WO2019086437A1

WO2019086437A1 - A compound comprising rna compounds and bcm moieties

Info

Publication number: WO2019086437A1
Application number: PCT/EP2018/079678
Authority: WO
Inventors: Modesto OROZCO LÓPEZ; Montserrat TERRAZAS MARTÍNEZ; Daniel SÁNCHEZ PÉREZ
Original assignee: Fundació Institut De Recerca Biomèdica (Irb Barcelona); Universitat De Barcelona
Priority date: 2017-10-31
Filing date: 2018-10-30
Publication date: 2019-05-09

Abstract

The invention refers to a compound comprising a compound A _n, a compound B _n and a compound C _n, wherein A _n comprises RNA compounds A _Tn and A _Bn and a first BCm moiety of the general formula (I) with the structure A _Tn -BCm-A _Bn, B _n comprises RNA compounds B Bn and B Tn and a second BCm moiety, with the structure B _Bn -BCm-B _Tn, and C _n comprises RNA compounds C _Tn a and C _Tn b, a third BCm moiety, with the structure C _Tn b-BCm-C _Tn a and a RNA compound C _Bn. The compound is useful as a medicament in the treatment of cancer and in a method for monitoring biological processes in vitro or in vivo. The invention also refers to a pharmaceutical composition comprising the compound. In addition, the invention refers to the use of BCm moieties for increasing stability of the RNA structure of the compound.

Description

DESCRIPTION

Title of Invention

A compound comprising RNA compounds and BCm moieties

Technical Field

The present invention relates to the provision of a novel compound, a bifunctional short hairpin RNA-based branched nanobinder. The compound can be functionalized to incorporate accessory molecules and/or sequences recognizing therapeutic targets. The compound may be used in the treatment of diseases, such as cancer, due to its ability to recognize and inhibit the expression of disease-related genes.

Background Art

Complex pathologies, such as cancer, are the result of the malfunctioning of the complex network of pathways controlling cellular life. Many crucial proteins in such pathways are known, whose inhibition can lead to the specific death of cancer cells, fueling the development of target-directed anticancer therapies as an alternative to poorly-performing traditional cytotoxic therapies. Unfortunately, clinical use of target-specific antineoplastic drugs has shown also some caveats, the most important ones the appearance of resistances.

Combined therapies, where two drugs are used to attack simultaneously primary and secondary targets, are promising alternatives to standard mono-drug therapies, as they can tackle resistance already before it appears. In fact, several drug combinations have been approved, the majority of them involving small molecules designed to interfere major and rescue pathways. However, these treatments rely on combinations of pre-existing drugs and face a series of problems related to: i) interesting protein targets might be undruggable, ii) cooperative toxicity effects linked to interaction of the two drugs, and iii) unpredictable changes in bio-ability, metabolism and pharmacokinetic properties derived from combination of drugs.

An alternative approach to combined therapies consists on the simultaneous inactivation of primary and alternative pathways by using small RNAs designed to inhibit the synthesis of target proteins by activating RISC-mediated cleavage of the corresponding mRNA. siRNAs and shRNAs are excellent candidates to accomplish this task. siRNAs are 19-bp double- stranded RNAS (dsRNAs) with 3'-dinucleotide overhangs that are generated in the cytoplasm by Dicer cleavage of longer RNAs. On the other hand, shRNAs with stem length of >19 base pairs are also processed by Dicer to give active siRNA duplexes. In both cases, the resulting siRNAs activate the RNA interference (RNAi) machinery and induce the RISC- mediated cleavage of the target mRNA, preventing the expression of genes of choice. This biological process can be used for therapeutic purposes. The advantages of this approach are clear: undruggable protein targets can be tackled and large specificity can be achieved by selecting suitable nucleotide sequences. The shortcomings are mostly related to poor bio-availability and to the difficulty of controlling the concentration of the two interference RNAs inside the cell.

Dumbbell-shaped RNAs have been created by introducing at terminal sites two 2'-deoxy-5- methylcytidine units linked together by a hexyl chain through the exocyclic amino group of the nucleobase (N-hexyl-N bridged). The resulting system was proven to be a good substrate of RISC, showing excellent interference RNA properties and an outstanding resistance to undesired degradation by intra and extra-cellular nucleases (Terrazas ef al. 2016). This document described the potential therapeutic effect of RNA nanostructures comprising two N-alkyl-N dimeric nucleotides (BCm dimers, wherein m is the number of carbon atoms of the alkyl chain). BC6-loop dumbbells were proven to target Renilla and Firefly mRNAs and grb7 mRNA as well. The BC6-loop RNA dumbbell with 29 bp stem silences the expression of grb7 in the HER2+ breast cancer cell line SKBR3 from 48 hours up to 6 days and it also reduces cell proliferation on culture.

WO2008094516 discloses short interfering RNA molecules (siRNA) that target two or more sequences so that each strand may be directed to separate targets. The bifunctional duplexes used are designed by computer algorithms in order to obtain RNA sequences from genes able to anneal between them. The document shows an example with RNAs designed against bcl6 and stat3 mRNAs, two B-cell lymphoma oncogenes. The different miRNAs of this document must anneal between them, forming a stable RNA helix, but being different enough to target two different cellular mRNAs with specificity.

Breast cancer is a very complex disease that represents the second leading cause of death in women in developed countries. For many years, its therapeutic treatment has been based on the use of cytotoxic chemotherapeutics, but recently targeted therapies have gained importance, especially those targeting Human Epidermal Growth factor (HER2), which is overexpressed in a significant proportion of breast cancers. The amplification of HER2 is observed in 25 to 30 % of all breast cancers (Slamon ef al. 2001 ). Patients with breast cancer with overexpression of HER2 have, originally, a poorer prognosis and shorter overall survival (OS). HER2 overexpression (HER2+) usually results in malignant transformations of cells and leads to tumorigenesis and metastasis. HER2 is a tyrosine kinase receptor which upon dimerization activates signaling cascades (such as PI3K/Akt), leading to antiapoptotic and growth promoting effects. The use of directed therapies which interfere with HER2 activity - monoclonal antibody trastuzumab (Herceptin®), dodecapeptide AHNP or the tyrosine kinase inhibitor, lapatinib (Tykerb®), reduces proliferation, leading to major improvements in patients' survival. Unfortunately, the majority of patients with metastatic HER2-overexpressing breast cancer, who initially respond to anti-HER2 therapy, develop resistance after long periods of treatment due mostly, to the induction of secondary pathways resulting in the overexpression of oncogenes involved in breast cancer metastatic spread. At present time, at least three proteins have been characterized as being involved in rescue pathways: Grb7, Stard3 and Hsp27.

Summary of Invention

The technical problem to be solved is to provide a compound comprising two shRNAs for simultaneous inhibition of the translation of two genes, with a stable double helix RNA structure.

In addition, another technical problem is to provide a stable RNA system comprising two shRNAs which can be functionalized to monitor biological processes.

The solution to the technical problem is solved by the compound defined in the claims. In a first aspect, the present invention provides a compound comprising a compound A_n, a compound B_n and a compound C_n, wherein:

A_n comprises RNA compounds Am and ΑΒΠ and a first BCm moiety of the general Formula (I)

Formula (I), wherein the first BCm moiety is covalently bonded to Am and ΑΒΠ, forming the structure Ατη-BCm-ABn, and wherein ΑΒΠ is base-paired with Am and with at least 5 nucleotides

B_n comprises RNA compounds ΒΒΠ and B and a second BCm moiety of Formula (I), wherein the second BCm is covalently bonded to ΒΒΠ and Bm, forming the structure

BBn-BCm-Βτη, and wherein B is base-paired with ΒΒΠ and with at least 5 nucleotides

Cn comprises RNA compounds Cma and Cmb, a third BCm moiety of Formula (I) and a RNA compound Cen, wherein the third BCm moiety is covalently bonded to Cmb and Cma, forming the structure Cmb-BCm-Cma, and wherein Cen is partly or completely base-paired with Cma and Cmb; wherein m is in the range from 2 to 8, Am is adjacently assembled to Cma; Bm is adjacently assembled to Cmb; ΑΒΠ is adjacently assembled to one side of CBn; and ΒΒΠ is adjacently assembled to the opposite side of CBn; Ri is selected from -CH2-OH, -CH2-SH or alkynyl; and is suitable to bond to chemically functionalized accessory molecules selected from the group consisting of biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes and peptide carriers; and

R2 is selected from -OH, -SH or alkynyl; and is suitable to bond to chemically functionalized accessory molecules selected from the group consisting of biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes and peptide carriers.

In a second aspect, the present invention provides a compound according to the first aspect of the invention, wherein the sequences of A_Tn and B_Tn are complementary to the sequences of messenger RNAs codifying for target genes.

Another embodiment is the compound according to the second aspect of the invention, wherein the target genes are selected from the group consisting of grb7, stard3 and/or hsp27.

In another embodiment, the sequences of Am and Bm are complementary to the sequences of messenger RNAs codifying for target genes grb7/stard3 or grb7/hsp27.

Another embodiment is the compound according to the second aspect of the invention, wherein Am is identified by the sequences SEQ ID NO: 14 or 20. Another embodiment is the compound according to the second aspect of the invention, wherein Bm is identified by the sequence SEQ I D NO: 17.

Another embodiment is the compound according to the first or second aspect of the invention, wherein the sequences C-mb and C-ma are separated by a BCm moiety flanked at 5' and 3' by an unpaired nucleotide.

Another embodiment is the compound according to the first or second aspect of the invention, wherein C-ma is identified by a sequence selected from the group consisting of SEQ I D NO: 12, 18, 22 and 26.

Another embodiment is the compound according to the first or second aspect of the invention, wherein Cmb is identified by the sequence GUUUUA or GCUUCGGAA.

Another embodiment is the compound according to the first or second aspect of the invention, wherein ΟΒΠ is identified by a sequence selected from the group consisting of SEQ I D NO: 19, 23 and 27.

Another preferred embodiment is the compound according to the first or second aspect of the invention, wherein m is 6.

Another embodiment is the compound according to the first or second aspect of the invention, wherein conjugation of the accessory molecules chemically functionalized with an azide or a maleimide group is made by click chemistry reactions with the alkynyl or the thiol moiety of Ri and/or R2 of the chemically functionalized BCm moiety selected from copper (l)-catalyzed azide-alkyne cycloaddition reaction or thiol-maleimide Michael addition reaction. Another embodiment is the compound according to the first or second aspect of the invention, wherein alkynyl is ethynyl.

Another embodiment is the compound according to the first or second aspect of the invention, wherein the chemically functionalized molecule is bonded to the chemically functionalized BCm moiety through a 1 ,2,3-triazole moiety or through a succinimide thioether moiety. In a third aspect, the present invention provides a pharmaceutical composition comprising the compound according to the first or second aspect of the invention and at least one pharmaceutically acceptable excipient or carrier.

In a fourth aspect, the present invention provides a compound according to the first or second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention for use as a medicament.

Another embodiment is the compound or pharmaceutical composition for use according to the fourth aspect of the invention in the treatment of cancer.

In another embodiment, said cancer is selected from the group consisting of breast cancer, breast cancer resistant to anti-HER2 therapy, breast carcinoma, breast adenocarcinoma, gastric carcinoma, gastric adenocarcinoma, colon carcinoma, colon adenocarcinoma, pancreas carcinoma, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, ovarian adenocarcinoma, ovarian carcinoma, endometrial carcinoma, carcinoma of the uterine cervix, lung carcinoma, lung adenocarcinoma, non- small-cell lung cancer, small-cell lung cancer, thyroid carcinoma, metastasizing papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, urine bladder carcinoma, transitional cell carcinoma of the urinary bladder, prostate carcinoma, cancer of glial lineage of the central nervous system (glioma), sarcomas, fibrosarcoma, malignant fibrous histiocytoma, Edwing sarcoma human, endometrial stromal sarcoma, osteosarcoma, rhabdomyosarcoma, melanoma, embryonal cancers, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, hematological cancers, B-cell or T-cell leukemia, non-Hodgkin lymphoma, non Hodgkin lymphoma B-cell or T-cell types, Burkitt lymphoma, Hodgkin lymphoma, leukemias, lymphoma B-cell or T- cell types, and multiple myeloma.

Particularly, the cancer is breast cancer. More particularly, the breast cancer has developed resistance to an anti-HER2 therapy.

In a fifth aspect, the present invention provides a compound according to the first or second aspect of the invention for use in a method for monitoring biological processes in vitro or in vivo.

In a sixth aspect, the present invention provides the use of the BCm moieties of general Formula (I) for increasing stability of the RNA structure of a compound comprising a compound A_n, a compound B_n and a compound C_n, according to first or second aspect of the invention. Detailed Description of Invention

In a first aspect, the present invention provides a compound comprising a compound A_n, a compound B_n and a compound C_n, wherein: A_n comprises RNA compounds Am and ΑΒΠ and a first BCm moiety of the general

Formula (I)

B_n comprises RNA compounds B_Bn and B_Tn and a second BCm moiety of Formula (I), wherein the second BCm is covalently bonded to ΒΒΠ and Bm, forming the structure BBn-BCm-Βτη, and wherein B is base-paired with ΒΒΠ and with at least 5 nucleotides

Cn comprises RNA compounds Cma and Cmb, a third BCm moiety of Formula (I) and a RNA compound Cen, wherein the third BCm moiety is covalently bonded to Cmb and Cma, forming the structure Cmb-BCm-Cma, and wherein Cen is partly or completely base-paired with Cma and Cmb; wherein m is in the range from 2 to 8, Am is adjacently assembled to Cma; Bm is adjacently assembled to Cmb; ΑΒΠ is adjacently assembled to one side of CBn; and ΒΒΠ is adjacently assembled to the opposite side of CBn;

Ri is selected from -CH2-OH, -CH2-SH or alkynyl; and is suitable to bond to chemically functionalized accessory molecules selected from the group consisting of biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes and peptide carriers; and R2 is selected from -OH, -SH or alkynyl; and is suitable to bond to chemically functionalized accessory molecules selected from the group consisting of biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes and peptide carriers. In an embodiment, any of the RNA compounds Am, ΑΒ_Π, ΒΒ_Π, Bm, Cma, Cmb and Cen may comprise modified ribonucleotides. In a preferred embodiment, said modified ribonucleotides are phosphorothioate RNA derivatives.

The structure Cmb-BCm-Cma is also referred herein as Cm.

Particularly, in the compound according to the first aspect of the invention, ΑΒ_Π is base- paired with Am and with 5 or 6 nucleotides of Cma.

Particularly, in the compound according to the first aspect of the invention, Bm is base- paired with ΒΒ_Π and with 5 or 6 nucleotides of ΟΒ_Π,

In the compound according to the first aspect, the BCm moiety is connected to its adjacent ribonucleotides by a natural phosphodiester bond.

A_n and B_n are, each of them, adjacently assembled to C_n, forming a two-arm branched structure.

The structure of the compound according to the first aspect of the invention has high flexibility degree and allows the incorporation of RNAs and the inhibition of gene expression of one or more targets, simultaneously.

A structural feature of the BCm moiety is the presence of free hydroxyl groups in their sugar rings, which offers the possibility to bond several molecules to the final branched nanostructure (biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes, peptide carriers) without affecting Dicer or RISC functions. Transformation of these free hydroxyl groups in the BCm moiety into other potentially reactive moieties (such as alkynyl or thiol groups), before RNA synthesis, might allow to selectively react the final BCm- RNA structure (via click chemistry or Michael addition) with the chemically functionalized molecule of interest (i.e. azido-derivative or maleimide-derivative). Such selective functionalization cannot be performed on natural RNA, as the 2'-OH groups of commercial phosphoramidites of natural ribonucleotides are protected in the same manner (by TBS or TOM groups), which avoids any class of selective conjugation after RNA deprotection.

In an embodiment, the peptide carrier is AHNP or octreotide. Examples of click chemistry reactions are copper (l)-catalyzed azide-alkyne cycloaddition reaction (Kolb ef al. 2001 ; Tornoe ef al. 2002) and thiol-maleimide Michael addition reaction (Belbekhouche ef al. 2016).

The term "accessory molecule" means a molecule which is bonded to the compound of the invention providing an additional function to the compound, such as an additional therapeutic effect, signalling properties or promoting delivery of the compound into cells.

The term "functionalization of accessory molecules or BCm moieties" means modification of the accessory molecules or BCm moieties for obtaining a reactive molecule that could chemoselectively react with another reactive molecule. This will allow conjugating one of the reactive molecules (i.e. containing an alkynyl group or a thiol group, as in the Ri or R2 moieties of functionalized BCm moieties) to the second reactive molecule (i.e. accessory molecules functionalized with an azido or a maleimide group, respectively). The term "chemically functionalized accessory molecules or BCm moieties" means chemically modified accessory molecules or BCm moieties that could react with another reactive molecule, allowing conjugation of said molecules.

In order to chemically functionalize the accessory molecules, a chemical group (i.e. azide or maleimide group) can be bonded to the aforesaid accessory molecule allowing the chemoselective reaction of said accessory molecule with the alkynyl (to react with the azide group), or the thiol group (to react with the maleimide group), from moieties Ri or R2 of the BCm moieties.

In a second aspect, the present invention provides a compound according to the first aspect of the invention, wherein the sequences of Am and B-m are complementary to the sequences of messenger RNAs codifying for target genes.

The compound according to the second aspect of the invention may target simultaneously two target genes. Thus, the target genes may be two different target genes.

Alternatively, the sequences of A_Tn and B_Tn are complementary to the sequences of messenger RNAs codifying for different isoforms of the same gene.

Human grb7 (Growth Factor Receptor-Bound Protein 7 or Epidermal Growth Factor Receptor 7, GCID: GC17P039744) is a gene encoding the growth factor receptor-binding protein Grb7 that interacts with epidermal growth factor receptor (EGFR) and ephrin receptors. Human grb7 is located on the long arm of chromosome 17, next to the erbb2 (alias HER2/neu) proto-oncogene. The protein is an adaptor protein involved in receptor tyrosine kinase signaling and plays a role in the integrin signalling pathway and cell migration by binding with focal adhesion kinase (FAK). Diseases associated with grb7 include Breast Cancer and Silver-Russell Syndrome. grb7 has a key role in HER2 signaling, promoting cell survival and migration. It has been reported that expression of grb7 in the HER2 overexpressed breast cancer subtype contributes to the aggressive nature of the tumor and that HER2 signaling inhibition causes grb7 upregulation. Moreover, knockdown of grb7 by RNA interference potentiates the activity of HER2-t.arget.ing drugs, leading to decreases in cell proliferation (Pradip et al. 2013).

Human stard3 (StAR Related Lipid Transfer Domain Containing 3 or Metastatic Lymph Node Gene 64 Protein Mln64, GCID: GC17P039637) is a gene codifying the protein Stard3, a late endosomal integral membrane protein involved in cholesterol transport. It has been described that targeted knockdown of stard3 leads to decreased cell proliferation (Kao ei al. 2006).

Human hsp27 (Heat shock protein 27 or heat shock protein beta-1 , HspB1 ) is a gene encoding the protein Hsp27 or HspB1 . Hsp27 is a chaperone of the sHsp (small heat shock protein) group, hsps, which are normally induced under environmental stress to serve as chaperons for maintenance of correct protein folding, are often overexpressed in many cancers, including breast cancer (Nagaraja ei al. 2012).

In another embodiment, the sequences of Am and B-m are complementary to the sequences of messenger RNAs codifying for target genes grb7/stard3 orgrb7/hsp27.

Another embodiment is the compound according to the second aspect of the invention, wherein A_Tn is identified by the sequences SEQ ID NO: 14 or 20.

Another embodiment is the compound according to the second aspect of the invention, wherein B_Tn is identified by the sequence SEQ ID NO: 17.

Another embodiment is the compound according to the first or second aspect of the invention, wherein the sequences Cmb and C-ma are separated by a BCm moiety flanked at 5' and 3' by an unpaired nucleotide.

Another embodiment is the compound according to the first or second aspect of the invention, wherein C-ma is identified by a sequence selected from the group consisting of SEQ ID NO: 12, 18, 22 and 26. Another embodiment is the compound according to the first or second aspect of the invention, wherein Cmb is identified by the sequence GUUUUA or GCUUCGGAA.

Another embodiment is the compound according to the first or second aspect of the invention, wherein ΟΒΠ is identified by a sequence selected from the group consisting of SEQ ID NO: 19, 23 and 27.

In another embodiment, A_n is identified by a compound selected from the group consisting of SEQ ID NO: 8 - BC6 - SEQ ID NO: 9, SEQ ID NO: 14 - BC6 - SEQ ID NO: 15, SEQ ID NO: 20 - BC6 - SEQ ID NO: 21 , SEQ ID NO: 24 - BC6 - SEQ ID NO: 25 and SEQ ID NO: 20 - BC6 - SEQ ID NO: 28.

In another embodiment, B_n is identified by a compound selected from the group consisting of SEQ ID NO: 10 - BC6 - SEQ ID NO: 1 1 and SEQ ID NO: 16 - BC6 - SEQ ID NO: 17.

In another embodiment, Cm is identified by a compound selected from the group consisting of GUUUUA - BC6 - SEQ ID NO: 12, GCUUCGGAA - BC6 - SEQ ID NO: 18, GCUUCGGAA- BC6 - SEQ ID NO: 22 and GCUUCGGAA - BC6 - SEQ ID NO: 26. Another embodiment is the compound according to the first or second aspect of the invention, wherein CBn is identified by a sequence selected from the group consisting of SEQ ID NO: 19, 23 and 27.

The sequences of Cma, Cmb and CBn for Ci , C2, C3 and C₄ are shown in Table 1.

Table 1

Another embodiment is the compound according to the second aspect of the invention, wherein the compounds Am and Bm are substrates for the enzyme Dicer.

Dicer, also known as endoribonuclease Dicer or as helicase with RNase motif, is an enzyme, which in humans is encoded by the DICER1 gene. Dicer cleaves double- stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA) and microRNA (miRNA), respectively. These processed RNAs are about 19-25 base pairs long with a two-base overhang on the 3' end. These RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA for degradation. Dicer facilitates the activation of RISC, which is essential for RNA interference. This enzyme has been described as advantageous in therapy due to the specificity and diversity of targets it can process, comparing to other therapeutically approaches to silencing genes, such as antibodies (only used against ligands or receptors) or small molecular inhibitors (low specificity and unendurable side effects). Injected siRNAs have poor stability in blood and cause stimulation of non-specific immunity.

The compound according to the first or second aspect of the invention allows administering two different shRNAs simultaneously, improving therapeutic effect in the treatment of complex diseases, that currently rely on the use of multiple drugs. The high degree of flexibility of BC6 allows its connection to two double-stranded RNA arms without perturbing the geometry of the double helixes, as judged by molecular dynamics (MD) simulation analysis of the present invention. Moreover, in silico studies of the present invention have also confirmed that the BC6 dimer adapts very well to the terminal loop positions.

The length of the RNA sequences of A_n and B_n is at least 29 nucleotides.

The length of the RNA sequences in C_n may be variable. Particularly, the length is within the range of 20-23 nucleotides.

BCm moiety wherein m is 6 is a linker with excellent results in terms of flexibility, length and tension. Thus, another preferred embodiment is the compound according to the first or second aspect of the invention, wherein m is 6.

Another embodiment is the compound according to the first or second aspect of the invention, wherein conjugation of the accessory molecules chemically functionalized with an azide or a maleimide group is made by click chemistry reactions with the alkynyl or the thiol moiety of Ri and/or R2 of the chemically functionalized BCm moiety selected from copper (l)-catalyzed azide-alkyne cycloaddition reaction or thiol-maleimide Michael addition reaction. Particularly, the azide or maleimide group comprises a spacer moiety.

The term "conjugation" means the combination of the compound of the invention with the accessory molecules. The term "spacer moiety" means any part of a molecule providing a connection between two other parts of a molecule. In another embodiment, click chemistry reactions with the alkynyl or the thiol moiety of Ri and/or R2 are selected from copper (l)-catalyzed azide-alkyne cycloaddition reaction or thiol-maleimide Michael addition reaction.

As used herein, fluorescent tags are fluorophores able to bond to an alkynyl group. Particularly, fluorescent tags are selected from the group consisting of 6-FAM (azide), ethidium bromide, fluorescein dT, CF dyes, ODN probes, FRET molecular probes, Cy5, Cy3, TAMRA, JOE, MAX, TET, Cy5.5, ROX, TYE 563, Ykima Yellow, HEX, TEX 615, TYE 665, TYE 705, Alexa Fluor dyes (350, 405, 488, 532, 546, 555, 568, 594, 647, 660, 680, 750), LI-COR IR dyes (700, 800, 800CW), ATTO Dyes (488, 532, 550, 565, Rho101 , 590, 633, 647N), Rhodamine dyes (Green-X, Red-X, 5-TAMRA), WellRED dyes (D4, D3, D2), Texas Red-X, Lightcycler 640, Dt 750, GFP, fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Orange, Pacific Green, Coumarinm tetramethylrhodamine (TRITC), BODIPY FL, Texas Red, Super Bright dyes (436. 600, 645, 702), DAPI, SYTOX Green, SYTO 9, TO- PRO-3, propidium iodide, Qdot probes (525, 565, 605, 655, 705, 800), R-Phycoerythrin (R- PE), Allophycocyanin (APC) or any derivatives thereof.

Alkynyl is preferentially ethynyl. Thus, another embodiment is the compound according to the first or second aspect of the invention, wherein alkynyl is ethynyl.

Another embodiment is the compound according to the first or second aspect of the invention, wherein the chemically functionalized accessory molecule is bonded to the chemically functionalized BCm moiety through a 1 ,2,3-triazole moiety or through a succinimide thioether moiety.

In embodiments, the chemically functionalized molecule is bonded to the BCm moiety through a 1 ,2,3-triazole moiety by reaction of the alkynyl group in Ri and/or R2 with the azido group of the chemically functionalized molecule. In embodiments, the chemically functionalized molecule is bonded to the BCm moiety through a succinimide thioether moiety by reaction of the thiol group in R1 and/or R2 with the maleimide group of the chemically functionalized molecule.

In a third aspect, the present invention provides a pharmaceutical composition comprising the compound according to the first or second aspect of the invention and at least one pharmaceutically acceptable excipient or carrier.

The pharmaceutical composition according to the third aspect of the invention may consist of sterile solutions in water, saline solutions or solutions buffered to physiological pH. The pharmaceutical composition may include various carrier agents, thickeners, buffers, preservatives, surfactants, nanoparticles, liposomes and others. The pharmaceutical composition may further include active ingredients such as antimicrobial agents, antiinflammatory agents, anaesthetic agents, etc.

The pharmaceutical composition according to the third aspect of the invention may be administered to a subject in several different ways, depending on whether the treatment is local or systemic, and depending on the area to be treated. Thus, for example, pharmaceutical composition according to the third aspect of the invention may be administered to a subject by ocular, vaginal, rectal, intranasal, oral, by inhalation, or by parenteral route, whether intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intra-arterial, intralymphatic, intravenous, intrathecal, intra-ocular, intracranial and intratracheal. Parenteral administration, if used, is generally performed by injection. The solutions for injection can be prepared in various ways, such as solutions or liquid suspensions, solid forms suitable for being dissolved or placed in suspension before the injection, or as emulsions. Other forms of parenteral administration use systems of slow or sustained release, so that a constant dose is achieved. The preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, and they may also contain buffers and diluent additives and others. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Examples of aqueous solvents are: water, alcohol-aqueous solutions, emulsions or suspensions, including saline and buffer solutions. Examples of parenteral vehicles are: sodium chloride solution, Ringer's dextrose, sodium chloride and dextrose, etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, anti-oxidants, chelating agents, inert gases, etc. The formulations for topical administration may include creams, lotions, gels, drops, suppositories, sprays, liquids and powders. Certain conventional pharmaceutical carriers, aqueous bases, oily bases or, in powder, thickeners, etc. may also be necessary. The compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous medium, capsules or tablets. It may be desirable to include thickening, flavouring, diluent, emulsifying, dispersant agents, etc.

The excipient or carrier may be an inert ingredient or ingredients. Said excipient or carrier may be a diluent, as a way of example. Such compositions can be in crystalline, powder, granular, compacted solid, liquid, solution, suspension, elixir, syrup, emulsion, cream, gel, droplet, mist, vapor or spray form. Conventional techniques for the preparation of pharmaceutical compositions may be used. For example, the pharmaceutical composition described herein may be comprised in a capsule, tablet, pill, caplet, ampoule, sachet, syringe, cartridge, nebulizer or other container.

In another embodiment, the compound for use according to the fourth aspect of the invention in the treatment of breast cancer that has developed resistance to an anti-HER2 therapy is combined with other drug. Examples of drugs which may be combined are Trastuzumab (Herceptin®), Pertuzumab, Ado-trastuzumab emtansine (T-DM1 ), Lapatinib, Neratinib, Bevacizumab, Palbociclib, Trastuzumab-maytansine, Ribociclib, Pazopanib and Afatinib, among others.

Another embodiment is a method of treatment of cancer wherein the compound according to the first or second aspect of the invention or the pharmaceutical composition according to the third aspect of the invention is administered to a subject. According to the present invention, the subject can be a mammal, including human.

Another embodiment is the use of the compound according to the first and second aspect of the invention for the preparation of a medicament for treatment of cancer.

In a particular embodiment, the breast cancer is a HER2+ breast cancer. Human epidermal growth factor receptor 2 (HER2) involved in the development of breast cancer. HER2- positive breast cancers tend to grow and spread faster than other breast cancers. Through dimerization with either other HER family members or itself, HER2 activates downstream signalling pathways that ultimately promote tumorigenesis, cellular proliferation, survival, invasion, and metastasis. Finding out the HER2 status of a breast tumor is important because there are treatments targeted at HER2-positive breast cancers. Currently, four targeted therapies have been approved for the treatment of HER2-positive breast cancer: the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab, the HER2/epidermal growth factor receptor (EGFR) kinase inhibitor lapatinib, and the antibody-drug conjugate trastuzumab emtansine (T-DM1 ). Despite these advances, many patients with HER2- positive breast cancer still succumb to their disease. The main reason behind this is tumor resistance to existing therapies. Early-stage tumors that resist adjuvant therapy will relapse in distant sites, and these metastatic lesions in turn ultimately evade the effects of HER2- targeting. Therefore, understanding the mechanisms by which HER2-positive breast cancers recur and develop therapeutic resistance is critical.

The compound according to the first or second aspect of the invention may be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier. When the carrier serves as a diluent, it may be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound. The compound according to the first or second aspect of the invention can be adsorbed on a granular solid carrier, for example in a sachet. Some examples of suitable carriers are solvents such as water, salt solutions such as isotonic saline, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil or olive oil, or other carriers such as lactose, terra alba, sucrose, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose, and polyvinylpyrrolidone. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The examples of the present invention show that Dicer enzyme process the dsRNAs comprised in the compound according to the first or second aspect of the invention. The enzyme's activity converts the shRNA into siRNAs whose RNA strands are separated by a helicase. The antisense strand is then recognized by protein factors which lead the ssRNA to the RISC complex. The ssRNA anneals to the target mRNA, whose translation will be inhibited. This cellular machinery allows inhibition of the synthesis of pathogenic proteins involved in diseases.

Labelled or tagged molecules are useful to monitor many biological processes and to study the internalization of drugs into their target cells (for example, a peptide-drug conjugate). The present invention discloses BC6 modification as a tool to functionalize the branched RNA BC6-2shRNA with a fluorescent tag. The modification consists on the transformation of at least one of the hydroxyl groups of any of the BC6 dimers into a potentially reactive species. The reactive species may chemoselectively react, after incorporation in the corresponding RNA strand, with another activated species in solution. In a preferred embodiment, the BC6 dimer modified is the central two-way junction element (C_n). In a more preferred embodiment, the modification is by click chemistry wherein at least one hydroxyl group is modified into an alkynyl group. The compound is treated with the Dess-Martin and Ohira reagents (Tsang ef al. 2012) and the free 5'-hydroxyl group of 3'-tert- butyldimethylsilylthymidine is converted to an ethynyl group to give the 5'-0-TBDMs- protected terminal alkyne. After 3'-0-TBDMS removal and 3'-OH acetylation, the resulting 3'-0-Ac-protected alkyne derivative may be converted to the 0⁴-triazolyl derivative, which was reacted with the 5'-0-DMT-3'-0-TBDMS-protected 0⁴-triazolyl nucleoside to give the ethynyl-bearing dimeric nucleoside This compound is deacetilated with methanolic ammonia and the resulting alcohol is then converted to the corresponding phosphoramidite derivative, which may be incorporated into the CT fragment of the C_n central structure of the nanobinder of the invention. RNA is added by automated oligonucleotide synthesis.

In a preferred embodiment, the resulting ethynyl-bearing RNA (d-alk) is treated with a twofold excess of azido-6-carboxyfluorescein (azido-FAM) in the presence of CuS0₄ and sodium ascorbate in a Tris-HCI-ACN (8.2) mixture to give the desired fluorescein-labelled RNA derivative (CT-FAM).

Document Terrazas ef al. 2016 discloses a compound acting as substrate of RISC, which showed interference RNA properties and resistance to undesired degradation by intra and extra-cellular nucleases. The document discloses that BC6 linker has high flexibility, allowing its use in many RNA constructs and the existence of anchoring points, which should allow the conjugation of fluorescent probes or accessory biomolecules to the RNA structure and without affecting Dicer and RISC activities (see below). The document does not provide any information or hint for a person skilled in the art to target simultaneously two targets and therefore, to inhibit the translation of two mRNAs using a RNA nanostructure.

As used herein, the target gene may include any nucleotide sequence including, without limitation, intergenic regions, non-coding regions, untranscribed regions, introns, exons and transgenes. The target gene can be a gene derived from a cell, an endogenous gene, a transgene or exogenous genes such as genes of a pathogen, for example a virus which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism. In a preferred embodiment, the cell containing the target gene may derive from pets (for example, cats, dogs, etc.), farm animals (for example, cows, horses, pigs, sheep, goats, etc.), laboratory animals (for example, mice, rabbits, guinea pigs, etc.) and birds. Preferably, the cell containing the target gene derives from a mammal such as a primate and, with greater preference, a human being.

The doses or quantities of the compounds of the invention must be sufficiently large to produce the desired effect. However, the dose must not be as large as to cause adverse secondary effects, for example unwanted cross reactions, anaphylactic reactions and such like. Generally, the dose will vary with age, condition, sex and degree of the subject's disease and can be determined by any person skilled in the art. The dose can be adjusted by each doctor, based on the clinical condition of the subject involved. The dose, dosage regime and administration route may vary.

The specific design of the compound according to the first or second aspect of the invention, which consists of four building blocks with complementary 3'-sticky ends designed to self- assemble after hybridization, protects the whole construct of the compound according to the first or second aspect of the invention from dissociation under physiological conditions, making possible the administration of its two shRNA components simultaneously once inside the cell. Thus, in a sixth aspect, the present invention provides the use of BCm moieties of general Formula (I) for increasing stability of the RNA structure of a compound comprising a compound A_n, a compound B_n and a compound C_n, according to first or second aspect of the invention. Definitions

As used herein, the term "complementary" refers to interactions between nucleobases: adenine, uracil (thymine in DNA), guanine and cytosine. Adenine and guanine are purines, while thymine, cytosine and uracil are pyrimidines. Both types of molecules complement each other and can only base pair with the opposing type of nucleobase. In nucleic acid, nucleobases are held together by hydrogen bonding, which only works efficiently between adenine and uracil and between guanine and cytosine.

As used herein, the term "target genes" refers to genes that are silenced via RNA interference by the use an RNA molecule.

As used herein, the term "base-paired" refers to interactions between nucleobases: adenine, uracil, guanine and cytosine. Nucleobases are held together by hydrogen bonding, which only works efficiently between adenine and uracil and between guanine and cytosine.

As used herein, the term "unpaired" refers to any nucleobase which is not interacting by hydrogen bonding with any other nucleobase.

As used herein, the term "cancer" can encompass all types of oncogenic processes and/or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, e.g., stages of invasiveness/severity, of a cancer. In embodiments, cancer includes relapsed and/or resistant cancer. The terms "cancer" and "tumor" can be used interchangeably.

As used herein, the term "biomarkers" refers to a traceable substance that is introduced into an organism as a means to examine organ function or other aspects of health. It can also be a substance whose detection indicates a particular disease state and can be typically utilized in the molecular diagnostics of diseases. Biomarkers can be used for personalized medicine and can be typically categorized as either prognostic or predictive. Prognostic biomarkers indicate the likelihood of patient outcome regardless of a specific treatment. Predictive biomarkers are used to help optimize ideal treatments, and indicates the likelihood of benefiting from a specific therapy. The terms "biomarkers" and "biological markers" can be used interchangeably.

As used herein, the term "drugs" refers to a substance used to treat, cure, prevent, or diagnose a disease. The terms "drugs" and "pharmeutical drugs" or "medicines" can be used interchangeably. As used herein, the term "labelled molecules" refers to molecules containing an atomic or molecular label added to the molecule or introduced by replacement of atoms of the molecule. Labelling may be by means of radiolabelling and fluorescence labelling, among others.

As used herein, the term "imaging probes" refers to probes used to help image particular targets or pathways. Imaging probes interact with their surroundings and in turn alter the image according to molecular changes occurring within the area of interest. Imaging refers to a variety of microscopy and nanoscopy techniques including magnetic resonance imaging, optical imaging, near Infrared imaging, single photon emission computed tomography, positron emission tomography, live-cell microscopy, Total Internal Reflection Fluorescence (TIRF)-microscopy, STimulated Emission Depletion (STED)-nanoscopy and Atomic Force Microscopy (AFM), among others.

As used herein, the term "peptide carriers" refers to peptides recognized by specific receptors overexpressed in a target cell and peptides able to cross biological membranes efficiently and to promote the delivery of active agents into cells. Examples of peptide carriers recognized by specific receptors are AHNP, recognized by HER2 and octreotride, recognized by somatostatin subtype 2 receptor (SSTR2), which is overexpressed in many tumor cells.

As used herein, the term "increasing stability of the RNA structure" refers to an increase of the robustness of the construct of the compound according to the first or second aspect of the invention.

Unless defined otherwise, all the technical and scientific terms have the same meaning as those commonly understood by a person skilled in the art in the field of the invention. Similar and equivalent methods and materials to those described here may be used in the practice of the present invention.

Throughout the description and the claims, the term "comprises", "comprising" and their variants are not limiting in nature and therefore do not aim to exclude other technical features.

The term "comprise", "comprising" and their variants, throughout the description and the claims, includes, specifically, the term "consisting" or "consisting of, when referred, particularly, to compounds or biological sequences. It means that the compounds of the invention may "comprise" any of the sequences along with other sequences or molecules or, in a preferred embodiment, the compounds of the invention may "consist of any of said sequences, the later case meaning that the compounds of the invention are precisely restricted to the fragment identified as such by the SEQ ID NO.

Brief description of Drawings

Figure 1 . (A) Synthetic scheme showing the structure of the bifunctional BCm-2shRNA nanobinder, composed of three RNA components: BCm-loop hairpins A_n and B_n and a central two-way junction (C_n), composed of a natural RNA strand (bottom strand; Cen) and its complementary counterpart (top strand; Cm). (B) Scheme showing the structure of the bifunctional BCm-2shRNA nanobinder and showing compounds A_n, B_n and C_n.

Figure 2. (A) Representative snapshot from the molecular dynamics (MD) trajectory of the branched RNA, showing how the internal BC6 bulge adapts its geometry to produce a well- defined branched architecture. The calculations were performed by dividing the branched RNA in three blunt-ended double-stranded fragments (fragments A', B' and C) and subjecting each of them to 200 ns MD simulations. The represented branched structure was constructed by superimposing (using Pymol) the average structures from the three simulations. (B) RMSD plots of fragments A', B' and C respect to the average structures along the MD simulations.

Figure 3. (A) Branched RNAs (BC6-2hRNAs) in which the sequence of each of the branches is complementary to a fragment of the target gene. This class of design involves different central building blocks (C_n) for each case (CTI :CBI ; CT2:CB2; CT3:CB3 and CT₄:CB4). BrRR BC6-2shRNA targets de same region of the Renilla luciferase gene (Ai, Bi ). BrRG, BrSG and BrHG systems target two different genes simultaneously: BrRG targets Renilia/grb7 (A1/B2), BrSG targets stard3lgrb7 (A₂/B₂) and BrHG targets hsp27lgrb7 (A3/B2)). (B) Branched RNAs (BC6-2hRNAs) in which the sticky ends and the central part are kept constant (CT3:CB3) and only the base-paired region of the A_n hairpins changes each time that a different gene is targeted. BrRG^* targets Renilla/grb7 (A4/B2) and BrHG^* targets hsp27/grb7 (A₅/B₂). The specific RNA sequence for each fragment of each construction in all constructions is indicated.

Figure 4. (A) Construction of the branched RNA BrSG by assembly of A₂, B2, CT3, and CB3 components (possessing complementary sticky ends). Native PAGE analysis of each of the four components and the BrSG branched product is shown (B) Native PAGE analysis of combination of building blocks with non-complementary sticky ends. This approach only produces dissociated parts (hybridized central part and hairpins), instead of branched product. Figure 5. Formation of fluorescently labelled BrSG-FAM branched structure via click chemistry. One of the hydroxyl groups of the dimer is converted into alkynyl, a reactive species, which reacts with azido-6-carboxyfluorescein (azido-FAM, N3-FAM) to give the desired fluorescein-labelled RNA derivative (CT3-FAM). This fluorescent RNA is used to incorporate a FAM tag into the BrSG branched system by hybridization of CT3-FAM with the other three RNA components (A₂, B₂ and CB3), obtaining BrSG-FAM.

Figure 6. (A) Treatment of branched BrSG with recombinant Dicer. This class of branched RNA architectures acts as substrate of the Dicer enzyme and is slowly digested to give sequences of about 19 bp (marked with an asterisk). (B) Incubation of the double stranded central part of the branched RNA BrSG formed by hybridization of CT3 and CB3 units, remains unaffected under the same digestion conditions. (C) Hairpin B2 is completely digested into 19-20 bp fragments after incubation with Dicer, suggesting that the enzyme recognizes only the two hairpin components (arms) of the branched RNA design, leading to the slow release of the two-active 19 bp short RNAs.

Figure 7. (A) Plot of specific inhibition of Renilla expression for BrRR, BrRG, BrRG^*, BrHG^* branched RNAs and unmodified siRNA I (20 nM) in He La cells. (B) Immunoblot for Grb7 and β-actin (internal control) from SK-BR-3 cells treated with BrRG branched RNA and unmodified siRNA II (40 nM). (C) Immunoblot for Stard3, Grb7 and β-actin (internal control) from SK-BR-3 cells treated with BrSG branched RNA, unmodified siRNA II (40 nM) and unmodified siRNA III (40 nM). (D) Immunoblot for Hsp27, Grb7 and β-actin (internal control) from SK-BR-3 cells treated with BrHG branched RNA, unmodified siRNA II (40 nM) and unmodified siRNA IV (40 nM).

Figure 8. (A) Immunoblot for Grb7, Hsp27 and β-actin (internal control) from SK-BR-3 cells treated with BrRG^* and BrHG^* branched RNAs and unmodified siRNA I (40 nM). (B) Immunoblot for Grb7, Stard3 and β-actin (internal control) from SK-BR-3 cells treated with fluorescently-labelled BrSG-FAM branched RNA. (C) Fluorescence (1 to 3) and bright-field microscopy images (4) of SK-BR-3 cells transfected with BrSG-FAM, taken 18 hours after transfection with a 4 OX objective. Co-staining with Hoechst (2, 3) and the fluorescent- labelled branched RNA (1 , 3) indicates that the RNA is located in the cytoplasm.

Figure 9. Viability of HER2+ breast cancer cell lines. (A) Cell lines Lapatinib-sensitive SK- BR-3 (upper panel) and BT-474 (lower panel) in the absence (plain bars) and in the presence of Lapatinib (0.1 μM and 1 μΜ respectively from stock solution in DMSO) (stripped bars) after transfection (using Lipofectamine 2000) with mixtures of siRNAs II (grb7) + III {stard3) and II + IV {hsp27), with branched RNAs BrSG (targeting stard3 and grb7) and BrHG^* (targeting hsp27 and grb7) and with non-targeting BrRR system (40 nM). (B) Cell line Lapatinib-resistant UACC-732 (upper panel) in the absence (plain bars) and in the presence (10 μΜ from stock solution in DMSO) (stripped bars) of Lapatinib after transfection (using Lipofectamine 2000) with mixtures of siRNAs II {grbT) + III (stard3) and II + IV (Hsp27), with branched RNAs BrSG (targeting stard3 and grb?) and BrHG^* (targeting hsp27 and grb7) and with non-targeting BrRR system (40 nM). The non-tumor cell line HEK-293 (lower panel) was used as negative control. The growth of the cells was assessed using crystal violet assay and plotted as a percentage of proliferation relative to the vehicle control cells. Vehicle: cells treated with Lipofectamine 2000 and DMSO alone. ^** P < 0.01 , ^*** P < 0.001 and ^**** P < 0.0001 versus indicated samples; # P < 0.05, ## P < 0.01 and #### P < 0.0001 versus vehicle in the absence of Lapatinib.

Description of Embodiments

Material and Methods

RNA synthesis

Oligonucleotide sequences that did not contain modified nucleotides were purchased from Sigma Aldrich. All modified sequences were synthesized at the 1 μηηοΙ scale via solid phase synthesis using standard phosphoramidite methods (Beaucage et al. 1981 ). Reagents for oligonucleotide synthesis including 2'-0-TBDMS-protected phosphoramidite monomers of A^Bz, C^Ac, G^dmf and U, the 5'-deblocking solution (3% TCA in CH₂CI₂), activator solution (0.3 M 5-benzylthio-1 -H-tetrazole in CH₃CN), CAP A solution (acetic anhydride/pyridine/THF), CAP B solution (THF//V-methylimidazole 84/16) and oxidizing solution (0.02 M iodine in tetrahydro-furan/pyridine/water (7:2:1 )) where obtained from commercial sources.

For the synthesis of RNA strands containing BC6 loops, commercially available 5'-0-DMT- A^Bz-3'-succinyl-LCAA-CPG, 5'-0-DMT-C^Ac-3'-succinyl-LCAA-CPG, 5'-0-DMT-G^dmf-3'- succinyl-LCAA-CPG and 5'-0-DMT-U-3'-succinyl-LCAA-CPG were used as the solid supports. The coupling time was 15 min. The coupling yields of natural and modified phosphoramidites were around 95%. Incorporation of the dimeric nucleoside modification did not have a negative effect in the yield. All oligonucleotides were synthesized in DMT- ON mode.

Deprotection and purification of unmodified RNA oligonucleotides

After the solid-phase synthesis, the solid support was transferred to a screw-cap vial and incubated at 55°C for 2 h with 1 .5 mL of NH₃ solution (33%) and 0.5 mL of ethanol. The vial was then cooled on ice and the supernatant was transferred into a 2 mL eppendorf tube. The solid support and vial were rinsed with 50% ethanol (2 x 0.25 ml_). The combined solutions were evaporated to dryness using an evaporating centrifuge. The residue that was obtained was dissolved in DMSO (115 μΙ_). After addition of 60 μΙ_ of triethylamine and 75 μΙ_ of triethylamine trihydrofloride, the resulting solution was incubated at 65°C for 2.5 h. Then, the oligonucleotides were purified using Glen-Pack Cartridges (Glen Research) according to manufacturer's instructions. The oligonucleotides were then purified by 20% polyacrylamide gel electrophoresis (DMT-OFF). After purification, the RNAs were isolated by the crush and soak method, dialyzed, quantified by absorption at 260 nm and confirmed by MALDI mass spectrometry.

For annealing of linear siRNAs, 20 μΜ single strands were incubated in siRNA buffer (100 mM KOAc, 30 mM HEPES-KOH at pH 7.4, 2 mM MgCI₂) for 1 min at 90°C followed by 1 h at 37°C.

Construction of branched RNAs

The construction of the branched nanostructures began by synthesizing the phosphoramidite of the BC6 dimeric nucleoside as described in Terrazas ef al. 2016, Terrazas ef al. 2013 and Noronha ef al. 2002. The resulting activated dimer was incorporated into the BC6-loop or the BC6-bulge internal position of a set of hairpins or single-stranded RNAs composing the central building blocks (A_n, B_n and Cm, respectively) by using an automated DNA/RNA synthesizer and 2'-0-TBDMS-protected phosphoramidites of natural ribonucleotides. For the formation of the branched nanostructures, a mixture of equimolar amounts of the four building blocks A_n, B_n, Cm and CBn were combined in HEPES buffer.

Synthesis of 5'-alkynyl-modified RNA Cr3-alk

Common chemicals and solvents in addition to 2-cyanoethyl diisopropyl- phosphoramidochloridite were purchased from commercial sources and used without further purification. Anhydrous solvents and deuterated solvents (CDC ) were obtained from reputable sources and used as received.

All reactions were carried out under argon atmosphere in oven-dried glassware. Thin-layer chromatography was carried out on aluminium-backed Silica-Gel 60 F254 plates. Column chromatography was performed using Silica Gel (60 A, 230 x 400 mesh). NMR spectra were measured on a Varian Mercury-400 instrument. Chemical shifts are given in parts per million (ppm); J values are given in hertz (Hz). All spectra were internally referenced to the appropriate residual undeuterated solvent. HRMS and ESI spectra were performed on a LC/MSD-TOF (Agilent Technologies) mass spectrometer.

MALDI-TOF spectra were performed using a Perspective Voyager DETMRP mass spectrometer, equipped with nitrogen laser at 337 nm using a 3ns pulse. The matrix used contained 2,4,6-trihydroxyacetophenone (THAP, 10 mg/mL in CI-hCN/water 1 :1 ) and ammonium citrate (50 mg/mL in water).

Analytical reversed-phase-HPLC analysis of the click reaction and the FAM-labeled product were performed on a Jupiter 10 Cie column (250x10 mm, flow rate: 3 mL/min), using linear gradients of 0.1 M aqueous TEAA (solvent A) and ACN (solvent B).

Synthesis of ethynyl-bearing N-hexyl-N dimeric nucleoside

4 3 R = Ac, 5

a

— R = H, 6

b

R = P(N'Pr2)(OCH2CH₂CN), 7 a) NH₃ MeOH

b) DIPEA, CIP(NⁱPr₂)(OCH₂CH₂CN), CH₂CI₂ Alkyne 1 was synthesized as previously described (Sharma etal.1978; Bu kamp etal. 2014)

1-(3-0-Acetyl-2,5,6-trideoxy- -D-erythro-hex-5-ynofuranosyl)-5-methyluracil (2)

AcO

2 DMAP (0.008 g, 0.06 mmol) and acetic anhydride (0.15 mL, 1.6 mmol) were added to a solution of alkyne 1 (0.150 g, 0.64 mmol) in anhydrous pyridine (4 mL). The reaction was stirred overnight at room temperature. The concentrated crude was purified by flash chromatography on silica gel (5% MeOH in CH2CI2) to obtain a yellow foam (2, 178 mg, quantitative yield).¹H NMR (400 MHz, CDCI₃) δ 9.93 (br, 1 H), 7.54 (d, J = 1.2 Hz, 1 H), 6.44 (dd, J = 8.6, 5.8 Hz, 1H), 5.38 (d, J = 4.7 Hz, 1H), 4.78 (m, 1H), 2.83 (d, J = 2.2 Hz, 1H), 2.62 (dd, J= 14.5, 5.8 Hz, 1H), 2.37-2.26 (m, 1H), 2.12 (s, 3H), 1.94 (d, J = 1.1 Hz, 3H). ¹³C NMR (101 MHz, CDC ) δ 170.1, 164.1, 150.8, 135.1, 111.7, 86.5, 79.4, 78.3, 77.7, 74.1, 37.1, 20.9, 12.8. HRMS (ESI+) m/z calcd. for Ci₃Hi₅N₂05 (M + H)⁺ 279.0975, found 279.0972. 1 -(3-0-Acetyl-2,5,6-trideoxy- -D-erythro-hex-5-ynofuranosyl)-5-methyl-4-(A-1 - triazolyl)uracil (3)

AcO

3

1 ,2,4-Triazole (0.476 g, 6.9 mmol) and triethylamine (1.37 mL, 9.9 mmol) were dissolved in anhydrous acetonitrile-C^C (1:1,6 mL) at 0 °C for 5 min, followed by the addition of POCI3 (0.1 mL, 1.08 mmol) at the same temperature. After stirring at 0 °C for 30 min, a solution of alkyne 2 (0.120 g, 0.431 mmol) in acetonitrile-Ch C (2 mL) was cannulated to the mixture and the reaction mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC (5% MeOH in CH2CI2). 5% NaHCC in water was added to the mixture and extracted with CH2CI2 three times. The organic phase was washed with brine, dried with MgS04, filtered and concentrated in vacuum to give 3 as a yellow foam in quantitative yield, which was used without further purification. ¹H NMR (400 MHz, CDCI₃) δ 9.29 (s, 1 H), 8.28 (m, 1 H), 8.12 (s, 1 H), 6.36 (dd, J = 7.6, 6.0 Hz, 1 H), 5.41 (d, J = 4.7 Hz, 1 H), 4.95 - 4.93 (m, 1 H), 3.09 (m, 1 H), 2.80 (d, J = 2.2 Hz, 1 H), 2.49 (d, J = 0.7 Hz, 3H), 2.33 (ddd, J = 14.8, 7.7, 4.9 Hz, 1 H), 2.13 (s, 3H). ¹³C NMR (101 MHz, CDCI₃) δ 170.0, 158.4, 153.9, 153.6, 146.3, 145.1 , 105.8, 89.6, 78.9, 78.3, 78.1 , 75.1 , 38.9, 20.9, 17.4. HRMS (ESI+) m/z calcd. for C15H16N5O4 (M+H)⁺ 330.1 197, found 330.1 191 .

HWM3'-0-fert-Butyldimethylsilyl-5'-OW

e-iA^-II -iS-O-Acetyl^.S.e-trideoxy- -D-erythro-hex-S-ynofuranosy l-S- methylcytosinyl]}hexane (5)

5 AcO

Aminonucleoside 4 (Terrazas ei al. 2016) (0.303 g, 0.40 mmol) and triazolyl derivative 3 (0.120 g, 0.36 mmol) were dissolved in anhydrous pyridine (9.3 mL) at room temperature under argon. Triethylamine (0.43 mL, 3.1 mmol) was added to the mixture and the reaction was stirred overnight at room temperature. The solvent was removed to dryness and the crude was purified by flash chromatography on silica gel (from 1 % MeOH in CH2CI2 to 5% MeOH in CH2CI2) to provide 5 as a yellow foam (300 mg, 81 %). ¹H NMR (400 MHz, CDCI₃) δ 7.64 (d, J = 0.9 Hz, 1 H), 7.49 (d, J = 1 .0 Hz, 1 H), 7.44 - 7.40 (m, 2H), 7.33 - 7.19 (m, 7H), 6.82 (dd, J = 9.0, 1 .2 Hz, 1 H), 6.52 (dd, J = 8.5, 5.7 Hz, 1 H), 6.36 (t, J = 6.1 Hz, 1 H), 6.25 (NH, br, 1 H), 5.93 (NH, br, 1 H), 5.37 (d, J = 4.3 Hz, 1 H), 4.78 (d, J = 2.1 Hz, 1 H), 4.47 (dt, J = 6.3, 4.7 Hz, 1 H), 3.93 (dt, J = 5.6, 2.9 Hz, 1 H), 3.78 (s, 6H), 3.49 (dd, J = 10.5, 2.7 Hz, 1 H), 3.45 - 3.35 (m, 4H), 3.24 (dd, J = 10.6, 3.1 Hz, 1 H), 2.74 (s, J = 2.2 Hz, 1 H), 2.75 - 2.69 (m, 1 H), 2.42 - 2.35 (m, 1 H), 2.24 - 2.13 (m, 2H), 2.1 1 (s, 3H), 2.10 (d, J = 0.7 Hz, 3H), 1 .65 (s, 3H), 1 .37 (m, 4H), 1 .14 (m, 4H), 0.80 (s, 9H), -0.02 (s, 3H), -0.08 (s, 3H). ¹³C NMR (101 MHz, CDCI3) δ 170.1 , 163.5, 163. 5, 158.7, 157.1 , 157.0, 144.6, 135.7, 135.6, 135.6, 135.3, 130.2, 130.1, 128.2, 128.0, 127.0, 113.3, 113.2, 105.5, 104.7, 87.4, 86.6, 86.2, 85.5, 79.9, 78.6, 77.3, 73.9, 71.7, 62.9, 55.3, 42.1, 42.0, 37.8, 28.6, 28.4, 27.0, 25.8, 21.0, 18.0, 14.0, 13.3, -4.6, -4.8. HRMS (ESI+) m/z calcd. for CseH/sNeOioSi (M+H)⁺ 1017.5152, found 1017.5146.

l-iA^-IS'-O-ferf-Butyldimethylsilyl-S'-O^^'-dimethoxytrity ^'-deoxy-S- methylcytidylyl]}-6-{W⁴-[1-(2,5,6-trideoxy- -D-erythro-hex-5-ynofuranosyl)]-5- methylcytosinyl]}hexane (6)

HO

6

Ammonium hydroxide (0.37 mL, 28.0-30% in water) was added to a solution of 5 (0.070 g, 0.069 mmol) in methanol (2.15 mL) and the reaction was allowed to stir for 15 h at room temperature. Solvent was removed under reduced pressure to give 6 in quantitative yield as a white foam.¹H NMR (400 MHz, CDCI₃) δ 7.67 (s, 1 H), 7.53 (s, 1 H), 7.42 (m, 2H), 7.33 -7.15 (m, 7H), 6.82 (m, 2H), 6.49 (dd, J = 7.4, 5.9 Hz, 1H), 6.36 (t, J = 6.1 Hz, 1H), 5.80 (br, 1H), 5.57 (br, 1H), 4.71 (s, 1H), 4.57 (m, 1H), 4.47 (m, 1H), 3.93 (m, 1H), 3.79 (s, 6H), 3.46 (m, 5H), 3.23 (dd, J= 10.5, 3.0 Hz, 1H), 2.69 (d, J = 2.2 Hz, 1H), 2.67 (m, 1H), 2.44- 2.37 (m, 1H), 2.19 (m, 2H), 2.03 (s, 3H), 1.48 (m, 4H), 1.26 (s, 3H), 1.26 (m, 4H), 0.80 (s, 9H), -0.01 (s, 3H), -0.08 (s, 3H).¹³C NMR (101 MHz, CDCI₃) δ 163.5, 163.4, 158.7, 157.1, 156.9, 144.6, 136.3, 136.1, 135.7, 135.6, 130.2, 130.2, 129.1, 128.3, 128.2, 128.0, 127.1, 125.4, 113.3, 113.3, 104.3, 103.8, 87.8, 86.7, 86.2, 85.6, 81.3, 77.0, 76.7, 76.5, 71.6, 62.8, 55.3, 50.6, 42.1, 41.6, 41.6, 40.8, 29.8, 28.8, 28.7, 26.7, 25.8, 21.5, 18.0, 13.8, 13.1, -4.6, - 4.8. HRMS (ESI+) m/z calcd. for C54H71N6O9S1 (M+H)⁺ 975.5046, found 975.5035. l-iA^-IS'-O-ferf-Butyldimethylsilyl-S'-O^^'-dimethoxytrity ^'-deoxy-S- methylcytidylyl]}-6-{A ⁴-[1 -(0-( -cyanoethyl-A ,A '-diisopropyl)phosphoramidite-2,5,6- trideoxy- -D-erythro-hex-5-ynofuranosyl)]-5-methylcytosinyl]}hexane (7)

Diisopropylethylamine (DIPEA, 42 μΙ_, 0.24 mmol) was added to a solution of 6 (0.067 g, 0.069 mmol) in anhydrous dichloromethane (3.3 mL) at 0 °C under argon atmosphere. After 15 min, 2-cyanoethyl-diisopropylphosphoramidochloridite (37 μΙ_, 0.17 mmol) was added and the mixture was stirred for 1 h at room temperature. Once the TLC (5% MeOH in CH2CI2) showed total conversion of the starting material, the mixture was poured into 5% NaHC03 (aqueous) and was extracted three times with CH2CI2. The organic phase was dried over MgS04, filtered and concentrated. The crude phosphoramidite was used without further purification. ³¹P NMR (CDCI3, 162 MHz) δ 149.1 , 148.7.

RNA synthesis

5'-Alkynyl-modified RNA d3-alk was synthesized on the 1 μηηοΙ scale with a K&A Laborgerate DNA RNA synthesizer. 2'-0-TBDMS-5'-0-DMT-protected-CE- phosphoramidites (A^Bz, G^dmf, C^Ac and U) and 5'-alkyne-5'-0-DMT-protected dimeric BC6- CE-phosphoramidite (7) were used. The coupling time was 15 min. The coupling yields of natural and modified phosphoramidites were around 95%. Incorporation of the 5'-alkyne nucleoside modification did not have a negative effect in the yield. The oligonucleotides were synthesized in DMT-OFF mode.

Deprotection and purification of 5'-alkynyl-modified RNAs

After the solid-phase synthesis, the solid support was transferred to a screw-cap vial and incubated at 55 °C for 1 h with 1.5 mL of NH3 solution (33%) and 0.5 mL of ethanol. The vial was then cooled on ice and the supernatant was transferred into a 2 mL eppendorf tube. The solid support and vial were rinsed with 50% ethanol (2 x 0.25 mL). The combined solutions were evaporated to dryness using an evaporating centrifuge. The residue that was obtained was dissolved in DMSO (115 μΙ_). After addition of 60 μΙ_ of triethylamine and 75 μΙ_ of triethylamine trihydrofluoride, the resulting solution was incubated at 65 °C for 2.5 h. Then, the oligonucleotide was purified using Glen-Pack Cartridges (Glen Research) according to manufacturer's instructions. The oligonucleotides were then purified by 20% polyacrylamide gel electrophoresis (DMT-OFF). After purification, the RNA was isolated by the crush and soak method, dialyzed, quantified by absorption at 260 nm and confirmed by MALDI mass spectrometry.

Synthesis of CT3-FAM by Cr3-3lk-FAM conjugation

Stock solutions of CuS0₄ (150 mM) and sodium ascorbate (150 mM), dry azido-FAM and 5-alkynyl-modified RNA and 0.1 mM Tris-HCI buffer (pH 7.5) were flushed with argon for 15 min prior any further treatment.

To an argon-flushed vial containing 0.3 μηηοΙ of azido-fluorescein, 120 μΙ_ of a freshly prepared CuS0₄/sodium ascorbate solution (prepared by addition of 15 μΙ_ of 150 mM CuS0₄ in 0.1 mM Tris-HCI pH 7.5/CH₃CN 8:2 and 15 μΙ_ of 150 mM sodium ascorbate in 0.1 mM Tris-HCI pH 7.5/CH₃CN 8:2 to a vial containing 90 μΙ_ of 0.1 mM Tris-HCI pH 7.5/CH3CN 8:2) were added. The resulting pale-blue solution was immediately added to an argon-flushed vial containing 0.15 μηηοΙ of dry 5'-alkynyl-bearing RNA (CT3-alk). The vial containing the fluorescent azide was rinsed with 30 μΙ_ of 0.1 mM Tris- HCI pH 7.5/CH₃CN 8:2 and the resulting solution was added to the RNA-peptide mixture. The resulting yellow- coloured solution was thoroughly shaken for 30 seconds and allowed to run at room temperature for 90 min.

The reaction was subsequently diluted with Milli-Q water and then purified by 20% polyacrylamide gel electrophoresis (DMT-OFF). After purification, the RNA was isolated by the crush and soak method, dialyzed, quantified by absorption at 260 nm and confirmed by MALDI mass spectrometry.

Cell culture

The SKBR3, BT-4T4, UACC-732, HEK-293T and HeLa cell lines were obtained from the American Type Culture Collection (ATCC). UACC-732 cells were grown on collagen-coated plates. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2. HeLa, BT-474, UACC-732 and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM/F-12; GIBCO) supplemented with glutamine (2 mmol L^"1), fetal bovine serum (FBS, 10%), penicillin (100 U mL^"1), and streptomycin (100 μg mL^"1). SKBR3 cells were cultured in McCoy's modified medium (GIBCO) supplemented with fetal bovine serum (FBS, 10%), penicillin (100 U mL^"1), and streptomycin (100 μg mL^"1).

Luciferase siRNA assays

HeLa cells were regularly passaged to maintain exponential growth. The cells were seeded one day prior to the experiment in a 24-well plate at a density of 150.000 cells/well in complete DMEM containing 10% FBS (500 μΙ_ per well). Following overnight culture, the cells were treated with luciferase plasmids and siRNAs. Two luciferase plasmids -Renilla luciferase (pRL-TK) and firefly luciferase (pGL3) from Promega- were used as a reporter and control. Co-transfection of plasmids and siRNAs was carried out with Lipofectamine 2000 (Life Technologies) as described by the manufacturer for adherent cell lines; pGL3- control (1 .0 μg), pRL-TK (0.1 μg), and RNAs (siRNA duplex or branched RNA) formulated into liposomes were added to each well with a final volume of 600 μί. After a 5-h incubation period, cells were rinsed once with PBS and fed with 600 μ\- of fresh medium (DMEM) containing 10% FBS. After a total incubation period time of 22 h, the cells were harvested and lysed with passive lysis buffer (100 μ\- per well) according to the instructions of the Dual-Luciferase Reporter Assay System (Promega). The luciferase activities of the samples were measured with a MicroLumaPlus LB 96V (Berthold Technologies) with a delay time of 2 s and an integration time of 10 s. The following volumes were used: 20 μ\- of sample and 30 μ\- of each reagent (Luciferase Assay Reagent II and Stop and Glo Reagent). The inhibitory effects generated by siRNAs or branched RNAs were expressed as normalized ratios between the activities of the reporter (Renilla or Firefly) luciferase gene and the control (Firefly or Renilla, respectively) luciferase gene.

Western blot

Analysis of Grb7, Stard3 and Hsp27 protein knockdown

SKBR3 cells were seeded 24 h before transfection in 60 mm dishes at a density of 800.000 cells/dish in medium containing 10% FBS. Following overnight culture, a mixture of two siRNA duplexes (45 nM each) or a branched RNAs (45 nM per dish) formulated into liposomes were added to each dish with a final volume of 2 mL. Co-transfection of RNAs was carried out using Lipofectamine 2000. After a 5-h incubation period, the transfection medium was changed to complete medium containing 10% FBS. After a 48-h incubation time, the cells were harvested with PBS and lysed by incubation in RIPA buffer containing protease inhibitors (Roche) at 4°C for 1 h. Cell debris were removed by centrifugation at 8000 x g for 20 min at 4°C, and protein concentration was determined using the BCA assay (Pierce). 30 μg of protein were resolved by SDS electrophoresis and transferred to a poly(vinylidene difluoride) membrane (Immobilon-P, Millipore). The membrane was blocked with 5% skim milk in TBS containing 0.1 % Tween for 1 h at room temperature and subsequently probed with anti-Grb7 monoclonal rabbit antibody (Santa Cruz Biotechnology) (diluted 1 :500 in blocking buffer), anti-Stard3 monoclonal rabbit antibody (diluted in 1 :3500 in blocking buffer) or Hsp27 monoclonal rabbit antibody (diluted 1 :500 in blocking buffer) overnight at 4°C. Anti-rabbit (goat) IgG HRP conjugated secondary antibody (Thermo Scientific, Rockford, IL) was incubated at 1 :5000 dilution in the blocking solution for 1 h at room temperature. β-Actin was selected as internal control and was detected by incubation with anti-p-actin HRP conjugated antibody (Abeam) (at a dilution of 1 :20.000 in blocking buffer) for 1 h at room temperature. The intensities of the bands were analyzed using I mageJ 1 .45 software.

Cell viability assay

Interference with in vitro growth rate of SKBR3, BT-474 and UACC-732 cells by natural siRNAs and branched RNAs in the presence or in the absence of Lapatinib was measured using crystal violet. 150.000 SKBR3, BT-474 and UACC-732 cells were plated in 24-well plates. Twenty-four hours after plating (0 hrs) cells were transfected with control mixtures of Renilla siRNA I (40 nM) + grb7 siRNA II (40 nM), stard3 siRNA III (40 nM) + grb7 siRNA II (40 nM) or hsp27 siRNA IV (40 nM) + grb7 siRNA II (40 nM), with control siRNA I {Renilla) (40 nM) alone or with branched RNAs BrRR, BrRG, BrSG, BrHG, BrRG^* or BrHG^* (40 nM each) using Lipofectamine 2000 (final volume: 500 μΙ_). 20 hours after transfection, 1 .2 μΙ_ Lapatinib stock solution in DMSO (430 nM, 43 nM or 4.3 mM in the case of SK-BR-3, BT- 474 and UACC-732 cells, respectively) or 1 .2 μΙ_ of DMSO (vehicle) were added. 72 hours later, cells were fixed with 4% formalin for 10 minutes, then washed twice with distilled water and stained with 0.1 % freshly prepared crystal violet for 30 minutes. After washing, the stain was dissolved with 10% acetic acid and subsequently quantified by absorbance at 570 nM. Viability of HEK-293 cells after treatment with branched RNAs and siRNAs was performed using the same protocol, with the exception of the step involving treatment with DMSO or Lapatinib. Cell viability was assessed by crystal violet assay 72 hours transfection with RNAs.

Statistical analysis

Data were analyzed by using the GraphPad Prism 5 program (GraphPad Software). Where appropriate, the results are expressed as mean ± standard deviation (SD). P-values of 0.05 or less were accepted as indicators of statistically significant data. Significant differences were assessed by Student's f-tests. Each experiment was performed in triplicate. Fluorescence microscopy

Imaging SK-BR-3 cells transfected with of BrSG-FAM by fluorescence microscopy

SKBR3 cells were seeded 24 h before transfection in 60 mm dishes at a density of 300.000 cells/dish in medium containing 10% FBS. Following overnight culture, fluorescently- labelled branched RNA BrSG-FAM (40 nM) formulated into liposomes (Lipofectamine 2000) was added to the dish with a final volume of 2 mL. Co-transfection of RNA was carried out using Lipofectamine 2000. 18 hours after transfection cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min, followed by permeabilization with 0.5% Triton for 5 min and nuclei staining with Hoechst (^g mL^-1) for 10 min at room temperature. Cells were then washed 3x with PBS and used for fluorescence microscopy.

Molecular dynamics simulations

The branched BrHG RNA was simulated as follows: the system was divided in 3 fragments (fragments A' (left arm), B' (right arm) and C (central part)) and 200 ns molecular dynamics simulations were performed for each of them. Simulations were done using AMBER14 package (Case ef al. 2014) using the parmbscO-OL3 force field. RNA structures were created using the make-na module (structure.usc.edu). The internal bulge was created by constructing a 5-nt internal bulge of natural ribonucleotides and replacing the three central nucleotides with the BC6 dimer, using the rnacomposer module. The structures were solvated with TIP3P water molecules and neutralized with K⁺ ions. Systems were minimized, annealed and equilibrated following the standard AMBER simulation protocol followed by a 10 ns of post equilibration time prior to the 200 ns production runs.

Examples

Example 1: Design and synthesis of branched RNA architectures

The structure of the BC6-2shRNA consists on a branched RNA architecture composed of three RNA building blocks: two BC6-loop RNA hairpins (forming part of the arms; hairpins A_n and B_n; Figures 1A and 1 B) with 5-6 nt 5'-/3'-terminal dangling ends (sticky ends) that form base-pairs with the 5V3'-terminal dangling ends of a central two-way junction (C_n). This central building block (C_n) is formed by a natural RNA strand (bottom strand; Cen) and its complementary counterpart (top strand; Cm), which possesses an internal 1 nt-BC6-1 nt bulge (acting as a central joint; were nt = natural ribonucleotide connected to the BC6 dimer by a natural phosphodiester bond). The construction of the bifunctional BC6-2shRNA nanobinder involves hybridization-induced assembly of the three components: BC6-loop hairpins A_n and B_n and the central two-way junction (C_n).

It was first investigated in silico the effect of the BC6-loops and BC6 internal bulge on the structure of the resulting branched RNA (BC6-2shRNA). With this aim, the branched structure was divided into three blunt-ended fragments: two BC6-loop 27 bp hairpins (A' and B') and a central 19 bp building block (C) 200 ns molecular dynamics (M D) simulations for each of them was performed. The sequences of the fragments are:

- Fragment A^*: SEQ ID NO: 1 - BC6 - SEQ ID NO: 2

- Fragment B': SEQ ID NO: 3 - BC6 - SEQ ID NO: 4

- Fragment C: Top strand: SEQ ID NO: 5 - BC6 - SEQ ID NO: 7

Bottom strand: SEQ ID NO: 7

Figure 2 shows the resulting simulated structure, constructed by superimposition of the average structure from the three independent simulations. MD simulations revealed that the presence of the BC6-loops and the BC6 internal bulge introduce little changes in the structure of the two double helixes that act as arms in the branched RNA architecture (Figure 2A and 2B). The BC6 bulge induces a local effect on the geometry of the branched structure, provoking a local bending around the internal bulge position generating a two- way branched architecture with very well-defined arms (Figure 2A).

The high degree of flexibility of the BC6 dimer prompted to investigate its ability to act as a joint between two double-stranded RNA fragments of different sequence in a branched nanostructure which might have great potential to inhibit two different genes simultaneously.

As a first approximation, it was constructed a group of four branched dsRNAs in which the sequence of the whole arms (from the BC6 loop of the hairpin to the BC6 internal bulge) was complementary to a fragment of a target gene (Figure 3A).

For the formation of the branched nanostructures, a mixture of equimolar amounts of the four building blocks A_n, B_n, Cm and Ce_n were combined in HEPES buffer. This class of design involves different sequences for each central building block (C_n) (d-d; each of them composed of C M :CBI , CT2:CB2, CT3:CB3 or CT4:CB₄ subunits, respectively). To investigate the ability of this class of structure to induce the inhibition of gene expression via an RNAi mechanism, it was first constructed a branched system with the two arms targeting the same region of the Renilla luciferase gene (BrRR; composed of Ai, Bi, Cn and CBI subunits). In order to study the potential of the branched design to silence two different genes simultaneously, it was also constructed a branched system: (i) BrRG (Renilla/grb7; composed of Ai, ?, Cu. and CB2 subunits), with the Ai and B2 hairpins targeting the Renilla luciferase gene and the grb7 oncogene, respectively, (ii) BrSG (stard3/grb7; composed of A2, B2, Cr3 and CB3 subunits), with the A2 and B2 hairpins targeting the stard3 and grb7 oncogenes, respectively, and (iii) BrHG (hsp27/grb7; composed of A3, B2, CT4 and CB4 subunits), with the A3 and B2 hairpins targeting the hsp27 and grb 7 oncogenes, respectively. Table 2 discloses the sequences of all constructions described above.

Table 2

In order to find an easy way to combine an unlimited number of sequences in the same scaffold, as a second approximation it was considered the possibility of keeping the sticky ends and the central part (C_n) constant and modifying only the base-paired sequence of the hairpins each time to target a different combination of genes. By using this approach, it wouid be able to use the same central two-way junction to test an unlimited number of target combinations, allowing the use of combinatorial approaches to design optimum constructs.

To explore this straightforward design, taking the BrSG (stard3lgrb7) as starting point, we constructed two branched derivatives differing only in the sequence of hairpin A_n (Figure 3B): (i) a first derivative in which A₂ (targeting stard3 in the original BrSG system) was replaced by a hairpin (A₄) with the base-paired region complementary to the Renilla mRNA and the sticky end identical to that of BrSG (branched RNA BrRG^*; Renilla/gr67) and (ii) a second derivative in which A₂ was replaced by another hairpin (A₅) with the base-paired region complementary to the hsp27 mRNA and the sticky end identical to the sticky end of BrSG (branched RNA BrHG^*; hsp27/grb7; where ^* means that the central part (ΟΤΠ:ΟΒΠ) and the building block B_n are kept constant and identical to the central part (CT3:CB3) and B₂ hairpin of BrSG).

Formation of the branched structures proceeds in a very straightforward manner by combination of the four building blocks of each of the branched RNAs, which possess complementary sticky ends specifically designed for each particular branched RNA. Figure 4A shows a relevant example of the construction of these class of nanostructures. Combination of the four RNA components of branched BrSG (A₂, CT3, CB3 and B₂) in HEPES buffer produces the desired product (BrSG) with high efficiency.

Native PAGE analysis of the RNA mixture reveals the formation of a single product (of about 80 bp) that migrates more slowly than each of the four separated RNA building blocks (A₂, CT3, CB3 and B₂) and that the double-stranded central part of the branched RNA BrSG (composed by hybridization of CT3 and CB3 units in the absence of the hairpins A₂ and B₂).

Additionally, as expected, combination of four RNA building blocks belonging to two different groups of branched RNAs and possessing non-complementary sticky ends did not afford the 80 bp branched product. After combination of fragments Ai and Bi (corresponding to the BrRR group) and CT3 and CB3 (corresponding to the BrSG group) were mixed under annealing conditions, native PAGE analysis revealed the presence of two separated RNA domains: one of them of about 20 bp, corresponding to the double-stranded central part formed by hybridization of the CT3 and CB3, and the other one of about 30 bp, corresponding to the separate hairpin components A₂ and B₂ (Figure 4B).

These results further confirm the specificity of the assembly approach. Example 2: Branched RNA architectures functionalized with a tag

Next, it was explored the possibility of using the BC6 modification as a tool to functionalize a branched RNA prodrug with a fluorescent tag, as labelling with fluorescent dyes is very useful to monitor many biological processes and to study the internalization of drugs into their target cells (for example, a peptide-drug conjugate).

With this aim, one of the hydroxyl groups of the dimer was converted into a potentially reactive species that could chemoselectively react (after incorporation into the RNA strand) with another activated species in solution. As the copper (l)-catalyzed azide-alkyne cycloaddition reaction (click chemistry) (Kolb et al. 2001 ; Tornoe et al. 2002) is one of the most selective and versatile (Hein ef al. 2010), the alkynyl functionality as the activating specie was chosen. By using this strategy, ethynyl-bearing RNA CT3-alk was synthesized, which successfully reacted with azido-6-carboxyfluorescein (azido-FAM) to give the desired fluorescein-labelled RNA derivative (CT3-FAM) as a single product (determined by MALDI, PAGE and HPLC analysis of the reaction, data available but not shown). This fluorescent RNA (CT3-FAM) was used to incorporate a FAM tag into the BrSG branched system by hybridization of CT3-FAM with the other three RNA components (A2, B2 and CB3) (Figure 5).

Example 3: Recognition of the branched RNAs by Dicer

In order to confirm that that this class of branched RNA architectures BC6-2shRNA structures can be substrate of Dicer enzyme, branched BrSG was treated with recombinant Dicer.

RNAs (0.91 μΜ) were mixed with Dicer enzyme (0.091 units/μΙ-.; Recombinant Human Turbo Dicer Enzyme Kit from Genlantis, USA) in the buffer system supplied. The mixtures were incubated at 37°C and aliquots (2.2 μΙ_) were taken from the mixture after 0, 1 , 6 and 20 h. They were analyzed by 15% non-denaturing PAGE. The gels were visualized with SYBR Gold.

Figure 6A shows that native PAGE analysis of the RNA mixture reveals the formation of a single product (of about 80 bp). The slow digestion with Dicer also give sequences of about 19 bp (marked with an asterisk).

In a separate experiment, it was observed that incubation of the double stranded central part of this branched RNA, formed by hybridization of CT3 and CB3 units, remained unaffected under the same conditions (Figure 6B), whereas hairpin B2 was completely digested into 19-20 bp fragments (Figure 6C), suggesting that Dicer recognizes only the two hairpin components (arms) of the branched RNA design, leading to the slow release of the two-active 19 bp short RNAs. Example 4: RNAi activity of branched RNAs

To investigate if the arms of the branched RNAs can also be processed by Dicer in vivo and induce inhibition of gene expression, their gene silencing activity in cell culture was analyzed. In a first series of studies, the activity of the first group of branched RNAs (BrRR, BrRG, BrSG, BrHG) was evaluated where the whole arms of the branched structure (from the BC6 terminal loop to the BC6 internal bulge) are complementary to a fragment of the target mRNA.

In a first experiment, HeLa cells were co-transfected with the dual reporter plasmids pRL- TK (Renilla) and pGL3 (Firefly), with the branched BrRR RNA (with each of the two arms targeting the same region of the Renilla luciferase mRNA) and the corresponding siRNA control (I; anti-Ren/7/a). The expression levels of the two luciferase genes were measured 24 h after transfection in the branched RNAs BrRR, BrRG, BrRG^* and BrHG^* disclosed in Example 1 (Figure 7A). Interestingly, the branched BrRR system displayed significant Renilla knockdown activity (95 ± 0.5% inhibition) very similar to the optimum 98 ± 0.1 % inhibition found for unmodified siRNA I. As negative control, BrHG^* system at 20 nM concentration showed expression levels of Renilla luciferase similar to those of untreated cells.

The next step was to evaluate the activity of branched systems targeting two different genes. It was studied first the BrRG RNA, which targets Renilla luciferase and the grb7 oncogene. To carry out this study two parallel experiments that involved treatment of two different cell lines with the BrRG system were performed. Treatment of HeLa cells with dual reporter plasmids pRL-TK and pGL3, the BrRG system, and the control siRNA I revealed a remarkable Renilla knockdown induced by BrRG (85%± 1.5%; Figure 7A). On the other hand, transfection of the HER2+ breast cancer cell line SK-BR-3 with the branched BrRG led to a strong decrease in grb7 expression (100% Grb7 knockdown for BrRG; Figure 7B).

Bifunctional branched systems targeting two different oncogenes simultaneously were evaluated. Interestingly, western blot analysis of SK-BR-3 cells treated with the stard3/grb7 system (BrSG) revealed strong inhibition of expression of both oncogenes, with activities comparable to those of the corresponding linear siRNAs (85% Stard3 knockdown for BrSG and 95% grb7 knockdown for BrSG; Figure 7C). Very interestingly, satisfactory results were also obtained for the branched RNA BrHG (targeting the hsp27 and grb7 genes), with excellent levels of inhibition of both hsp27 and grb7 genes (100% hsp27 knockdown for BrHG and 95% grb7 knockdown for BrHG) (Figure 7D). The branched RNAs BrRG^* and BrHG^* were further studied, which involve invariable central building blocks and variable sequence of the base-paired region of the hairpins. As previously shown in Figure 8A, very encouraging results were obtained for the BrRG^* system, since 86 ± 0.5% inhibition of Renilla expression after treatment of HeLa cell with a 20 nM RNA dose was observed. Moreover, western blot analysis of SK-BR-3 cells treated with the same system showed a 92% of grb7 knockdown 48 hours after transfection. Remarkably, levels of Hsp27 protein levels after treatment of this cell line (SK-BR-3) with the BrRG^* RNA remained unaffected, confirming the specificity of our branched design. Satisfactory results were also obtained for BrHG^*, with 100% and 91 % hsp27 and grb7 knockdown (respectively) after treatment of SK-BR-3 cells, further confirming the efficiency of this second design (Figure 8A).

Moreover, RNAi activity of branched RNAs tagged with a fluorescent label to the central bulge of the branched structure did not interfere with RNAi activity, as the FAM-labelled BrSG-FAM system displayed good levels of dual grb7 and stard3 silencing (95% and 85% grb7 and stard3 knockdown, respectively; Figure 8B), as observed for its unlabelled analogue BrSG (Figure 7C). Furthermore, the label allowed excellent visualization of the BrSG-FAM system in the cytoplasm after transfection. Fluorescence (1 -3) and bright-field microscopy (4) images of SK-BR-3 cells transfected with BrSG-FAM (Figure 8C). Co- staining with Hoechst (2, 3) and fluorescent-labelled branched RNA (1 , 3) indicates that the RNA is located in the cytoplasm. These results thus confirming that the bulges can be used for tracking therapeutic analogues inside the cell without disruption of the RNAi machinery. Using similar chemistry, it is possible to attach the branched structure to carrier molecules to facilitate targeting to specific cells.

Taken together, the results obtained from these experiments are in good agreement with the digestion pattern observed for in vitro Dicer cleavage, suggesting that Dicer only cleaves the double-stranded region of the hairpins contained in the branched structure to produce the functional RNAs. The nature of central part (Cr_n:CBn) and sticky ends, on the other hand, serves only as a carrier of the two RNA drugs and does not interfere with the RNAi process.

These results are of great importance in the design and biological evaluation of new branched analogues for therapeutic applications, as they verify the effectiveness of the propose second approximation, thus allowing the use of the same central two-way junction to test an unlimited number of target combinations. Example 5: Cell viability of SK-BR-3, BT-474 and Lapatinib-resistant UACC-732 cell lines in the presence of combination of naturals siRNAs and branched RNAs.

RNAi-mediated silencing of grb7, stard3 and hsp27 (independently) is known to decrease the viability of HER2+ breast cancer cell lines such as SK-BR-3 and BT-474. Moreover, it has been found that suppression of grb7 by siRNA transfection increases the activity of Lapatinib in these cell lines (SK-BR-3 and BT-474; Lapatinib-sensitive, Ramsey 201 1 ) and that hsp27 removal increases the susceptibility of HER2+ drug-resistant breast cancer cell lines to HER2 inhibitors. There are also some examples about the positive effect of the dual silencing of relevant target oncogenes— overexpressed in resistance processes in complex malignant diseases such as prostate cancer— on the efficacy of conventional anticancer therapies (Kim 2013).

In order to investigate the potential beneficial effect of the branched-mediated knockdown of grb7lstard3 and grb7lhsp27 gene combinations on breast cancer therapy, it was carried out a detailed cell viability study, quantified 72 h after transfection using the crystal violet cell viability assay in the presence and in the absence of Lapatinib.

The investigations started with the Lapatinib-sensitive HER2+ breast cancer cell lines SK- BR-3 and BT-474.

Figure 9A, upper panel, shows the viability profile of SK-BR-3 cells transfected with two of the most relevant branched RNAs developed in this invention, BrSG (targeting stard3 and grb7) and BrHG^* (targeting hsp27 and grb7), with mixtures of the corresponding natural siRNA analogues: II (grb7) + III (stard3) and II + IV (hsp27), and with non-targeting BrRR branched RNA. Very interestingly, 72 h after transfection, viability was significantly lower in cells transfected with branched systems targeting the grb7lstard3 and grb7lhsp27 combinations compared with cells transfected with mixtures of natural targeting RNAs II {grb7) + III (stard3) and II (grb7) + hsp27 (IV), with 48 ± 2% and 54 ± 2% cell proliferation for BrSG and BrHG^* versus 68 ± 2%, 75 ± 5% for mixtures of siRNAs II (grb7) + III (stard3) and II + IV (hsp27) (P < 0.001 and P < 0.01 ), respectively. Remarkably, independently of the nature of the RNA design (mixtures of natural siRNAs or branched RNAs), the grb7lstard3 combination caused stronger antiproliferative effect than the grb7lhsp27 combination. Very interestingly, when SK-BR-3 cells previously transfected with siRNA grb7 + siRNA stard3 or siRNA grb7 + siRNA hsp27 combinations were treated with Lapatinib (1 μΜ), proliferation was significantly diminished, compared with cells treated with the corresponding mixtures of siRNAs alone (68 ± 2% and 75 ± 5% cell proliferation for mixtures of siRNAs II {grb7) + III (stard3) and II + IV (hsp27) respectively in the absence of Lapatinib, versus 36 ± 1 % and 45 ± 1 % for the same mixtures in the presence of Lapatinib (P < 0.0001 and P < 0.01 ), respectively).

The combination of Lapatinib with the branched oligos lead to a dramatic decrease on cell proliferation (48 ± 2% and 54 ± 2% cell proliferation for BrSG and BrHG^* in the absence of Lapatinib versus 29 ± 1 % and 40 ± 1 % for the same branched RNAs in the presence of Lapatinib (P < 0.0001 and P < 0.001 ), respectively), outperforming treatments based on the combination of Lapatinib and siRNAs. Similar, or even better results were obtained with the BT-474 cell line (Figure 9A, lower panel), thus confirming the higher anti-proliferative effect of the branched systems, both alone, or in combination with Lapatinib. Again, in all cases, branched systems are more efficient than standard siRNAs.

At this point, the potential of the branched RNAs in a drug-resistant model was explored. With this aim, we used the HER2+ breast cancer cell line UACC-732, which displays a marked resistance to anti-HER2 inhibitors such as Lapatinib. Very interestingly, treatment of this cell line with the branched RNAs BrSG and BrHG^* caused a significant decrease in cell proliferation, compared with untreated cells and cells treated with non-targeting BrRR (P < 0.0001 in both cases; Figure 9B, upper panel). In contrast, the decrease in proliferation caused by the corresponding mixtures of individual siRNAs (siRNA II, grb7 + siRNA III, stard3 or siRNA II + siRNA IV, hsp27) was significantly smaller (P < 0.01 and no significant, respectively). Lapatinib which alone does not have effect in this cell line recovers antiproliferative activity when combined with BrHG^* and BrSG branched structures.

In contrast to what had been observed for the HER2+ breast cancer cell lines SK-BR-3, BT- 474 and UACC-732, non-malignant HEK-293 cells, that express very low levels of grb7, were unaffected when treated with combinations of grb7lstard3 and grb7lhsp27 siRNAs or branched RNAs (Figure 9B, lower panel), confirming the low toxicity of the branched RNAs. Taken together, the results reveal that the ability of the BC6-branched RNAs (BC6-2shRNA) to administer simultaneously two targeting shRNAs inside the cell is translated in a more efficient anti-proliferative effect, compared with the administration of mixtures of separated units of the corresponding siRNA analogues.

In summary, in this specification it is described a new RNA tool of great potential in the treatment of complex diseases that rely on the use of multiple drugs. Importantly, the fact that the two arms of the RNA architecture of the invention are hybridized with the central part makes the branched structure a co-delivery system able to administer two different shRNA precursors simultaneously, improving therapeutic efficacy. Moreover, the possibility to functionaiize the dimeric nucleoside with potentially reactive groups and the fact that the branched structure possesses three BC6 modifications (two loops and one internal bulge) offers an avenue for the conjugation, not only of fluorescent tags, but up to three biomolecules of interest such as peptide carriers -recognized by specific receptors- in the final branched nanostructure. By using this strategy, it might be possible to monitor the selective receptor mediated delivery of this bifunctional RNA prodrug inside a specific cell line. Moreover, as the BC6 modification is located at the ends and at the central part of the branched structure, it could be compatible with conventional oligonucleotide chemistries at internal positions (like phosphorothioate chemistries) to give rise to therapeutic RNA analogues with even higher biostability.

Sequence Listing Free Text

SEQ ID NO: 1. Sequence A' top

SEQ ID NO: 2. Sequence A' bottom

SEQ ID NO: 3. Sequence B' bottom

SEQ ID NO: 4. Sequence B' top

SEQ ID NO: 5. Sequence C top b

SEQ ID NO: 6. Sequence C top a

SEQ ID NO: 7. Sequence C bottom

SEQ ID NO: 8. Sequence AT1

SEQ ID NO: 9. Sequence AB1

SEQ ID NO: 10 . Sequence BB1

SEQ ID NO: 1 1 . Sequence BT1

SEQ ID NO: 12 . Sequence CT1 a

SEQ ID NO: 13 . Sequence CT1 b

SEQ ID NO: 14 . Sequence AT2. stard3 gene

SEQ ID NO: 15 . Sequence AB2

SEQ ID NO: 16 . Sequence BB2

SEQ ID NO: 17 . Sequence BT2. grb7 gene

SEQ ID NO: 18 . Sequence CT2a

SEQ ID NO: 19 . Sequence CB2

SEQ ID NO: 20 . Sequence AT3 and AT5. hsp27 gene

SEQ ID NO: 21 . Sequence AB3

SEQ ID NO: 22 . Sequence CT3a

SEQ ID NO: 23 . Sequence CB3

SEQ ID NO: 24 . Sequence AT4 SEQ ID NO: 25. Sequence AB4

SEQ ID NO: 26. Sequence CT4a

SEQ ID NO: 27. Sequence CB4

SEQ ID NO: 28. Sequence AB5

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Claims

1. A compound comprising a compound A_n, a compound B_n and a compound C_n, characterised in that:

B_n comprises RNA compounds ΒΒΠ and B and a second BCm moiety of Formula (I), wherein the second BCm is covalently bonded to ΒΒΠ and Bm, forming the structure BBn-BCm-Βτη, and wherein B_Tn is base-paired with B_Bn and with at least 5 nucleotides of C_Bn;

Cn comprises RNA compounds C a and Cmb, a third BCm moiety of Formula (I) and a RNA compound Cen, wherein the third BCm moiety is covalently bonded to Cmb and Cma, forming the structure Cmb-BCm-Cma, and wherein Cen is partly or completely base-paired with Cma and Cmb; wherein m is in the range from 2 to 8, A_Tn is adjacently assembled to Cma; B_Tn is adjacently assembled to Cmb; A_Bn is adjacently assembled to one side of

and B_Bn is adjacently assembled to the opposite side of

RI is selected from -CH2-OH, -CH2-SH or alkynyl; and is suitable to bond to chemically functionalized accessory molecules selected from the group consisting of biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes and peptide carriers; and R2 is selected from -OH, -SH or alkynyl; and is suitable to bond to chemically functionalized accessory molecules selected from the group consisting of biomarkers, drugs, antibodies, labelled molecules, fluorescent tags, imaging probes and peptide carriers.

2. The compound according to claim 1 , characterised in that the sequences of Am and Bm are complementary to the sequences of messenger RNAs codifying for target genes.

3. The compound according to any of claims 1 or 2, characterised in that the target genes are selected from the group consisting of grb7, stard3 and/or hsp27.

4. The compound according to claims 1 to 3, characterised in that A_Tn is identified by the sequences SEQ ID NO: 14 or 20.

5. The compound according to claims 1 to 4, characterised in that Bm is identified by the sequence SEQ ID NO: 17.

6. The compound according to any of claims 1 to 5, characterised in that Cmb and Cma are separated by a BCm moiety flanked at 5' and 3' by an unpaired nucleotide.

7. The compound according to any of claims 1 to 6, characterised in that Cma is identified by a sequence selected from the group consisting of SEQ ID NO: 12, 18, 22 and 26.

8. The compound according to any of claims 1 to 7, characterised in that Cmb is identified by the sequence GUUUUA or GCUUCGGAA.

9. The compound according to any of claims 1 to 8, characterised in that CBn is identified by a sequence selected from the group consisting of SEQ ID NO: 19, 23 and 27.

10. The compound according to any of claims 1 to 9, characterised in that m is 6.

1 1. The compound according to any of claims 1 to 10, characterised in that conjugation of the accessory molecules chemically functionalized with an azide or a maleimide group is made by click chemistry reactions with the alkynyl or the thiol moiety of Ri and/or R20f the chemically functionalized BCm moiety selected from copper (l)-catalyzed azide-alkyne cycloaddition reaction or thiol-maleimide Michael addition reaction.

12. A pharmaceutical composition comprising the compound of claims 1 to 1 1 and at least one pharmaceutically acceptable excipient or carrier.

13. The compound according to any of claims 1 to 12 or the pharmaceutical composition according to claim 15 for use as a medicament.

14. The compound or pharmaceutical composition for use, according to claim 13, in the treatment of cancer.

15. The compound or pharmaceutical composition for use, according to claim 14, characterised in that said cancer is selected from the group consisting of breast cancer, breast cancer resistant to anti-HER2 therapy, breast carcinoma, breast adenocarcinoma, gastric carcinoma, gastric adenocarcinoma, colon carcinoma, colon adenocarcinoma, pancreas carcinoma, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, ovarian adenocarcinoma, ovarian carcinoma, endometrial carcinoma, carcinoma of the uterine cervix, lung carcinoma, lung adenocarcinoma, non- small-cell lung cancer, small-cell lung cancer, thyroid carcinoma, metastasizing papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, urine bladder carcinoma, transitional cell carcinoma of the urinary bladder, prostate carcinoma, cancer of glial lineage of the central nervous system (glioma), sarcomas, fibrosarcoma, malignant fibrous histiocytoma, Edwing sarcoma human, endometrial stromal sarcoma, osteosarcoma, rhabdomyosarcoma, melanoma, embryonal cancers, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, hematological cancers, B-cell or T-cell leukemia, non-Hodgkin lymphoma, non Hodgkin lymphoma B-cell or T-cell types, Burkitt lymphoma, Hodgkin lymphoma, leukemias, lymphoma B-cell or T- cell types, and multiple myeloma.

16. The compound according to any of claims 1 to 15 for use in a method for monitoring biological processes in vitro or in vivo.

17. Use of the BCm moieties of general Formula (I), as defined in claim 1 , for increasing stability of the RNA structure of a compound comprising a compound A_n, a compound B_n and a compound C_n, according to any one of claims 1 to 1 1.