DNA origami encoding for gene expression and contransfection

1 . A nucleic acid nanostructure for expression of a target gene in a eukaryotic cell comprising:

(a) a single-stranded DNA (ssDNA) scaffold strand that comprises an expression cassette comprising at least one nucleic acid sequence encoding the target gene, a promoter, a terminator, and a polyadenylation signal sequence, and

(b) at least one enhancer staple that comprises a sequence that binds to the ssDNA scaffold strand and upon binding enhances expression of the target gene when the nucleic acid nanostructure is inserted into the eukaryotic cell, as compared to a nucleic acid nanostructure without the enhancer staple,

wherein the at least one enhancer staple binds to the ssDNA scaffold strand at a start region of the promoter and at the polyadenylation signal sequence; whereby when the at least one enhancer staple binds to both the start region of the promoter and the polyadenylation signal sequence, the single enhancer staple acts as a splint that brings together the start region of the promoter and the polyadenylation signal sequence.

2 . The nucleic acid nanostructure according to claim 1 , wherein said ssDNA scaffold strand comprises a plurality of nucleic acid sequences encoding a plurality of target genes.

3 . The nucleic acid nanostructure according to claim 1 , wherein said nucleic acid nanostructure comprises a first subunit and a second subunit that each independently comprise a nucleic acid sequence encoding a first target gene and a second target gene, respectively.

4 . The nucleic acid nanostructure according to claim 1 , wherein the at least one enhancer staple has a length ranging from about 60 to about 250 nucleic acid bases.

5 . The nucleic acid nanostructure according to claim 1 , wherein said ssDNA scaffold strand further comprises (a) an inverted terminal repeat nucleic acid that forms a hairpin, wherein the inverted terminal repeat nucleic acid sequence is positioned upstream or downstream of the expression cassette; or (b) an inverted terminal repeat nucleic acid sequence binding site positioned upstream and downstream of the expression cassette.

6 . The nucleic acid nanostructure according to claim 5 , wherein said ssDNA scaffold strand further comprises said inverted terminal repeat nucleic acid sequence that forms the hairpin, wherein formation of said hairpin results in increased expression of the target gene, as compared to an amount of expression in an otherwise comparable nanostructure that comprises an ssDNA scaffold strand without the inverted terminal repeat nucleic acid sequence that forms the hairpin.

7 . The nucleic acid nanostructure according to claim 5 , wherein said ssDNA scaffold strand further comprises an intron, a DNA nuclear targeting sequence, a Kozak sequence, or a woodchuck hepatitis virus posttranscriptional regulatory element.

8 . The nucleic acid nanostructure according to claim 1 , wherein said ssDNA scaffold strand comprises a nuclear targeting sequence.

9 . The nucleic acid nanostructure according to claim 8 , wherein said ssDNA scaffold strand further comprises an intron, a kozak sequence, or a woodchuck hepatitis virus posttranscriptional regulatory element.

10 . The nucleic acid nanostructure of claim 1 , wherein the nucleic acid nanostructure comprises a first enhancer staple that binds to the ssDNA scaffold strand at the start region of the promoter and a second enhancer staple that binds to the ssDNA scaffold strand at the polyadenylation signal sequence.

11 . A composition comprising the nucleic acid nanostructure according to claim 1 .

12 . A plasmid encoding the nucleic acid nanostructure according to claim 1 .

13 . A method of expressing a gene from a nucleic acid nanostructure comprising:

i) providing the nucleic acid nanostructure of claim 1 ;

ii) delivering said nucleic acid nanostructure provided in step i) to a cell; wherein said delivering comprises transfecting or transforming said cell; and

iii) allowing said cell to express said gene.