RNA-nanostructured double robots and methods of use thereof

1. An RNA nanostructure robot having the sequence of (R 3 ) n —NR 1 -L-NR 2 —(R 4 ) m , wherein:

NR 1 and NR 2 independently represent an RNA nanostructure each comprising a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:1 or SEQ ID NO: 9 that self-assembles into respective first and second scaffolds;

L is a linker which operably links NR 1 to NR 2 ;

wherein R 3 and R 4 are independently selected from a pair of fastener strands, an aptamer, a cargo molecule, a capture strand, a targeting strand, and H;

n is an integer from 1 to 20; and

m is an integer from 0 to 20.

2. An RNA nanostructure robot having the sequence of (R 3 ) n —NR 1 -L-NR 2 —(R 4 ) m , wherein:

NR 1 and NR 2 independently represent an RNA nanostructure each comprising a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:1 or SEQ ID NO: 9 that self-assembles into respective first and second scaffolds;

L is a linker which operably links NR 1 to NR 2 ;

wherein R 3 is selected from a pair of fastener strands, an aptamer, a cargo molecule, a capture strand, a targeting strand, and H;

n is an integer from 1 to 20; and

m is an integer from 0 to 20,

wherein R 4 is a targeting strand operably linked to a targeting moiety and to NR 2 ,

wherein the targeting moiety is an aptamer that specifically binds nucleolin.

3. The RNA nanostructure robot of claim 2 , wherein the aptamer that specifically binds nucleolin is the F50 AS1411 aptamer having the sequence: 5′-GGTGGTGGTGGTTGTGGTGG TGGTGG-3′ (SEQ ID NO: 38).

4. The RNA nanostructure robot of claim 1 , wherein the RNA nanostructure comprises a nucleic acid sequence wherein the nucleic acid sequence has at least about 95% sequence identity to SEQ ID NO:1 or SEQ ID NO: 9.

5. The RNA nanostructure robot of claim 1 , wherein the RNA nanostructure comprises a nucleic acid sequence wherein the nucleic acid sequence comprises SEQ ID NO:1 or SEQ ID NO: 9.

6. An RNA nanostructure robot having the sequence of (R 3 ) n —NR 1 -L-NR 2 —(R 4 ) m , wherein:

NR 1 and NR 2 independently represent an RNA nanostructure each comprising a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:1 or SEQ ID NO: 9 that self-assembles into respective first and second scaffolds;

L is a linker which operably links NR 1 to NR 2 ;

wherein R 3 and R 4 are independently selected from a pair of fastener strands, an aptamer, a cargo molecule, a capture strand, a targeting strand, and H;

n is an integer from 1 to 20;

m is an integer from 0 to 20,

wherein NR 1 or NR 2 further comprises at least one operably linked therapeutic agent, and wherein the therapeutic agent is a peptide or a polypeptide.

7. The RNA nanostructure robot of claim 6 , wherein the polypeptide therapeutic agent is a positively-charged moiety comprising 10 lysine residues.

8. The RNA nanostructure robot of claim 6 , wherein the peptide therapeutic agent is a tumor targeting peptide (TTP) or a human cancer peptide.

9. The RNA nanostructure robot of claim 6 , wherein the polypeptide therapeutic agent is calreticulin.

10. The RNA nanostructure robot of claim 9 , wherein the calreticulin protein engages interactions between tumor cells and macrophages or dendritic cells for enhanced antigen presentation and stimulation of antigen-specific T cells.

11. The RNA nanostructure robot of claim 8 , wherein the human cancer peptide is a human NY-ESO-1 or Muc1 peptide.

12. The RNA nanostructure robot of claim 8 , wherein the TTP is CTKD-K10 having the sequence: CTKDNNLLGRFELSGGGSKKKKKKKKKK (SEQ ID NO: 3).

13. The RNA nanostructure robot of any of claim 1 , 2 , or 6 , wherein the RNA nanostructure robot is a TLR3 agonist.

14. The RNA nanostructure robot of claim 1 , wherein R 3 is a pair of DNA fastener strands configured to fasten the first or second scaffold into an origami structure.

15. The RNA nanostructure robot of claim 1 , wherein R 4 is a pair of DNA fastener strands selected from the following DNA oligonucleotide pairs:

5′-FITC-labeled F50 and 3′-BHQ1-labeled Comp15;

FITC-F50-48 and Comp15-48-Q;

FITC-F50-73 and Comp15-73-Q;

FITC-F50-97 and Comp15-97-Q;

FITC-F50-120 and Comp15-120-Q;

FITC-F50-144 and, Comp15-144-Q; and

FITC-F50-169 and Comp15-169-Q;

wherein the aforementioned oligonucleotides have the following sequences:

5′-FITC-labeled F50:

(SEQ ID NO: 10)

5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCG

TGAATTGCG-3′;

3′-BHQ1-labeled Comp15:

(SEQ ID NO: 11)

5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′;

FITC-F50-48:

(SEQ ID NO: 12)

5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCG

TGAATTGCG-3′;

Comp15-48-Q:

(SEQ ID NO: 13)

5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′;

FITC-F50-73

(SEQ ID NO: 14)

5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGG

AAATCAAAA-3′;

Comp15-73-Q:

(SEQ ID NO: 15)

5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′;

FITC-F50-97:

(SEQ ID NO: 16)

5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGA

ACAAACTAT-3′;

Comp15-97-Q:

(SEQ ID NO: 17)

5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′;

FITC-F50-120:

(SEQ ID NO: 18)

5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTA

CAAACAGTT-3′;

Comp15-120-Q:

(SEQ ID NO: 19)

5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′;

FITC-F50-144:

(SEQ ID NO: 20)

5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTT

CCTATTATT-3;;

Comp15-144-Q:

(SEQ ID NO: 21)

5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-BHQ1′-3′;

FITC-F50-169:

(SEQ ID NO: 22)

5′FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTC

ACCTGAAA-3′;

Comp15-169-Q:

(SEQ ID NO: 23)

5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-BHQ1-

3′[[.]];

F50 and Comp15;

F50-48 and Comp15-48;

F50-73 and Comp15-73;

F50-97 and Comp15-97;

F50-120 and Comp15-120;

F50-144 and, Comp15-144; and

F50-169 and Comp15-169;

wherein the aforementioned oligonucleotides have the following sequences:

F50:

(SEQ ID NO: 24)

GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAATTGC

G-3′;

Comp15:

(SEQ ID NO: 25)

5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′;

F50-48:

(SEQ ID NO: 26)

5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAA

TTGCG-3′;

Comp15-48:

(SEQ ID NO: 27)

5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′;

FF50-73

(SEQ ID NO: 28)

5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAAT

CAAAA-3′;

Comp15-73:

(SEQ ID NO: 29)

5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-3′;

F50-97:

(SEQ ID NO: 30)

5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAA

ACTAT-3′;

Comp15-97:

(SEQ ID NO: 31)

5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-3′;

F50-120:

(SEQ ID NO: 32)

5′-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAAA

CAGTT-3′;

Comp15-120:

(SEQ ID NO: 33)

5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-3′;

F50-144:

(SEQ ID NO: 34)

5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTA

TTATT-3;;

Comp15-144:

(SEQ ID NO: 35)

5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-3′;

F50-169:

(SEQ ID NO: 36)

5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCACC

TGAAA-3′;

Comp15-169:

(SEQ ID NO: 37)

5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-3′.