent described in Item 18, wherein R1 or R2 is the following substituted (C1-C6) alkyl: -(OCH2-CH2) q OP1; -(OCH2-CH2) q -NHP1; -(SCH2-CH2) q-SP1; -(OCH2-CH2) r -OH; -(OCH2-CH2) r -NH2; -(OCH2-CH2) r -NHC(NH)NH2; or -(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2; wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50.

[0334] Item 20. The gene recognition reagent described in Item 18, wherein R5 is -SH, -OH, or -SS-R8, wherein R8 is one or more amino acid residues, amino acid side chains, guanidine-containing, unsubstituted or substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene or (C3-C8) cycloalkyl (C1-C6) alkylene.

[0335] Item 21. The gene recognition reagent described in Item 18, wherein R1 is H and R2 is not H, or R2 is H and R1 is not H.

[0336] Item 22. The gene recognition reagent described in Item 18, wherein R1 is H and R2 includes a polyoxyethylene portion.

[0337] Item 23. The gene recognition reagent described in Item 18, wherein R5 is -SH; and X is S.

[0338] Item 24. The gene recognition reagent described in Item 18, wherein R5 includes a lysine residue, an arginine residue or a guanidine group.

[0339] Item 25. The gene recognition reagent described in Item 18, wherein R4 is methylene, R5 is -SH, X is S, and R5 is a hydroxy-substituted (C1-C8) alkyl group, such as -CH2-CH2-OH.

[0340] Item 26. The gene recognition reagent described in any one of items 2-14, wherein the first linking group and the second linking group are independently 2 to 5 ring fused polycyclic aromatic parts, wherein when the recognition reagent hybridizes with the adjacent sequence of the target nucleic acid, the part stacks with the aromatic part of the adjacent recognition reagent.

[0341] Item 27. The gene identification agent described in Item 26, wherein the 2- to 5-ring fused polycyclic aromatic moiety is unsubstituted or substituted pentene, indene, naphthalene, azulene, heptene, biphenyl, as-indenene, s-indenene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, acetophenanthren, acetothracene, triphenylphenanthren, pyrene, naphthacene / tetracene, septene, chrysene or perylene, optionally substituted by one or more heteroatoms such as O, N and / or S, for example xanthene, riboflavin (vitamin B2), mangiferin or mangiferin.

[0342] Item 28. The gene recognition agent described in Item 26 or 27, wherein the 2 to 5-ring condensed polycyclic aromatic moieties are the same.

[0343] Item 29. The gene recognition reagent according to Item 2, wherein the recognition reagent has the following structure:

[0344]

[0345] wherein each instance of R is independently a nucleobase, and each instance of R may be the same or a different nucleobase;

[0346] N is an integer ranging from 1 to 6, such as 1, 2, 3, 4, 5 or 6;

[0347] R1 and R2 are each independently: H; containing guanidine, for example

[0348]

[0349] Where n = 1, 2, 3, 4 or 5; amino acid side chains, such as

[0350]

[0351] Unsubstituted or substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(OCH2-CH2-0) q -SP1; -CH2-(SCH2-CH2) q -SP1, -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r-NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; r and s are each independently an integer from 1 to 50, inclusive;

[0352] R 10 or R 11 One of the 12 , R 13 or R 14 wherein one of the above is -L-R3, wherein each instance of R3 is independently a 2-5 ring fused polycyclic aromatic moiety, such as unsubstituted or substituted pentene, indene, naphthalene, azulene, heptene, biphenyl, as-indenene, s-indenene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, acetophenanthrenene, acetothracene, triphenylphenanthrenene, pyrene, naphthacene / tetracene, septobalene, chrysene or perylene, optionally substituted with one or more heteroatoms such as O, N and / or S, for example xanthene, riboflavin (vitamin B2), mangiferin or mangiferin, wherein each is capable of stacking with the R3 group of an adjacent recognition agent when the recognition agent hybridizes to an adjacent sequence of the target nucleic acid;

[0353] L is a linker; and

[0354] R 10 , R 11 , R 12 , R 13 or R 14 The remainder of each independently is H, one or more adjacent amino acid residues, such as one or more Arg residues, containing a guanidine group, such as

[0355]

[0356] Where n = 1, 2, 3, 4 or 5; amino acid side chains, such as

[0357]

[0358] Unsubstituted or substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene; -CH2-(OCH2-CH2)q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(OCH2-CH2-0) q -SP1; -CH2-(SCH2-CH2) q -SP1, -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; r and s are each independently an integer from 1 to 50;

[0359] or a pharmaceutically acceptable salt thereof.

[0360] Item 30. The gene recognition reagent described in Item 29, wherein R1, R2, R 10 , R 11 , R 12 , R 13 or R 14 One or more of the following is substituted (C1-C6) alkyl: -(OCH2-CH2) q OP1; -(OCH2-CH2) q -NHP1; -(SCH2-CH2) q -SP1; -(OCH2-CH2) r -OH; -(OCH2-CH2) r -NH2; -(OCH2-CH2) r -NHC(NH)NH2; or -(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2; wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50.

[0361] Item 31. The gene recognition reagent described in Item 29, wherein R4 and R7 are -L-R3.

[0362] Item 32. The gene recognition reagent described in Item 29, wherein the linker contains about 5 to 25 atoms, such as 5-20, 5-10, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms, or a total of 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C and heteroatoms, such as O, P, N or S atoms.

[0363] Item 33. The gene recognition agent described in Item 29, wherein both instances of R3 contain the same 2 to 5-ring fused polycyclic aromatic moiety.

[0364] Item 34. The gene recognition reagent described in any one of Items 1 to 33, comprising a guanidine group, such as

[0365]

[0366] Where n=1, 2, 3, 4 or 5.

[0367] Item 35. A method for binding a nucleic acid comprising an expanded repeat associated with an expanded repeat disease, comprising contacting the nucleic acid comprising the expanded repeat with a gene recognition reagent of one of Items 1-32.

[0368] Item 36. The method of Item 35, wherein the nucleic acid comprises (GAA) on both strands n 、(CGG) n 、(CCG) n 、(CAG) n 、(CTG) n 、(CCTG) n 、(ATTCT) n , or (GGGGCC) n A unit target sequence formed by continuous iteration of, or a sequence complementary to any of the aforementioned sequences.

[0369] Item 37. The method of Item 35, wherein the nucleic acid comprises (CAG) on both strands. n A unit target sequence formed by continuous iteration of, or a sequence complementary to any of the aforementioned sequences.

[0370] Item 38. The method of Item 35, wherein the nucleic acid is obtained from a patient suffering from a repeat expansion disease.

[0371] Item 39. A method for identifying the presence of a nucleic acid containing an expanded repeat associated with an expanded repeat disease in a sample obtained from a patient, the method comprising contacting the nucleic acid sample obtained from the patient with a gene recognition reagent as described in one of Items 2-34, and determining whether binding and connection of the gene recognition reagent occurs, indicating that the sample contains a nucleic acid containing an expanded repeat associated with an expanded repeat disease, and optionally treating the expanded repeat disease in the patient.

[0372] Item 40. A composition comprising the gene recognition reagent described in any one of Items 1 to 34 and a pharmaceutically acceptable carrier.

[0373] Item 41. A method for knocking down the expression of a gene containing an expanded repeat associated with an expanded repeat disease in a cell, comprising contacting a nucleic acid, such as RNA, containing the expanded repeat with a gene recognition reagent as described in any one of Items 1-34.

[0374] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, one of ordinary skill in the art will recognize that various substitutions, modifications or combinations may be made to any exemplary embodiment without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the description of the exemplary embodiments, but rather by the attached original claims as filed. Sequence Listing <110> Carnegie Mellon University <120> Bivalent nucleic acid ligands and their uses <130> 6526-1807597 <150> US 62 / 708,783 <151> 2017-12-21 <150> US 62 / 710,262 <151> 2018-02-14 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> RNA <213> people <400> 1 cgccagcgca gcagcgcagg cg 22

Claims

1. A gene recognition reagent comprising (a) a nucleic acid analog backbone comprising (i) three or more nucleic acid analog backbone residues; (ii) a first end and a second end; and (iii) a first linking group connected to the first end of the backbone and a second linking group connected to the second end, wherein the first linking group and the second linking group are each independently a 2- to 5-ring fused polycyclic aromatic moiety, which is xanthene, riboflavin, mangiferin or mangiferin; and (b) a sequence of a divalent nucleobase linked to the three or more nucleic acid analog backbone residues, wherein the sequence of the divalent nucleobase binds to a unit target sequence on a nucleic acid strand, or one or more consecutive iterations of the unit target sequence on two nucleic acid strands, wherein the one or more consecutive iterations of the unit target sequence are expanded repeats of a repeat expansion disease, wherein the sequence of the divalent nucleobase comprises the following unit recognition reagent sequence: 3-6-4、 6-4-3、 4-3-6、 4-6-3、 6-3-4、 or 3-4-6, in: Each of the 3 independent Each of the 4 independent Each of the 6 independent wherein R is a nucleic acid analog backbone residue, and Wherein R1 is a protecting group or H.

2. The gene recognition reagent of claim 1, wherein the sequence of the divalent nucleobase is: JB3 nucleobase, JB6 nucleobase, and JB4 nucleobase; JB6 nucleobase, JB4 nucleobase, and JB3 nucleobase; JB4 nucleobase, JB3 nucleobase, and JB6 nucleobase; JB3 or JB3b; JB6 or JB6b; and JB4, JB4b, JB4c, JB4d, or JB4e; JB6 or JB6b; JB4, JB4b, JB4c, JB4d or JB4e; and JB3 or JB3b; JB4, JB4b, JB4c, JB4d or JB4e; JB3 or JB3b; and JB6 or JB6b, JB3, JB6 and JB4; JB6, JB4 and JB3; or JB4, JB3 and JB6, or two or more consecutive iterations of the foregoing. The gene recognition reagent according to claim 1 , wherein the two nucleic acid chains are both RNA. 4 . The gene recognition reagent according to claim 1 , wherein the nucleic acid analog backbone residues are γPNA residues.

5. The gene recognition reagent of claim 1, wherein the recognition reagent has the following structure: in, Each instance of R is independently a nucleobase having the sequence of said divalent nucleobase; n is an integer ranging from 1 to 6; R1 and R2 are each independently: H; guanidine-containing; amino acid side chain; unsubstituted or substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(OCH2-CH2-0) q -SP1; -CH2-(SCH2-CH2) q -SP1, -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; r and s are each independently an integer from 1 to 50, inclusive; R 10 or R 11 One of the 12 , R 13 or R 14 One of the following is -L-R3, wherein each instance of R3 is independently a 2- to 5-ring fused polycyclic aromatic moiety; L is a linker; and R 10 , R 11 , R 12 , R 13 or R 14 The remainder of is each independently H, one or more consecutive amino acid residues, guanidine-containing, amino acid side chains, unsubstituted or substituted (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C1-C8) hydroxyalkyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl (C1-C6) alkylene, (C3-C8) cycloalkyl (C1-C6) alkylene; -CH2-(OCH2-CH2) q OP1; -CH2-(OCH2-CH2) q -NHP1; -CH2-(OCH2-CH2-0) q -SP1; -CH2-(SCH2-CH2) q -SP1, -CH2-(OCH2-CH2) r -OH; -CH2-(OCH2-CH2) r -NH2; -CH2-(OCH2-CH2) r -NHC(NH)NH2; or -CH2-(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2, wherein P1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50, inclusive; r and s are each independently an integer from 1 to 50; or a pharmaceutically acceptable salt thereof.

6. The gene recognition reagent according to claim 5, wherein R1, R2, R 10 , R 11 , R 12 , R 13 or R 14 One or more of the following is substituted (C1-C6) alkyl: -(OCH2-CH2) q OP1; -(OCH2-CH2) q -NHP1; -(SCH2-CH2) q -SP1; -(OCH2-CH2) r -OH; -(OCH2-CH2) r -NH2; -(OCH2-CH2) r -NHC(NH)NH2; or -(OCH2-CH2) r -SS[CH2CH2] s NHC(NH)NH2; among them, P 1 is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)aryl, (C3-C8)cycloalkyl, (C3-C8)aryl(C1-C6)alkylene or (C3-C8)cycloalkyl(C1-C6)alkylene; q is an integer from 0 to 50; r is an integer from 1 to 50, and s is an integer from 1 to 50. The gene recognition reagent according to claim 5 , comprising a guanidine-containing group.

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