Nucleic acid nanostructures with core motifs

Abstract

Optimized inhibitory nucleic acids are provided. The nucleic acids have sequences which include an optimal inhibitory motif, such as GGG. Related methods are also described.

We claim: 
     
         1 . A spherical nucleic acid (SNA) comprising, a dense configuration of oligonucleotides radially positioned around a lipidated structure, wherein the oligonucleotides have a length of 8-200 nucleotides and include at least one GGG. 
     
     
         2 . The SNA of  claim 1 , wherein the oligonucleotides have the following structure:
   5′ X 1 GGGX 2  3′
   wherein X 1  and X 2  are independently of one another any nucleotide.   
     
     
         3 . The SNA of  claim 2 , wherein X 1  is selected from the group consisting of G and A. 
     
     
         4 . The SNA of  claim 2  or  3 , wherein 5′ X 1 GGGX 1  3′ is selected from the group consisting of GGGG, GGGT, AGGG and GGGC. 
     
     
         5 . The SNA of any one of  claims 1 - 4 , wherein the oligonucleotides are antisense oligonucleotides. 
     
     
         6 . The SNA of any one of  claims 1 - 5 , wherein the oligonucleotides include 2-5 GGG motifs. 
     
     
         7 . The SNA of any one of  claims 1 - 6 , wherein the SNA structure contains 2-10,000 oligonucleotides. 
     
     
         8 . The SNA of any one of  claims 1 - 7 , wherein the SNA is not a dendrimer. 
     
     
         9 . The SNA of any one of  claims 1 - 8 , wherein the oligonucleotides are linked to a core. 
     
     
         10 . The SNA of  claim 9 , wherein the core is the lipidated structure and is a liposomal core. 
     
     
         11 . The SNA of  claim 10 , wherein the liposomal core is comprised of one type of lipid. 
     
     
         12 . The SNA of  claim 10 , wherein the liposomal core is comprised of 2-10 different lipids. 
     
     
         13 . The SNA of  claim 9 , wherein the core is a solid or hollow core. 
     
     
         14 . The SNA of  claim 9 , wherein the core is inert, paramagnetic or supramagnetic. 
     
     
         15 . The SNA of  claim 9 , wherein the core is a solid core. 
     
     
         16 . The SNA of  claim 15 , wherein the solid core is comprised of noble metals, including gold and silver, transition metals including iron and cobalt, metal oxides including silica, polymers or combinations thereof. 
     
     
         17 . The SNA of  claim 16 , wherein the core is a polymeric core and wherein the polymeric core is comprised of amphiphilic block copolymers, hydrophobic polymers including polystyrene, poly(lactic acid), poly(lactic co-glycolic acid), poly(glycolic acid), poly(caprolactone) and other biocompatible polymers. 
     
     
         18 . The SNA of any one of  claims 1 - 17 , wherein the oligonucleotides have 5′-terminus exposed to the outside surface of the SNA. 
     
     
         19 . The SNA of any one of  claims 1 - 17 , wherein the oligonucleotides have 3′-terminus exposed to the outside surface of the SNA. 
     
     
         20 . The SNA of any one of  claims 1 - 17 , wherein the oligonucleotides are directly linked to the core. 
     
     
         21 . The SNA of any one of  claims 1 - 17 , wherein the oligonucleotides are indirectly linked to the core through a linker. 
     
     
         22 . The SNA of any one of  claims 1 - 17 , wherein the oligonucleotides are nucleolipids. 
     
     
         23 . The SNA of any one of  claim 1 - 4  or  6 - 17 , wherein the oligonucleotides are siRNAs. 
     
     
         24 . The SNA of any one of  claims 1 - 23 , wherein the oligonucleotides are single stranded. 
     
     
         25 . The SNA of any one of  claims 1 - 24 , wherein the oligonucleotides are linear. 
     
     
         26 . The SNA of any one of  claims 1 - 24 , wherein the oligonucleotides are double stranded and do not have an overhang. 
     
     
         27 . The SNA of any one of  claims 1 - 26 , wherein the at least one GGG is positioned at the 5′ end of the oligonucleotide within the first 10 nucleotides. 
     
     
         28 . The SNA of any one of  claims 1 - 26 , wherein the at least one GGG is positioned at the 3′ end of the oligonucleotide within the 10 nucleotides at the 3′ end. 
     
     
         29 . The SNA of any one of  claims 1 - 26 , wherein the at least one GGG is positioned in the center of the oligonucleotide. 
     
     
         30 . A method for reducing gene expression, comprising
 contacting a cell with a nanostructure comprising a nucleotide amphiphile having an inhibitory oligonucleotide having at least one GGG motif, wherein gene expression is reduced to a greater extent than when the cell is contacted with free inhibitory oligonucleotide.   
     
     
         31 . The method of  claim 30 , wherein the nucleotide amphiphile is a spherical nucleic acid (SNA) 
     
     
         32 . The method of  claim 31 , wherein the SNA is an SNA of any of  claims 1 - 29 . 
     
     
         33 . The method of  claim 30  or  31 , wherein the inhibitory oligonucleotide is an antisense oligonucleotide. 
     
     
         34 . The method of  claim 30  or  31 , wherein the inhibitory oligonucleotide has the following structure:
   5′ X 1 GGGX 2  3′
 
 wherein X 1  and X 2  are independently of one another any nucleotide. 
 
     
     
         35 . The method of  claim 34 , wherein X 1  is selected from the group consisting of G and A. 
     
     
         36 . The method of  claim 34  or  35 , wherein 5′ X 1 GGGX 2  3′ is selected from the group consisting of GGGG, GGGT, AGGG and GGGC. 
     
     
         37 . The method of any of  claims 30 - 36 , wherein the inhibitory oligonucleotide is complementary to a target sequence selected from the group consisting of TNF, Bcl-2, EGFR, mdm2, MyD88, PCSK9, survivin, VEGF, developmental genes (e.g. adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth or differentiation factors and their receptors, neurotransmitters and their receptors), oncogenes (e.g. ABLI, BCL1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES), tumor suppresser genes (e.g. APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT1), enzymes (e.g. ACP desaturases and hycroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehycrogenases, amylases, amyloglucosidases, catalases, cellulases, cyclooxygenases, decarboxylases, dextrinases, esterases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, GTPases, helicases, hemicellulases, integrases, invertases, isomersases, kinases, lactases, lipases, lipoxygenases, lysozymes, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, polygalacturonases, proteinases and peptideases, pullanases, recombinases, reverse transcriptases, topoisomerases, xylanases), and a TLR (e.g. TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9). 
     
     
         38 . The method of any of  claims 30 - 36 , wherein the inhibitory oligonucleotide is a therapeutic agent selected from the group consisting of anti-viral agents, anti-tumor agents, and agents for treating inherited disorders. 
     
     
         39 . The method of any of  claims 30 - 36 , wherein the method is a method for treating a disorder selected from the group consisting of an autoimmune disease, an infectious disease, transplant rejection or graft-versus-host disease, malignancy, a pulmonary disorder, an intestinal disorder, a cardiac disorder, sepsis, a spondyloarthropathy, a metabolic disorder, anemia, pain, a hepatic disorder, a skin disorder, a nail disorder, rheumatoid arthritis, psoriasis, psoriasis in combination with psoriatic arthritis, ulcerative colitis, Crohn's disease, vasculitis, Behcet's disease, ankylosing spondylitis, asthma, chronic obstructive pulmonary disorder (COPD), idiopathic pulmonary fibrosis (IPF), restenosis, diabetes, anemia, pain, a Crohn's disease-related disorder, juvenile rheumatoid arthritis (JRA), a hepatitis C virus infection, psoriatic arthritis, and chronic plaque psoriasis. 
     
     
         40 . An inhibitory oligonucleotide comprising a nucleolipid comprising a lipid linked to an oligonucleotide having a nucleotide sequence that includes at least one GGG, wherein the oligonucleotide has a length of 8-200 nucleotides and wherein 30%-90% of the nucleotides are modified. 
     
     
         41 . The inhibitory oligonucleotide of  claim 40 , wherein the oligonucleotide has the following structure:
   5′ X 1 GGGX 2  3′
   wherein X 1  and X 2  are independently of one another any nucleotide.   
     
     
         42 . The inhibitory oligonucleotide of  claim 41 , wherein X 1  is selected from the group consisting of G and A. 
     
     
         43 . The inhibitory oligonucleotide of  claim 41  or  claim 42 , wherein 5′ X 1 GGGX 2  3′ is selected from the group consisting of GGGG, GGGT, AGGG and GGGC. 
     
     
         44 . The inhibitory oligonucleotide of any one of  claims 40 - 43 , wherein the oligonucleotide is an antisense oligonucleotide. 
     
     
         45 . The inhibitory oligonucleotide of any one of  claims 40 - 44 , wherein the oligonucleotide includes 2-5 GGG motifs. 
     
     
         46 . The inhibitory oligonucleotide of any one of  claims 40 - 45 , wherein the lipid is stearyl or cholesterol. 
     
     
         47 . The inhibitory oligonucleotide of any one of  claims 40 - 46 , wherein the nucleotide modifications are phosphorothioate modifications. 
     
     
         48 . The inhibitory oligonucleotide of any one of  claims 40 - 46 , wherein the nucleotide modifications are 2′O Methyl modifications. 
     
     
         49 . The inhibitory oligonucleotide of any one of  claims 40 - 46 , wherein 40%-70% of the nucleotides are modified. 
     
     
         50 . The inhibitory oligonucleotide of any one of  claims 40 - 49 , wherein the oligonucleotide is comprised of at least 40% G. 
     
     
         51 . The inhibitory oligonucleotide of any one of  claims 40 - 49 , wherein the oligonucleotide is comprised of at least 50% G.