Dynamic nanomechanical amplification antenna and uses

Abstract

The present disclosure relates to DNA-based structures and methods of using the same for evaluating different DNA binding interactions including bending kinetics and dynamics of DNA caused by small molecules, DNA-binding proteins or DNA-binding nucleic acid molecules (e.g., tRNAs, long non-coding RNAs etc)).

Description

DYNAMIC NANOMECHANICAL AMPLIFICATION ANTENNA AND USESTHEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Appl. No. 63 / 538,032, filed September 12, 2023, which is incorporated herein by reference in its entirety for any and all purposes.TECHNICAL FIELD

[0002] The present disclosure relates to DNA-based structures and methods of using the same for evaluating different DNA binding interactions including bending kinetics and dynamics of DNA caused by small molecules, DNA-binding proteins or DNA-binding nucleic acid molecules (e.g., tRNAs, long non-coding RNAs etc.). The methods of the present technology enable simultaneous imaging and screening of hundreds of single molecule / protein / DNA / RNA binding observations in a single field of view.BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Mechanical interactions between proteins and DNA are the foundation of life's mechanics. These interactions control the expression of genes, maintain genomic stability, shape the structure of chromatin, enable DNA replication and repair, and underpin the function of molecular machines in the cell. Exploring the biophysics of these interactions not only advances the understanding of fundamental biology but also has profound implications for medicine, biotechnology, and the ability to manipulate and harness the mechanics of life.

[0005] Currently, bending kinetics and dynamics of DNA within the smaller length scales of drug / protein interactions are hard to measure with current optical microscopy techniques due to the diffraction limit of light (-250-300 nm). Accordingly, there is an urgent need for accurate and sensitive methods for evaluating different DNA binding interactions caused by small molecules, DNA-binding proteins or DNA-binding nucleic acid.SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides a DNA-based structure (e.g., DNAntenna) comprising (a) a component that anchors the DNA-based structure to a surface, (b) a double-stranded DNA target region, wherein the double-stranded DNA target region is configured to physically interact with at least one DNA-binding agent, and (c) a high aspect ratio component (e.g., helix bundle), wherein the high aspect ratio component amplifies movement caused by physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region when the high aspect ratio component is tagged with a first plurality of fluorescent molecules, wherein the double-stranded DNA target region is located between the component that anchors the DNA-based structure to the surface and the high aspect ratio component. The at least one DNA-binding agent may be a DNA-binding protein, a DNA-binding nucleic acid molecule, a DNA intercalator, or a small molecule. In some embodiments, the DNA-binding protein is selected from the group consisting of transcription factors, polymerases, ligases, nucleases, restriction enzymes, and helicases. In certain embodiments, the small molecule is a groove binder, a DNA intercalator, a singlestrand binder, and a multi-mode binder. Examples of small molecules include, but are not limited to, distamycin-A, methyl Green, Berenil, netropsin, CC-1065, pentamidine, furamidine, duocarmycins, Hoechst 33342, Hoechst 333580, Hoechst 333258, ethidium bromide, doxorubicin, daunorubicin, daunomycin, cisplatin, carboplatin, oxaliplatin, Polynuclear platinum complexes (e.g., BBR 3464), bleomycin, neocarzinostatin, esperamicins, calichemicins, Dynemicin A, actinomycin D, proflavine, berberine, acridine orange, alkylators, lexitropsins, polyamides, ethyleneimines, nitrogen mustards, methane sulfonates, or thalidomide. In other embodiments, the DNA-binding nucleic acid molecule is an aptamer, an RNA, or a peptide nucleic acid. In any of the above embodiments, the DNA- based structure further comprises a barcode.

[0007] Additionally or alternatively, in some embodiments, the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region are detected by measuring a shift in position of a fluorescent molecule tagging the high aspect ratio component. In certain embodiments, the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region comprise bending kinetics. In some embodiments, the physical interactions between the at least one DNA-binding agentand the double-stranded DNA target region comprise DNA dynamics selected from among DNA extension, DNA contraction, and DNA rotation.

[0008] Additionally or alternatively, in certain embodiments, the component that anchors the DNA-based structure to the surface has a height of about 30 nm to about 33 nm. In some embodiments, the component that anchors the DNA-based structure to the surface has a height of about 30 nm, about 30.1 nm, about 30.2 nm, about 30.3 nm, about 30.4 nm, about 30.5 nm, about 30.6 nm, about 30.7 nm, about 30.8 nm, about 30.9 nm, about 31 nm, about31.1 nm, about 31.2 nm, about 31.3 nm, about 31.4 nm, about 31.5 nm, about 31.6 nm, about31.7 nm, about 31.8 nm, about 31.9 nm, about 32 nm, about 32.1 nm, about 32.2 nm, about32.3 nm, about 32.4 nm, about 32.5 nm, about 32.6 nm, about 32.7 nm, about 32.8 nm, about32.9 nm, or about 33 nm.

[0009] Additionally or alternatively, in some embodiments, the component that anchors the DNA-based structure to the surface has a width of about 30 nm to about 33 nm. In certain embodiments, the component that anchors the DNA-based structure to the surface has a width of about 30 nm, about 30.1 nm, about 30.2 nm, about 30.3 nm, about 30.4 nm, about 30.5 nm, about 30.6 nm, about 30.7 nm, about 30.8 nm, about 30.9 nm, about 31 nm, about 31.1 nm, about 31.2 nm, about 31.3 nm, about 31.4 nm, about 31.5 nm, about 31.6 nm, about 31.7 nm, about 31.8 nm, about 31.9 nm, about 32 nm, about 32.1 nm, about 32.2 nm, about 32.3 nm, about 32.4 nm, about 32.5 nm, about 32.6 nm, about 32.7 nm, about 32.8 nm, about 32.9 nm, or about 33 nm.

[0010] Additionally or alternatively, in some embodiments, the length of the high aspect ratio component is about 70 nm to about 85 nm. In certain embodiments, the length of the high aspect ratio component is about 70 nm, about 70.1 nm, about 70.2 nm, about 70.3 nm, about 70.4 nm, about 70.5 nm, about 70.6 nm, about 70.7 nm, about 70.8 nm, about 70.9 nm, about 71 nm, about 71.1 nm, about 71.2 nm, about 71.3 nm, about 71.4 nm, about 71.5 nm, about 71.6 nm, about 71.7 nm, about 71.8 nm, about 71.9 nm, about 72 nm, about 72.1 nm, about 72.2 nm, about 72.3 nm, about 72.4 nm, about 72.5 nm, about 72.6 nm, about 72.7 nm, about 72.8 nm, about 72.9 nm, about 73 nm, about 73.1 nm, about 73.2 nm, about 73.3 nm, about 73.4 nm, about 73.5 nm, about 73.6 nm, about 73.7 nm, about 73.8 nm, about 73.9 nm, about 74 nm, about 74.1 nm, about 74.2 nm, about 74.3 nm, about 74.4 nm, about 74.5 nm,about 74.6 nm, about 74.7 nm, about 74.8 nm, about 74.9 nm, about 75 nm, about 75.1 nm, about 75.2 nm, about 75.3 nm, about 75.4 nm, about 75.5 nm, about 75.6 nm, about 75.7 nm, about 75.8 nm, about 75.9 nm, about 76 nm, about 76.1 nm, about 76.2 nm, about 76.3 nm, about 76.4 nm, about 76.5 nm, about 76.6 nm, about 76.7 nm, about 76.8 nm, about 76.9 nm, about 77 nm, about 77.1 nm, about 77.2 nm, about 77.3 nm, about 77.4 nm, about 77.5 nm, about 77.6 nm, about 77.7 nm, about 77.8 nm, about 77.9 nm, about 78 nm, about 78.1 nm, about 78.2 nm, about 78.3 nm, about 78.4 nm, about 78.5 nm, about 78.6 nm, about 78.7 nm, about 78.8 nm, about 78.9 nm, about 79 nm, about 79.1 nm, about 79.2 nm, about 79.3 nm, about 79.4 nm, about 79.5 nm, about 79.6 nm, about 79.7 nm, about 79.8 nm, about 79.9 nm, about 80 nm, about 80.1 nm, about 80.2 nm, about 80.3 nm, about 80.4 nm, about 80.5 nm, about 80.6 nm, about 80.7 nm, about 80.8 nm, about 80.9 nm, about 81 nm, about 81.1 nm, about 81.2 nm, about 81.3 nm, about 81.4 nm, about 81.5 nm, about 81.6 nm, about 81.7 nm, about 81.8 nm, about 81.9 nm, about 82 nm, about 82.1 nm, about 82.2 nm, about 82.3 nm, about 82.4 nm, about 82.5 nm, about 82.6 nm, about 82.7 nm, about 82.8 nm, about 82.9 nm, about 83 nm, about 83.1 nm, about 83.2 nm, about 83.3 nm, about 83.4 nm, about 83.5 nm, about 83.6 nm, about 83.7 nm, about 83.8 nm, about 83.9 nm, about 84 nm, about 84.1 nm, about 84.2 nm, about 84.3 nm, about 84.4 nm, about 84.5 nm, about 84.6 nm, about 84.7 nm, about 84.8 nm, about 84.9 nm, or about 85 nm.

[0011] Additionally or alternatively, in some embodiments, the width of the high aspect ratio component is about 5 nm to about 10 nm. In some embodiments, the width of the high aspect ratio component is about 5 nm, about 5.1 nm, about 5.2 nm, about 5.3 nm, about 5.4 nm, about 5.5 nm, about 5.6 nm, about 5.7 nm, about 5.8 nm, about 5.9 nm, about 6 nm, about 6.1 nm, about 6.2 nm, about 6.3 nm, about 6.4 nm, about 6.5 nm, about 6.6 nm, about 6.7 nm, about 6.8 nm, about 6.9 nm, about 7 nm, about 7.1 nm, about 7.2 nm, about 7.3 nm, about 7.4 nm, about 7.5 nm, about 7.6 nm, about 7.7 nm, about 7.8 nm, about 7.9 nm, about 8 nm, about 8.1 nm, about 8.2 nm, about 8.3 nm, about 8.4 nm, about 8.5 nm, about 8.6 nm, about 8.7 nm, about 8.8 nm, about 8.9 nm, about 9 nm, about 9.1 nm, about 9.2 nm, about 9.3 nm, about 9.4 nm, about 9.5 nm, about 9.6 nm, about 9.7 nm, about 9.8 nm, about 9.9 nm, or about 10 nm.

[0012] In any of the preceding embodiments, the double-stranded DNA target region is about 15 bp to about 25 kb in length. In some embodiments, the double-stranded DNA targetregion is about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 125 bp, about 150 bp, about 175 bp, about 200 bp, about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, about 600 bp, about 625 bp, about 650 bp, about 675 bp, about 700 bp, about 725 bp, about 750 bp, about 775 bp, about 800 bp, about 825 bp, about 850 bp, about 875 bp, about 900 bp, about 925 bp, about 950 bp, about 975 bp, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3 kb, about 3.1 kb, about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb, about 3.9 kb, about 4 kb, about 4.1 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, about 5 kb, about 5.1 kb, about 5.2 kb, about 5.3 kb, about 5.4 kb, about 5.5 kb, about 5.6 kb, about 5.7 kb, about 5.8 kb, about 5.9 kb, about 6 kb, about 6.1 kb, about 6.2 kb, about 6.3 kb, about 6.4 kb, about 6.5 kb, about 6.6 kb, about 6.7 kb, about 6.8 kb, about 6.9 kb, about 7 kb, about 7.1 kb, about7.2 kb, about 7.3 kb, about 7.4 kb, about 7.5 kb, about 7.6 kb, about 7.7 kb, about 7.8 kb, about 7.9 kb, about 8 kb, about 8.1 kb, about 8.2 kb, about 8.3 kb, about 8.4 kb, about 8.5 kb, about 8.6 kb, about 8.7 kb, about 8.8 kb, about 8.9 kb, about 9 kb, about 9.1 kb, about 9.2 kb, about 9.3 kb, about 9.4 kb, about 9.5 kb, about 9.6 kb, about 9.7 kb, about 9.8 kb, about 9.9 kb, about 10 kb, about 10.1 kb, about 10.2 kb, about 10.3 kb, about 10.4 kb, about 10.5 kb, about 10.6 kb, about 10.7 kb, about 10.8 kb, about 10.9 kb, about 11 kb, about 11.1 kb, about11.2 kb, about 11.3 kb, about 11.4 kb, about 11.5 kb, about 11.6 kb, about 11.7 kb, about 11.8 kb, about 11.9 kb, about 12 kb, about 12.1 kb, about 12.2 kb, about 12.3 kb, about 12.4 kb, about 12.5 kb, about 12.6 kb, about 12.7 kb, about 12.8 kb, about 12.9 kb, about 13 kb, about 13.1 kb, about 13.2 kb, about 13.3 kb, about 13.4 kb, about 13.5 kb, about 13.6 kb, about 13.7 kb, about 13.8 kb, about 13.9 kb, about 14 kb, about 14.1 kb, about 14.2 kb, about 14.3 kb, about 14.4 kb, about 14.5 kb, about 14.6 kb, about 14.7 kb, about 14.8 kb, about 14.9 kb, or about 15 kb. Additionally or alternatively, in some embodiments, the double-stranded DNAtarget region comprises a promoter, an enhancer, an intron, an exon, a telomere, a centromere, a silencer, an insulator, a Long noncoding RNA, a tRNA, or a microRNA.

[0013] Additionally or alternatively, in some embodiments, the surface is a slide, a well, a plastic plate, a microtiter plate, a microfluidic array, or a gel.

[0014] In any of the foregoing embodiments, the first plurality of fluorescent molecules comprise 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino- N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"- dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6- dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6- carb oxy fluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6- carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4(Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); ATTO 488 or VIC®.

[0015] In any and all embodiments of the DNA-based structures disclosed herein, the component that anchors the DNA-based structure to the surface is tagged with a second plurality of fluorescent molecules. The second plurality of fluorescent molecules may comprise 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino- N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"- dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6- dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6- carb oxy fluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6- carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine;IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); ATTO 488 or VIC®.

[0016] In another aspect, the present disclosure provides a method for evaluating DNA binding interactions comprising contacting any and all embodiments of the DNA-based structures disclosed herein with at least one DNA-binding agent, wherein the DNA-based structure is anchored to a surface and wherein the at least one DNA-binding agent binds to the double-stranded DNA target region of the DNA-based structure, and detecting physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region by determining a shift in position of the first plurality of fluorescent molecules relative to the second plurality of fluorescent molecules. The surface may be a slide, a well, a plastic plate, a microtiter plate, a microfluidic array, or a gel. In certain embodiments, the shift in position of the first plurality of fluorescent molecules relative to the second plurality of fluorescent molecules is determined via atomic force microscopy (AFM) or single molecule total internal reflection fluorescence (smTIRF) microscopy.

[0017] Additionally or alternatively, in some embodiments of the methods described herein, the at least one DNA-binding agent is a DNA-binding protein, a DNA-binding nucleic acid molecule, a DNA intercalator, or a small molecule. In some embodiments of the methods disclosed herein, the DNA-binding protein is selected from the group consisting of transcription factors, polymerases, ligases, nucleases, restriction enzymes, and helicases. In certain embodiments, the small molecule is a groove binder, a DNA intercalator, a singlestrand binder, and a multi-mode binder. Examples of small molecules include, but are notlimited to, distamycin-A, methyl Green, Berenil, netropsin, CC-1065, pentamidine, furamidine, duocarmycins, Hoechst 33342, Hoechst 333580, Hoechst 333258, ethidium bromide, doxorubicin, daunorubicin, daunomycin, cisplatin, carboplatin, oxaliplatin, Polynuclear platinum complexes (e.g., BBR 3464), bleomycin, neocarzinostatin, esperamicins, calichemicins, Dynemicin A, actinomycin D, proflavine, berberine, acridine orange, alkylators, lexitropsins, polyamides, ethyleneimines, nitrogen mustards, methane sulfonates, or thalidomide. In other embodiments, the DNA-binding nucleic acid molecule is an aptamer, an RNA, or a peptide nucleic acid.

[0018] In any and all embodiments of the methods described herein, the double-stranded DNA target region comprises nicked DNA or non-nicked DNA. The double-stranded DNA target region may comprise A-form DNA, B-form DNA, or Z-form DNA. Additionally or alternatively, in some embodiments of the methods disclosed herein, the double-stranded DNA target region is about 15 bp to about 25 kb in length. In some embodiments, the double-stranded DNA target region is about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 125 bp, about 150 bp, about 175 bp, about 200 bp, about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, about 600 bp, about 625 bp, about 650 bp, about 675 bp, about 700 bp, about 725 bp, about 750 bp, about 775 bp, about 800 bp, about 825 bp, about 850 bp, about 875 bp, about 900 bp, about 925 bp, about 950 bp, about 975 bp, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3 kb, about 3.1 kb, about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb, about 3.9 kb, about 4 kb, about 4.1 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about4.8 kb, about 4.9 kb, about 5 kb, about 5.1 kb, about 5.2 kb, about 5.3 kb, about 5.4 kb, about 5.5 kb, about 5.6 kb, about 5.7 kb, about 5.8 kb, about 5.9 kb, about 6 kb, about 6.1 kb, about 6.2 kb, about 6.3 kb, about 6.4 kb, about 6.5 kb, about 6.6 kb, about 6.7 kb, about 6.8 kb, about 6.9 kb, about 7 kb, about 7.1 kb, about 7.2 kb, about 7.3 kb, about 7.4 kb, about 7.5 kb,about 7.6 kb, about 7.7 kb, about 7.8 kb, about 7.9 kb, about 8 kb, about 8.1 kb, about 8.2 kb, about 8.3 kb, about 8.4 kb, about 8.5 kb, about 8.6 kb, about 8.7 kb, about 8.8 kb, about 8.9 kb, about 9 kb, about 9.1 kb, about 9.2 kb, about 9.3 kb, about 9.4 kb, about 9.5 kb, about 9.6 kb, about 9.7 kb, about 9.8 kb, about 9.9 kb, about 10 kb, about 10.1 kb, about 10.2 kb, about 10.3 kb, about 10.4 kb, about 10.5 kb, about 10.6 kb, about 10.7 kb, about 10.8 kb, about 10.9 kb, about 11 kb, about 11.1 kb, about 11.2 kb, about 11.3 kb, about 11.4 kb, about 11.5 kb, about 11.6 kb, about 11.7 kb, about 11.8 kb, about 11.9 kb, about 12 kb, about 12.1 kb, about 12.2 kb, about 12.3 kb, about 12.4 kb, about 12.5 kb, about 12.6 kb, about 12.7 kb, about 12.8 kb, about 12.9 kb, about 13 kb, about 13.1 kb, about 13.2 kb, about 13.3 kb, about 13.4 kb, about 13.5 kb, about 13.6 kb, about 13.7 kb, about 13.8 kb, about 13.9 kb, about 14 kb, about 14.1 kb, about 14.2 kb, about 14.3 kb, about 14.4 kb, about 14.5 kb, about 14.6 kb, about 14.7 kb, about 14.8 kb, about 14.9 kb, or about 15 kb. Additionally or alternatively, in some embodiments, the double-stranded DNA target region comprises a promoter, an enhancer, an intron, an exon, a telomere, a centromere, a silencer, an insulator, a Long noncoding RNA, a tRNA, or a microRNA.

[0019] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the double-stranded DNA target region comprises an epigentic modification selected from among methylation, acetylation, deacetylation, phosphorylation, ubiquitination, and sumoylation. In certain embodiments, the double-stranded DNA target region comprises an AT -rich region, an AC-rich region, an AG-rich region, a GC-rich region, a poly-A region, or a GG-rich region. Additionally or alternatively, in some embodiments of the methods disclosed herein, the double-stranded DNA target region comprises simple plectonemes (e.g., two-start right-handed helix with terminal loops), hyperplectonemes, or toroids (e.g., one- start left-handed helix).

[0020] In any and all embodiments of the methods described herein, the double-stranded DNA target region has a persistence length of about 46.5 nm to about 55 nm. In certain embodiments, the double-stranded DNA target region has a persistence length of about 46.5 nm, about 46.6 nm, about 46.7 nm, about 46.8 nm, about 46.9 nm, about 47.0 nm, about 47.1 nm, about 47.2 nm, about 47.3 nm, about 47.4 nm, about 47.5 nm, about 47.6 nm, about 47.7 nm, about 47.8 nm, about 47.9 nm, about 48.0 nm, about 48.1 nm, about 48.2 nm, about 48.3 nm, about 48.4 nm, about 48.5 nm, about 48.6 nm, about 48.7 nm, about 48.8 nm, about 48.9nm, about 49.0 nm, about 49.1 nm, about 49.2 nm, about 49.3 nm, about 49.4 nm, about 49.5 nm, about 49.6 nm, about 49.7 nm, about 49.8 nm, about 49.9 nm, about 50.0 nm, about 50.1 nm, about 50.2 nm, about 50.3 nm, about 50.4 nm, about 50.5 nm, about 50.6 nm, about 50.7 nm, about 50.8 nm, about 50.9 nm, about 51.0 nm, about 51.1 nm, about 51.2 nm, about 51.3 nm, about 51.4 nm, about 51.5 nm, about 51.6 nm, about 51.7 nm, about 51.8 nm, about 51.9 nm, about 52.0 nm, about 52.1 nm, about 52.2 nm, about 52.3 nm, about 52.4 nm, about 52.5 nm, about 52.6 nm, about 52.7 nm, about 52.8 nm, about 52.9 nm, about 53.0 nm, about 53.1 nm, about 53.2 nm, about 53.3 nm, about 53.4 nm, about 53.5 nm, about 53.6 nm, about 53.7 nm, about 53.8 nm, about 53.9 nm, about 54.0 nm, about 54.1 nm, about 54.2 nm, about 54.3 nm, about 54.4 nm, about 54.5 nm, about 54.6 nm, about 54.7 nm, about 54.8 nm, about 54.9 nm, or about 55 nm.

[0021] Additionally or alternatively, in some embodiments of the methods disclosed herein, the double-stranded DNA target region has a melting temperature of about 50°C to about 100°C. In certain embodiments, the double-stranded DNA target region has a melting temperature of about 50°C, about 50.5°C, about 51°C, about 51.5°C, about 52°C, about 52.5°C, about 53°C, about 53.5°C, about 54°C, about 54.5°C, about 55°C, about 55.5°C, about 56°C, about 56.5°C, about 57°C, about 57.5°C, about 58°C, about 58.5°C, about 59°C, about 59.5°C, about 60°C, about 60.5°C, about 61°C, about 61.5°C, about 62°C, about 62.5°C, about 63°C, about 63.5°C, about 64°C, about 64.5°C, about 65°C, about 65.5°C, about 66°C, about 66.5°C, about 67°C, about 67.5°C, about 68°C, about 68.5°C, about 69°C, about 69.5°C, about 70°C, about 70.5°C, about 71°C, about 71.5°C, about 72°C, about 72.5°C, about 73°C, about 73.5°C, about 74°C, about 74.5°C, about 75°C, about 75.5°C, about 76°C, about 76.5°C, about 77°C, about 77.5°C, about 78°C, about 78.5°C, about 79°C, about 79.5°C, about 80°C, about 80.5°C, about 81°C, about 81.5°C, about 82°C, about 82.5°C, about 83°C, about 83.5°C, about 84°C, about 84.5°C, about 85°C, about 85.5°C, about 86°C, about 86.5°C, about 87°C, about 87.5°C, about 88°C, about 88.5°C, about 89°C, about 89.5°C, about 90°C, about 90.5°C, about 91°C, about 91.5°C, about 92°C, about 92.5°C, about 93°C, about 93.5°C, about 94°C, about 94.5°C, about 95°C, about 95.5°C, about 96°C, about 96.5°C, about 97°C, about 97.5°C, about 98°C, about 98.5°C, about 99°C, about 99.5°C, or about 100°C.

[0022] In any and all embodiments of the methods described herein, the double-stranded DNA target region has a diameter of about 18 A to about 23 A. In some embodiments, the double-stranded DNA target region has a diameter of about 18 A, about 18.1 A, about 18.2 A, about 18.3 A, about 18.4 A, about 18.5 A, about 18.6 A, about 18.7 A, about 18.8 A, about 18.9 A, about 19 A, about 19.1 A, about 19.2 A, about 19.3 A, about 19.4 A, about 19.5 A, about 19.6 A, about 19.7 A, about 19.8 A, about 19.9 A, about 20 A, about 20.1 A, about 20.2 A, about 20.3 A, about 20.4 A, about 20.5 A, about 20.6 A, about 20.7 A, about 20.8 A, about 20.9 A, about 21 A, about 21.1 A, about 21.2 A, about 21.3 A, about 21.4 A, about 21.5 A, about 21.6 A, about 21.7 A, about 21.8 A, about 21.9 A, about 22 A, about 22.1 A, about 22.2 A, about 22.3 A, about 22.4 A, about 22.5 A, about 22.6 A, about 22.7 A, about 22.8 A, about 22.9 A, or about 23 A.

[0023] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the double-stranded DNA target region comprises at least one bend angle that ranges from about 15° to about 180°. In some embodiments, the double-stranded DNA target region comprises at least one bend angle that ranges from about 15°, about 16°, about 17°, about 18°, about 19°, about 20°, about 21°, about 22°, about 23°, about 24°, about 25°, about 26°, about 27°, about 28°, about 29°, about 30°, about 31°, about 32°, about 33°, about 34°, about 35°, about 36°, about 37°, about 38°, about 39°, about 40°, about 41°, about 42°, about 43°, about 44°, about 45°, about 46°, about 47°, about 48°, about 49°, about 50°, about 51°, about 52°, about 53°, about 54°, about 55°, about 56°, about 57°, about 58°, about 59°, about 60°, about 61°, about 62°, about 63°, about 64°, about 65°, about 66°, about 67°, about 68°, about 69°, about 70°, about 71°, about 72°, about 73°, about 74°, about 75°, about 76°, about 77°, about 78°, about 79°, about 80°, about 81°, about 82°, about 83°, about 84°, about 85°, about 86°, about 87°, about 88°, about 89°, about 90°, about 91°, about 92°, about 93°, about 94°, about 95°, about 96°, about 97°, about 98°, about 99°, about 100°, about 101°, about 102°, about 103°, about 104°, about 105°, about 106°, about 107°, about 108°, about 109°, about 110°, about 111°, about 112°, about 113°, about 114°, about 115°, about 116°, about 117°, about 118°, about 119°, about 120°, about 121°, about 122°, about 123°, about 124°, about 125°, about 126°, about 127°, about 128°, about 129°, about 130°, about 131°, about 132°, about 133°, about 134°, about 135°, about136°, about 137°, about 138°, about 139°, about 140°, about 141°, about 142°, about 143°, about 144°, about 145°, about 146°, about 147°, about 148°, about 149°, about 150°, about 151°, about 152°, about 153°, about 154°, about 155°, about 156°, about 157°, about 158°, about 159°, about 160°, about 161°, about 162°, about 163°, about 164°, about 165°, about 166°, about 167°, about 168°, about 169°, about 170°, about 171°, about 172°, about 173°, about 174°, about 175°, about 176°, about 177°, about 178°, about 179°, or about 180°.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows exemplary design of DNAntenna (Dynamic nanomechanical amplification antenna).

[0025] FIG. 2 shows construction of DNAntenna. The tripod base was designed with M13mpl8 scaffold, and the 10 helix bundle (HB) was designed with the p3031 scaffold.

[0026] FIG. 3 shows calibration of DNAntenna brownian motion.

[0027] FIG. 4 shows single molecule total internal reflection fluorescence (TIRF) microscopy (smTIRF) data analysis pipeline. Total internal reflection fluorescence (TIRF) microscopy is widely used to examine the localization and dynamics of molecular events near the coverslips.

[0028] FIG. 5 shows exemplary smTIRF trajectories.

[0029] FIG. 6 shows exemplary images of 10HB lever arm (e.g., high aspect ratio component) of DNAntenna. RMSF stands for root mean square fluctuation, which is a numerical measurement similar to RMSD, but instead of indicating positional differences between entire structures over time, RMSF is a calculation of individual residue flexibility, or how much a particular residue moves (fluctuates) during a simulation.

[0030] FIG. 7 shows exemplary images of tripod base structure of DNAntenna.

[0031] FIG. 8 shows structural configuration of ligated DNAntenna.

[0032] FIG. 9 shows exemplary DNA-protein interaction-induced bending or kinetics that are observable in DNAntenna platform e.g., vertical extension / contraction, rotation, and bending of lever arm of DNAntenna.DETAILED DESCRIPTION

[0033] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0034] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0035] The large helix bundle of the compositions disclosed herein acts as a lever arm to amplify DNA binding interactions in a way that is measurable with optical microscopy using the shift in position of the fluorescent tags on the long helix bundle compared to the position of the fluorescent tags on the base (FIG. 8). Leveraging this amplification the DNAntenna can be used to measure DNA bending dynamics and kinetics in an optical microscopy set up(FIG. 9). Further, many compositions of the present technology can be DNA barcoded in conjunction with different DNA target regions, enabling simultaneous imaging and screening of different single molecule / protein / DNA / RNA binding observations in a single field of view (FOV).Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0037] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0038] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptideare in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0039] The term “barcode” refers to a sequence of nucleotides within a polynucleotide that is used to identify a nucleic acid molecule. For example, a barcode can be used to identify molecules when the molecules from several groups are combined for processing or sequencing in a multiplexed fashion. A barcode can be located at a certain position within a polynucleotide (e.g., at the 3'-end, 5'-end, or middle of the polynucleotide) and can comprise sequences of any length (e.g., 1-100 or more nucleotides). Additionally, a barcode can comprise one or more pre-defined sequences. The term “pre-defined” means that sequence of a barcode is predetermined or known prior to identifying or without the need to identify the entire sequence of the nucleic acid comprising the barcode. In some cases, pre-defined barcodes can be attached to nucleic acids for sorting the nucleic acids into groups. In some embodiments, a barcode can comprise artificial sequences, e.g., designed or engineered sequences that are not present in the unaltered (wild-type) genome of a subject. In other embodiments, a barcode can comprise an endogenous sequence, e.g., sequences that are present in the unaltered (wildtype) genome of a subject. In certain embodiments, a barcode can be an endogenous barcode. An endogenous barcode can be a sequence of a genomic nucleic acid, where the sequence is used as a barcode or identifier for the genomic nucleic acid. One or more sequences of the genomic DNA fragment can be an endogenous barcode. Different types of barcodes can be used in combination. For example, an endogenous genomic nucleic acid fragment can be attached to an artificial sequence, which can be used as a unique identifier of the genomic nucleic acid fragment. A "sample-specific barcode" refers to a polynucleotide sequence that is used to identify the origin or source of a nucleic acid molecule.

[0040] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thusstabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0041] As used herein, the term “DNA origami” refers to the nanoscale folding of DNA to create arbitrary two- and three-dimensional shapes at the nanoscale. The specificity of the interactions between complementary base pairs make DNA a useful construction material, through design of its base sequences.

[0042] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0043] The terms “helix bundle,” “HB,” and “high aspect ratio component” are synonymous with each other and refer to the lever arm component of the DNAntenna disclosed herein.

[0044] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0045] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.DNAntenna Structures of the Present Technology and Uses Thereof

[0046] Disclosed herein is a DNA origami-based structure that is configured to measure interactions on DNA. The DNAntenna (Dynamic Nanomechanical Amplification Antenna) is designed to amplify and measure bends imparted on a DNA helix.

[0047] In one aspect, the present disclosure provides a DNA-based structure (e.g., DNAntenna) comprising (a) a component that anchors the DNA-based structure to a surface, (b) a double-stranded DNA target region, wherein the double-stranded DNA target region is configured to physically interact with at least one DNA-binding agent, and (c) a high aspect ratio component (e.g., helix bundle), wherein the high aspect ratio component amplifies movement caused by physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region when the high aspect ratio component is tagged with a first plurality of fluorescent molecules, wherein the double-stranded DNA target region is located between the component that anchors the DNA-based structure to the surface and the high aspect ratio component. The at least one DNA-binding agent may be a DNA-bindingprotein, a DNA-binding nucleic acid molecule, a DNA intercalator, or a small molecule. In some embodiments, the DNA-binding protein is selected from the group consisting of transcription factors, polymerases, ligases, nucleases, restriction enzymes, and helicases. In certain embodiments, the small molecule is a groove binder, a DNA intercalator, a singlestrand binder, and a multi-mode binder. Examples of small molecules include, but are not limited to, distamycin-A, methyl Green, Berenil, netropsin, CC-1065, pentamidine, furamidine, duocarmycins, Hoechst 33342, Hoechst 333580, Hoechst 333258, ethidium bromide, doxorubicin, daunorubicin, daunomycin, cisplatin, carboplatin, oxaliplatin, Polynuclear platinum complexes (e.g., BBR 3464), bleomycin, neocarzinostatin, esperamicins, calichemicins, Dynemicin A, actinomycin D, proflavine, berberine, acridine orange, alkylators, lexitropsins, polyamides, ethyleneimines, nitrogen mustards, methane sulfonates, or thalidomide. In other embodiments, the DNA-binding nucleic acid molecule is an aptamer, an RNA, or a peptide nucleic acid. In any of the above embodiments, the DNA- based structure further comprises a barcode.

[0048] Additionally or alternatively, in some embodiments, the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region are detected by measuring a shift in position of a fluorescent molecule tagging the high aspect ratio component. In certain embodiments, the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region comprise bending kinetics. In some embodiments, the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region comprise DNA dynamics selected from among DNA extension, DNA contraction, and DNA rotation.

[0049] Additionally or alternatively, in certain embodiments, the component that anchors the DNA-based structure to the surface has a height of about 30 nm to about 33 nm. In some embodiments, the component that anchors the DNA-based structure to the surface has a height of about 30 nm, about 30.1 nm, about 30.2 nm, about 30.3 nm, about 30.4 nm, about 30.5 nm, about 30.6 nm, about 30.7 nm, about 30.8 nm, about 30.9 nm, about 31 nm, about31.1 nm, about 31.2 nm, about 31.3 nm, about 31.4 nm, about 31.5 nm, about 31.6 nm, about31.7 nm, about 31.8 nm, about 31.9 nm, about 32 nm, about 32.1 nm, about 32.2 nm, about32.3 nm, about 32.4 nm, about 32.5 nm, about 32.6 nm, about 32.7 nm, about 32.8 nm, about32.9 nm, or about 33 nm.

[0050] Additionally or alternatively, in some embodiments, the component that anchors the DNA-based structure to the surface has a width of about 30 nm to about 33 nm. In certain embodiments, the component that anchors the DNA-based structure to the surface has a width of about 30 nm, about 30.1 nm, about 30.2 nm, about 30.3 nm, about 30.4 nm, about 30.5 nm, about 30.6 nm, about 30.7 nm, about 30.8 nm, about 30.9 nm, about 31 nm, about 31.1 nm, about 31.2 nm, about 31.3 nm, about 31.4 nm, about 31.5 nm, about 31.6 nm, about 31.7 nm, about 31.8 nm, about 31.9 nm, about 32 nm, about 32.1 nm, about 32.2 nm, about 32.3 nm, about 32.4 nm, about 32.5 nm, about 32.6 nm, about 32.7 nm, about 32.8 nm, about 32.9 nm, or about 33 nm.

[0051] Additionally or alternatively, in some embodiments, the length of the high aspect ratio component is about 70 nm to about 85 nm. In certain embodiments, the length of the high aspect ratio component is about 70 nm, about 70.1 nm, about 70.2 nm, about 70.3 nm, about 70.4 nm, about 70.5 nm, about 70.6 nm, about 70.7 nm, about 70.8 nm, about 70.9 nm, about 71 nm, about 71.1 nm, about 71.2 nm, about 71.3 nm, about 71.4 nm, about 71.5 nm, about 71.6 nm, about 71.7 nm, about 71.8 nm, about 71.9 nm, about 72 nm, about 72.1 nm, about 72.2 nm, about 72.3 nm, about 72.4 nm, about 72.5 nm, about 72.6 nm, about 72.7 nm, about 72.8 nm, about 72.9 nm, about 73 nm, about 73.1 nm, about 73.2 nm, about 73.3 nm, about 73.4 nm, about 73.5 nm, about 73.6 nm, about 73.7 nm, about 73.8 nm, about 73.9 nm, about 74 nm, about 74.1 nm, about 74.2 nm, about 74.3 nm, about 74.4 nm, about 74.5 nm, about 74.6 nm, about 74.7 nm, about 74.8 nm, about 74.9 nm, about 75 nm, about 75.1 nm, about 75.2 nm, about 75.3 nm, about 75.4 nm, about 75.5 nm, about 75.6 nm, about 75.7 nm, about 75.8 nm, about 75.9 nm, about 76 nm, about 76.1 nm, about 76.2 nm, about 76.3 nm, about 76.4 nm, about 76.5 nm, about 76.6 nm, about 76.7 nm, about 76.8 nm, about 76.9 nm, about 77 nm, about 77.1 nm, about 77.2 nm, about 77.3 nm, about 77.4 nm, about 77.5 nm, about 77.6 nm, about 77.7 nm, about 77.8 nm, about 77.9 nm, about 78 nm, about 78.1 nm, about 78.2 nm, about 78.3 nm, about 78.4 nm, about 78.5 nm, about 78.6 nm, about 78.7 nm, about 78.8 nm, about 78.9 nm, about 79 nm, about 79.1 nm, about 79.2 nm, about 79.3 nm, about 79.4 nm, about 79.5 nm, about 79.6 nm, about 79.7 nm, about 79.8 nm, about 79.9 nm, about 80 nm, about 80.1 nm, about 80.2 nm, about 80.3 nm, about 80.4 nm, about 80.5 nm, about 80.6 nm, about 80.7 nm, about 80.8 nm, about 80.9 nm, about 81 nm, about 81.1 nm, about 81.2 nm, about 81.3 nm, about 81.4 nm, about 81.5 nm, about 81.6 nm, about 81.7 nm,about 81.8 nm, about 81.9 nm, about 82 nm, about 82.1 nm, about 82.2 nm, about 82.3 nm, about 82.4 nm, about 82.5 nm, about 82.6 nm, about 82.7 nm, about 82.8 nm, about 82.9 nm, about 83 nm, about 83.1 nm, about 83.2 nm, about 83.3 nm, about 83.4 nm, about 83.5 nm, about 83.6 nm, about 83.7 nm, about 83.8 nm, about 83.9 nm, about 84 nm, about 84.1 nm, about 84.2 nm, about 84.3 nm, about 84.4 nm, about 84.5 nm, about 84.6 nm, about 84.7 nm, about 84.8 nm, about 84.9 nm, or about 85 nm.

[0052] Additionally or alternatively, in some embodiments, the width of the high aspect ratio component is about 5 nm to about 10 nm. In some embodiments, the width of the high aspect ratio component is about 5 nm, about 5.1 nm, about 5.2 nm, about 5.3 nm, about 5.4 nm, about 5.5 nm, about 5.6 nm, about 5.7 nm, about 5.8 nm, about 5.9 nm, about 6 nm, about 6.1 nm, about 6.2 nm, about 6.3 nm, about 6.4 nm, about 6.5 nm, about 6.6 nm, about 6.7 nm, about 6.8 nm, about 6.9 nm, about 7 nm, about 7.1 nm, about 7.2 nm, about 7.3 nm, about 7.4 nm, about 7.5 nm, about 7.6 nm, about 7.7 nm, about 7.8 nm, about 7.9 nm, about 8 nm, about 8.1 nm, about 8.2 nm, about 8.3 nm, about 8.4 nm, about 8.5 nm, about 8.6 nm, about 8.7 nm, about 8.8 nm, about 8.9 nm, about 9 nm, about 9.1 nm, about 9.2 nm, about 9.3 nm, about 9.4 nm, about 9.5 nm, about 9.6 nm, about 9.7 nm, about 9.8 nm, about 9.9 nm, or about 10 nm.

[0053] In any of the preceding embodiments, the double-stranded DNA target region is about 15 bp to about 25 kb in length. In some embodiments, the double-stranded DNA target region is about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 125 bp, about 150 bp, about 175 bp, about 200 bp, about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, about 600 bp, about 625 bp, about 650 bp, about 675 bp, about 700 bp, about 725 bp, about 750 bp, about 775 bp, about 800 bp, about 825 bp, about 850 bp, about 875 bp, about 900 bp, about 925 bp, about 950 bp, about 975 bp, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3 kb, about 3.1 kb, about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7kb, about 3.8 kb, about 3.9 kb, about 4 kb, about 4.1 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, about 5 kb, about 5.1 kb, about 5.2 kb, about 5.3 kb, about 5.4 kb, about 5.5 kb, about 5.6 kb, about 5.7 kb, about5.8 kb, about 5.9 kb, about 6 kb, about 6.1 kb, about 6.2 kb, about 6.3 kb, about 6.4 kb, about 6.5 kb, about 6.6 kb, about 6.7 kb, about 6.8 kb, about 6.9 kb, about 7 kb, about 7.1 kb, about7.2 kb, about 7.3 kb, about 7.4 kb, about 7.5 kb, about 7.6 kb, about 7.7 kb, about 7.8 kb, about 7.9 kb, about 8 kb, about 8.1 kb, about 8.2 kb, about 8.3 kb, about 8.4 kb, about 8.5 kb, about 8.6 kb, about 8.7 kb, about 8.8 kb, about 8.9 kb, about 9 kb, about 9.1 kb, about 9.2 kb, about 9.3 kb, about 9.4 kb, about 9.5 kb, about 9.6 kb, about 9.7 kb, about 9.8 kb, about 9.9 kb, about 10 kb, about 10.1 kb, about 10.2 kb, about 10.3 kb, about 10.4 kb, about 10.5 kb, about 10.6 kb, about 10.7 kb, about 10.8 kb, about 10.9 kb, about 11 kb, about 11.1 kb, about11.2 kb, about 11.3 kb, about 11.4 kb, about 11.5 kb, about 11.6 kb, about 11.7 kb, about 11.8 kb, about 11.9 kb, about 12 kb, about 12.1 kb, about 12.2 kb, about 12.3 kb, about 12.4 kb, about 12.5 kb, about 12.6 kb, about 12.7 kb, about 12.8 kb, about 12.9 kb, about 13 kb, about 13.1 kb, about 13.2 kb, about 13.3 kb, about 13.4 kb, about 13.5 kb, about 13.6 kb, about 13.7 kb, about 13.8 kb, about 13.9 kb, about 14 kb, about 14.1 kb, about 14.2 kb, about 14.3 kb, about 14.4 kb, about 14.5 kb, about 14.6 kb, about 14.7 kb, about 14.8 kb, about 14.9 kb, or about 15 kb. Additionally or alternatively, in some embodiments, the double-stranded DNA target region comprises a promoter, an enhancer, an intron, an exon, a telomere, a centromere, a silencer, an insulator, a Long noncoding RNA, a tRNA, or a microRNA.

[0054] Additionally or alternatively, in some embodiments, the surface is a slide, a well, a plastic plate, a microtiter plate, a microfluidic array, or a gel.

[0055] In any of the foregoing embodiments, the first plurality of fluorescent molecules comprise 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino- N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"- dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6- dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6- carb oxy fluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6- carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); ATTO 488 or VIC®.

[0056] In any and all embodiments of the DNA-based structures disclosed herein, the component that anchors the DNA-based structure to the surface is tagged with a second plurality of fluorescent molecules. The second plurality of fluorescent molecules may comprise 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647(Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino- N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"- dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6- dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6- carb oxy fluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6- carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); ATTO 488 or VIC®.

[0057] In another aspect, the present disclosure provides a method for evaluating DNA binding interactions comprising contacting any and all embodiments of the DNA-based structures disclosed herein with at least one DNA-binding agent, wherein the DNA-based structure is anchored to a surface and wherein the at least one DNA-binding agent binds to the double-stranded DNA target region of the DNA-based structure, and detecting physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region by determining a shift in position of the first plurality of fluorescent molecules relative to the second plurality of fluorescent molecules. The surface may be a slide, a well, a plastic plate, a microtiter plate, a microfluidic array, or a gel. In certain embodiments, the shift in position of the first plurality of fluorescent molecules relative to the second plurality of fluorescent molecules is determined via atomic force microscopy (AFM) or single molecule total internal reflection fluorescence (smTIRF) microscopy.

[0058] Additionally or alternatively, in some embodiments of the methods described herein, the at least one DNA-binding agent is a DNA-binding protein, a DNA-binding nucleic acid molecule, a DNA intercalator, or a small molecule. In some embodiments of the methods disclosed herein, the DNA-binding protein is selected from the group consisting of transcription factors, polymerases, ligases, nucleases, restriction enzymes, and helicases. In certain embodiments, the small molecule is a groove binder, a DNA intercalator, a singlestrand binder, and a multi-mode binder. Examples of small molecules include, but are not limited to, distamycin-A, methyl Green, Berenil, netropsin, CC-1065, pentamidine, furamidine, duocarmycins, Hoechst 33342, Hoechst 333580, Hoechst 333258, ethidium bromide, doxorubicin, daunorubicin, daunomycin, cisplatin, carboplatin, oxaliplatin, Polynuclear platinum complexes (e.g., BBR 3464), bleomycin, neocarzinostatin, esperamicins, calichemicins, Dynemicin A, actinomycin D, proflavine, berberine, acridine orange, alkylators, lexitropsins, polyamides, ethyleneimines, nitrogen mustards, methane sulfonates, or thalidomide. In other embodiments, the DNA-binding nucleic acid molecule is an aptamer, an RNA, or a peptide nucleic acid.

[0059] In any and all embodiments of the methods described herein, the double-stranded DNA target region comprises nicked DNA or non-nicked DNA. The double-stranded DNA target region may comprise A-form DNA, B-form DNA, or Z-form DNA. Additionally or alternatively, in some embodiments of the methods disclosed herein, the double-strandedDNA target region is about 15 bp to about 25 kb in length. In some embodiments, the double-stranded DNA target region is about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 125 bp, about 150 bp, about 175 bp, about 200 bp, about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, about 600 bp, about 625 bp, about 650 bp, about 675 bp, about 700 bp, about 725 bp, about 750 bp, about 775 bp, about 800 bp, about 825 bp, about 850 bp, about 875 bp, about 900 bp, about 925 bp, about 950 bp, about 975 bp, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3 kb, about 3.1 kb, about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb, about 3.9 kb, about 4 kb, about 4.1 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, about 5 kb, about 5.1 kb, about 5.2 kb, about 5.3 kb, about 5.4 kb, about 5.5 kb, about 5.6 kb, about 5.7 kb, about 5.8 kb, about 5.9 kb, about 6 kb, about 6.1 kb, about6.2 kb, about 6.3 kb, about 6.4 kb, about 6.5 kb, about 6.6 kb, about 6.7 kb, about 6.8 kb, about 6.9 kb, about 7 kb, about 7.1 kb, about 7.2 kb, about 7.3 kb, about 7.4 kb, about 7.5 kb, about 7.6 kb, about 7.7 kb, about 7.8 kb, about 7.9 kb, about 8 kb, about 8.1 kb, about 8.2 kb, about 8.3 kb, about 8.4 kb, about 8.5 kb, about 8.6 kb, about 8.7 kb, about 8.8 kb, about 8.9 kb, about 9 kb, about 9.1 kb, about 9.2 kb, about 9.3 kb, about 9.4 kb, about 9.5 kb, about 9.6 kb, about 9.7 kb, about 9.8 kb, about 9.9 kb, about 10 kb, about 10.1 kb, about 10.2 kb, about 10.3 kb, about 10.4 kb, about 10.5 kb, about 10.6 kb, about 10.7 kb, about 10.8 kb, about 10.9 kb, about 11 kb, about 11.1 kb, about 11.2 kb, about 11.3 kb, about 11.4 kb, about 11.5 kb, about 11.6 kb, about 11.7 kb, about 11.8 kb, about 11.9 kb, about 12 kb, about 12.1 kb, about12.2 kb, about 12.3 kb, about 12.4 kb, about 12.5 kb, about 12.6 kb, about 12.7 kb, about 12.8 kb, about 12.9 kb, about 13 kb, about 13.1 kb, about 13.2 kb, about 13.3 kb, about 13.4 kb, about 13.5 kb, about 13.6 kb, about 13.7 kb, about 13.8 kb, about 13.9 kb, about 14 kb, about 14.1 kb, about 14.2 kb, about 14.3 kb, about 14.4 kb, about 14.5 kb, about 14.6 kb, about 14.7 kb, about 14.8 kb, about 14.9 kb, or about 15 kb. Additionally or alternatively, in someembodiments, the double-stranded DNA target region comprises a promoter, an enhancer, an intron, an exon, a telomere, a centromere, a silencer, an insulator, a Long noncoding RNA, a tRNA, or a microRNA.

[0060] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the double-stranded DNA target region comprises an epigentic modification selected from among methylation, acetylation, deacetylation, phosphorylation, ubiquitination, and sumoylation. In certain embodiments, the double-stranded DNA target region comprises an AT -rich region, an AC-rich region, an AG-rich region, a GC-rich region, a poly-A region, or a GG-rich region. Additionally or alternatively, in some embodiments of the methods disclosed herein, the double-stranded DNA target region comprises simple plectonemes (e.g., two-start right-handed helix with terminal loops), hyperplectonemes, or toroids (e.g., one- start left-handed helix).

[0061] In any and all embodiments of the methods described herein, the double-stranded DNA target region has a persistence length of about 46.5 nm to about 55 nm. In certain embodiments, the double-stranded DNA target region has a persistence length of about 46.5 nm, about 46.6 nm, about 46.7 nm, about 46.8 nm, about 46.9 nm, about 47.0 nm, about 47.1 nm, about 47.2 nm, about 47.3 nm, about 47.4 nm, about 47.5 nm, about 47.6 nm, about 47.7 nm, about 47.8 nm, about 47.9 nm, about 48.0 nm, about 48.1 nm, about 48.2 nm, about 48.3 nm, about 48.4 nm, about 48.5 nm, about 48.6 nm, about 48.7 nm, about 48.8 nm, about 48.9 nm, about 49.0 nm, about 49.1 nm, about 49.2 nm, about 49.3 nm, about 49.4 nm, about 49.5 nm, about 49.6 nm, about 49.7 nm, about 49.8 nm, about 49.9 nm, about 50.0 nm, about 50.1 nm, about 50.2 nm, about 50.3 nm, about 50.4 nm, about 50.5 nm, about 50.6 nm, about 50.7 nm, about 50.8 nm, about 50.9 nm, about 51.0 nm, about 51.1 nm, about 51.2 nm, about 51.3 nm, about 51.4 nm, about 51.5 nm, about 51.6 nm, about 51.7 nm, about 51.8 nm, about 51.9 nm, about 52.0 nm, about 52.1 nm, about 52.2 nm, about 52.3 nm, about 52.4 nm, about 52.5 nm, about 52.6 nm, about 52.7 nm, about 52.8 nm, about 52.9 nm, about 53.0 nm, about 53.1 nm, about 53.2 nm, about 53.3 nm, about 53.4 nm, about 53.5 nm, about 53.6 nm, about 53.7 nm, about 53.8 nm, about 53.9 nm, about 54.0 nm, about 54.1 nm, about 54.2 nm, about 54.3 nm, about 54.4 nm, about 54.5 nm, about 54.6 nm, about 54.7 nm, about 54.8 nm, about 54.9 nm, or about 55 nm.

[0062] Additionally or alternatively, in some embodiments of the methods disclosed herein, the double-stranded DNA target region has a melting temperature of about 50°C to about 100°C. In certain embodiments, the double-stranded DNA target region has a melting temperature of about 50°C, about 50.5°C, about 51°C, about 51.5°C, about 52°C, about 52.5°C, about 53°C, about 53.5°C, about 54°C, about 54.5°C, about 55°C, about 55.5°C, about 56°C, about 56.5°C, about 57°C, about 57.5°C, about 58°C, about 58.5°C, about 59°C, about 59.5°C, about 60°C, about 60.5°C, about 61°C, about 61.5°C, about 62°C, about 62.5°C, about 63°C, about 63.5°C, about 64°C, about 64.5°C, about 65°C, about 65.5°C, about 66°C, about 66.5°C, about 67°C, about 67.5°C, about 68°C, about 68.5°C, about 69°C, about 69.5°C, about 70°C, about 70.5°C, about 71°C, about 71.5°C, about 72°C, about 72.5°C, about 73°C, about 73.5°C, about 74°C, about 74.5°C, about 75°C, about 75.5°C, about 76°C, about 76.5°C, about 77°C, about 77.5°C, about 78°C, about 78.5°C, about 79°C, about 79.5°C, about 80°C, about 80.5°C, about 81°C, about 81.5°C, about 82°C, about 82.5°C, about 83°C, about 83.5°C, about 84°C, about 84.5°C, about 85°C, about 85.5°C, about 86°C, about 86.5°C, about 87°C, about 87.5°C, about 88°C, about 88.5°C, about 89°C, about 89.5°C, about 90°C, about 90.5°C, about 91°C, about 91.5°C, about 92°C, about 92.5°C, about 93°C, about 93.5°C, about 94°C, about 94.5°C, about 95°C, about 95.5°C, about 96°C, about 96.5°C, about 97°C, about 97.5°C, about 98°C, about 98.5°C, about 99°C, about 99.5°C, or about 100°C.

[0063] In any and all embodiments of the methods described herein, the double-stranded DNA target region has a diameter of about 18 A to about 23 A. In some embodiments, the double-stranded DNA target region has a diameter of about 18 A, about 18.1 A, about 18.2 A, about 18.3 A, about 18.4 A, about 18.5 A, about 18.6 A, about 18.7 A, about 18.8 A, about 18.9 A, about 19 A, about 19.1 A, about 19.2 A, about 19.3 A, about 19.4 A, about 19.5 A, about 19.6 A, about 19.7 A, about 19.8 A, about 19.9 A, about 20 A, about 20.1 A, about 20.2 A, about 20.3 A, about 20.4 A, about 20.5 A, about 20.6 A, about 20.7 A, about 20.8 A, about 20.9 A, about 21 A, about 21.1 A, about 21.2 A, about 21.3 A, about 21.4 A, about 21.5 A, about 21.6 A, about 21.7 A, about 21.8 A, about 21.9 A, about 22 A, about 22.1 A, about 22.2 A, about 22.3 A, about 22.4 A, about 22.5 A, about 22.6 A, about 22.7 A, about 22.8 A, about 22.9 A, or about 23 A.

[0064] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the double-stranded DNA target region comprises at least one bend angle that ranges from about 15° to about 180°. In some embodiments, the double-stranded DNA target region comprises at least one bend angle that ranges from about 15°, about 16°, about 17°, about 18°, about 19°, about 20°, about 21°, about 22°, about 23°, about 24°, about 25°, about 26°, about 27°, about 28°, about 29°, about 30°, about 31°, about 32°, about 33°, about 34°, about 35°, about 36°, about 37°, about 38°, about 39°, about 40°, about 41°, about 42°, about 43°, about 44°, about 45°, about 46°, about 47°, about 48°, about 49°, about 50°, about 51°, about 52°, about 53°, about 54°, about 55°, about 56°, about 57°, about 58°, about 59°, about 60°, about 61°, about 62°, about 63°, about 64°, about 65°, about 66°, about 67°, about 68°, about 69°, about 70°, about 71°, about 72°, about 73°, about 74°, about 75°, about 76°, about 77°, about 78°, about 79°, about 80°, about 81°, about 82°, about 83°, about 84°, about 85°, about 86°, about 87°, about 88°, about 89°, about 90°, about 91°, about 92°, about 93°, about 94°, about 95°, about 96°, about 97°, about 98°, about 99°, about 100°, about 101°, about 102°, about 103°, about 104°, about 105°, about 106°, about 107°, about 108°, about 109°, about 110°, about 111°, about 112°, about 113°, about 114°, about 115°, about 116°, about 117°, about 118°, about 119°, about 120°, about 121°, about 122°, about 123°, about 124°, about 125°, about 126°, about 127°, about 128°, about 129°, about 130°, about 131°, about 132°, about 133°, about 134°, about 135°, about 136°, about 137°, about 138°, about 139°, about 140°, about 141°, about 142°, about 143°, about 144°, about 145°, about 146°, about 147°, about 148°, about 149°, about 150°, about 151°, about 152°, about 153°, about 154°, about 155°, about 156°, about 157°, about 158°, about 159°, about 160°, about 161°, about 162°, about 163°, about 164°, about 165°, about 166°, about 167°, about 168°, about 169°, about 170°, about 171°, about 172°, about 173°, about 174°, about 175°, about 176°, about 177°, about 178°, about 179°, or about 180°.EXAMPLES

[0065] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: Methods

[0066] DNAntenna Component Design: All components of the DNAntenna were designed with caDNAno software (ref. 6). Preliminary structures were computed with CanDo software, a finite element analysis simulation that predicts equilibrium shape and physical properties of custom DNA nanostructures in solution (ref. 5). The tripod base was designed with M13mpl8 scaffold (single-stranded, New England Biolabs), and the 10HB was designed with the p3031 scaffold (single-stranded, provided by Konlin Shen). See FIGs. 6-7

[0067] DNAntenna Component Folding. All unlabeled DNA oligomer staple strands were purchased from Integrated DNA Technologies (IDT) in 96-well plates at 150 pM per oligo, and all labeled staples were purchased in tubes at 100 pM per oligo. The staple strands (unlabeled and labeled) and appropriate scaffold strand were mixed in a folding buffer consisting of 10 mM Tris, pH 8.0, 1 mM EDTA, and 9 mM MgCh. The concentrations of DNAs in each reaction were 10 nM scaffold strand, 100 nM unlabeled staple strands, 200 nM biotin primaries, 500 nM biotin secondaries, 200 nM Cy3 primaries, 3pM Cy3 secondaries, 200 nM Cy5 primaries, 4pM Cy5 secondaries. The junction oligos were at either 200 nM, 250 nM, or IpM, depending on the linker schematic. All origami samples were heated and annealed starting at 80°C for 5 minutes, 80-65°C in 1°C steps every minute, 65-25°C in 1°C steps every 20 minutes, then held at 4°C (~14 hours total folding time).

[0068] DNAntenna Component Purification: DNA origami were purified by 2% agarose gel electrophoresis, run over an ice bath for 1.5 hours at 100 V, in running buffer consisting of 89 mM Tris pH 8.0, 89 mM borate, 2 mM EDTA, and 10 mM MgCh. The appropriate bands were excised from the gel and samples were extracted with Freeze n’ Squeeze spin columns (Biorad), then were centrifuged at 1000 rpm for 60 minutes at 4°C. After the centrifuge, the samples were pipetted into Amicon Ultra 0.5 mL, 100K filters and spun lx for 12 min at 3000 rpm at 4°C. All samples were stored frozen at -20°C until needed for further use. Note that use of the agarose gel is important when folding a new origami structure to evaluate self assembly products, but not necessary for later preps as samples can be purified with Amicon filters immediately after removal from the thermocycler.

[0069] Construction of DNAntennas via Ligation: After being resuspended in T4 ligation buffer (New England Biolabs), the tripod base origami and 10HB origami were incubated with T4 DNA Ligase (New England Biolabs) for 1 hour at room temperature with 10 mM ATP. The reaction mixture was treated by Proteinase K (New England Biolabs) for 1 hour at room temperature to degrade the ligase. See FIG. 2. The origami sample was then purified by agarose gel electrophoresis and was Amicon filtered as mentioned previously to concentrate the DN Antenna origami.

[0070] Atomic Force Microscopy (AFM) Sample Preparation & Imaging: AFM images were obtained using either a Veeco Scanning Probe system. A 5 pL droplet of Amicon purified origami sample and then a 30 pL drop of folding buffer with 10 mM NiC12 were applied to a freshly cleaved mica surface and left to incubate for at least 4 minutes. Images were acquired under liquid tapping mode, with SNL-10 (silicon nitride, Bruker) C-type triangular tips (resonant frequency, ) = 40-75 kHz; spring constant, k = 0.24 N / m).

[0071] smTIRF Optical Calibration: All smTIRF imaging was conducted using a Nikon Eclipse Ti inverted microscope body with a 60x 1.4 NA oil objective (Nikon), a high speed scientific CMOS camera (Hamamatsu Orca-Flash 4.0 v2), and a liquid cooling system (Koolance Revl.5, EX2-1055). The camera field of view was cropped as necessary to achieve high frame rates. Each camera pixel corresponded to 107 nm in the sample plane. The microscope hardware was controlled with custom software written in Python. To calibrate the microscope optics, 0.1 pm fluorescent Tetraspeck Microspheres (Invitrogen, T7279) were pipetted onto a slide, and a glass coverslip was placed on top. Using the 532 and 647 nm lasers, a colocalization matrix was computed with chromatic aberration correction. For each laser, custom Python code was used to create a localization precision vs intensity curve to complete the optical calibration. Note that optical calibration only needs to be performed once for a particular setup.

[0072] smTIRF Fluidic Chamber Preparation: After optical calibration, origami were imaged in a flow chamber consisting of a glass coverslip (VWR, No. 1.5) attached to a microscope slide with double sided tape. The edges of the coverslip were sealed with epoxy, liquid was pulled through the chamber via a negative pressure from a Kimwipe on the opposite side of the channel from the liquid inlet. The coverslips were cleaned by sonicationin 95% ethanol, rinsing in water, and drying thoroughly with compressed nitrogen. Between uses, slides were soaked in acetone and water to facilitate disassembly, then scrubbed with alconox, sonicated in 1 M KOH, then rinsed in water.

[0073] smTIRF Slide Surface Preparation: With a clean smTIRF fluidic chamber, the channel was washed thoroughly with lx Tris-EDTA buffer (lx TE, ThermoFisher Scientific). 2x channel volume of 1 mg / mL BSA-Biotin was added to the channel and left to incubate for 2 minutes. The channel was washed thoroughly with lx TE buffer, then 2x channel volume of 0.01 mg / mL Streptavidin was added to the channel and left to incubate for 2 minutes. The channel was washed thoroughly with lx TE buffer, and DN Antenna was added to the channel and incubated for 5 minutes. After incubation, the channel was thoroughly washed with imaging buffer, which contained 50 mM Tris, 0.5 mM EDTA, 3 mM Trolox, 50 pM Trolox quinone (prepared using UV irradiation and quantified by UV-Vis absorbance spectroscopy before adding to buffer), 10% w / v glucose, and 10 mM MgCh.

[0074] smTIRF DN Antenna Brownian Motion Experiments: After finishing slide surface preparation, an aliquot of imaging buffer with fresh glucose oxidase and catalase were added to the channel. Immediately after adding, both sides of the channel were sealed with Parafilm. All experiments were conducted at room temperature (~23°C). See FIG. 3.

[0075] smTIRF DNAntenna Protein / Small Molecule Experiments: All experiments were conducted at room temperature (~23°C).

[0076] Custom Python code to 1) calibrate an optical microscopy setup suitable for DNAntenna assays and 2) process data through analysis pipeline and visualize singlemolecule trajectories taken in parallel.Example 2: DNAntenna and Uses Thereof

[0077] Using caDNAno, we designed the DNAntenna as a structure composed of three parts: a fluorescently tagged tripod base, a DNA target region, and a fluorescently tagged long DNA helix bundle. The target region is modular and is a platform to test different DNA binding interactions. These include bending kinetics and dynamics of DNA caused by small molecules, protein binding or DNA / RNA binding. The large helix bundle acts as a lever arm to amplify these interactions in a way that is measurable with optical microscopy using the shift in position of the fluorescent tags on the long helix bundle compared to the position ofthe fluorescent tags on the tripod base. Leveraging this amplification, our DNAntenna can be used to measure DNA bending dynamics and kinetics in an optical microscopy set up.

[0078] A singular DNAntenna is the minimum functional unit for the platform technology. However, the full utility of this method lies in a high-throughput platform with hundreds of DNAntennas per field of view, deposited onto the surface of a simple microfluidic channel. Upon TIRF illumination, hundreds of DNAntenna trajectories are observed and imaged simultaneously. See FIGs. 4-5. DNAntenna assays are an exceptionally robust platform for investigating a wide array of DNA binding interactions and biophysical properties with unparalleled precision. What sets the DNAntenna platform apart is the unique combination of the specificity inherent in single-molecule techniques and the high-throughput parallel capabilities that enhance experimental convenience and efficiency. By allowing scientists to delve into the nuances of individual molecular interactions while also enabling it to be done on a larger scale, the DNAntenna platform can enable exploration of complex molecular phenomena with a level of detail and throughput that was previously unattainable.

[0079] The platform captures intermediate states and motion over a continuous time interval instead of a single snapshot in time; Choice of imaging framerate makes timescale of measurements flexible and easily adjustable for new applications.

[0080] The platform may be useful for:

[0081] Evaluating bending / binding kinetics and dynamics of DNA induced by small molecules, DNA intercalators, DNA binding proteins (Examples including but not limited to: transcription factors, polymerases, ligases, nucleases, restriction enzymes)

[0082] Length and sequence dependent physical properties of dsDNA permit higher accuracy nucleic acid computational simulations and modeling for evaluating gene therapy outcomes

[0083] Determining effect of epigenetic modifications of DNA on transcription factor binding

[0084] Determining physical properties of nicked vs non-nicked DNA

[0085] Diagnostic strand detection via DNA / RNA strand displacement circuits

[0086] Microfluidic arrays of barcoded DNAntennas may be useful for personal health diagnostics in the future, specifically for multiplexed screening of various medical conditions that are dependent on combinations of diagnostic biomarkersEQUIVALENTS

[0087] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0088] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0089] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0090] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.REFERENCES1. Bohlin, J.; Matthies, M.; Poppleton, E.; Procyk, J.; Mallya, A.; Yan, H.; Sulc, P. Design and Simulation of DNA, RNA and Hybrid Protein-Nucleic Acid Nanostructures with Ox View. Nat Protoc 2022, 17 (8), 1762-1788.2. Cai, X.; Arias, D. S.; Velazquez, L. R.; Vexler, S.; Bevier, A. L.; Fygenson, D. K. DNA Nunchucks: Nanoinstrumentation for Single-Molecule Measurement of Stiffness and Bending. Nano Lett. 2020, 20 (2), 1388-1395.3. Dey, S.; Fan, C.; Gothelf, K. V.; Li, J.; Lin, C.; Liu, L.; Liu, N.; Nijenhuis, M. A. D.; Sacca, B.; Simmel, F. C.; Yan, H.; Zhan, P. DNA Origami. Nat Rev Methods Primers 2021, 7(1), 1-24.4. Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440 (7082), 297-302.5. Kim, D.-N.; Kilchherr, F.; Dietz, H.; Bathe, M. Quantitative Prediction of 3D Solution Shape and Flexibility of Nucleic Acid Nanostructures. Nucleic Acids Res 2012, 40 (7), 2862-2868.6. Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Rapid Prototyping of 3D DNA-Origami Shapes with CaDNAno. Nucleic Acids Research 2009, 37 (15), 5001-5006.7. Kosuri, P.; Altheimer, B. D.; Dai, M.; Yin, P.; Zhuang, X. Rotation Tracking of Genome-Processing Enzymes Using DNA Origami Rotors. Nature 2019, 572 (7767), 136— 140.8. Douglas, S. M.; Dietz, H.; Liedl, T.; Hogberg, B.; Graf, F.; Shih, W. M. SelfAssembly of DNA into Nanoscale Three-Dimensional Shapes. Nature 2009, 459 (7245), 414-418.9. Pitikultham, P.; Wang, Z.; Wang, Y.; Shang, Y.; Jiang, Q.; Ding, B. Stimuli- Responsive DNA Origami Nanodevices and Their Biological Applications. ChemMedChem 2022, 77 (1), e202100635.

Claims

WHAT IS CLAIMED IS1. A DNA-based structure comprising a component that anchors the DNA-based structure to a surface, a double-stranded DNA target region, wherein the double-stranded DNA target region is configured to physically interact with at least one DNA- binding agent, and a high aspect ratio component, wherein the high aspect ratio component amplifies movement caused by physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region when the high aspect ratio component is tagged with a first plurality of fluorescent molecules, wherein the double-stranded DNA target region is located between the component that anchors the DNA-based structure to the surface and the high aspect ratio component.

2. The DNA-based structure of claim 1, wherein the at least one DNA-binding agent is a DNA-binding protein, a DNA-binding nucleic acid molecule, a DNA intercalator, or a small molecule.

3. The DNA-based structure of claim 2, wherein the DNA-binding protein is selected from the group consisting of transcription factors, polymerases, ligases, nucleases, restriction enzymes, and helicases.

4. The DNA-based structure of claim 2, wherein the small molecule is a groove binder, a DNA intercalator, a single-strand binder, and a multi-mode binder.

5. The DNA-based structure of claim 2 or 4, wherein the small molecule is distamycin- A, methyl Green, Berenil, netropsin, CC-1065, pentamidine, furamidine, duocarmycins, Hoechst 33342, Hoechst 333580, Hoechst 333258, ethidium bromide, doxorubicin, daunorubicin, daunomycin, cisplatin, carboplatin, oxaliplatin, Polynuclear platinum complexes (e.g., BBR 3464), bleomycin, neocarzinostatin, esperamicins, calichemicins, Dynemicin A, actinomycin D, proflavine, berberine, acridine orange, alkylators, lexitropsins, polyamides, ethyleneimines, nitrogen mustards, methane sulfonates, or thalidomide.

6. The DNA-based structure of claim 2, wherein the DNA-binding nucleic acid molecule is an aptamer, an RNA, or a peptide nucleic acid.

7. The DNA-based structure of any one of claims 1-6, wherein the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region are detected by measuring a shift in position of a fluorescent molecule tagging the high aspect ratio component.

8. The DNA-based structure of any one of claims 1-7, wherein the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region comprise bending kinetics.

9. The DNA-based structure of any one of claims 1-8, wherein the physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region comprise DNA dynamics selected from among DNA extension, DNA contraction, and DNA rotation.

10. The DNA-based structure of any one of claims 1-9, wherein the component that anchors the DNA-based structure to the surface has a height of about 30 nm to about 33 nm.

11. The DNA-based structure of any one of claims 1-10, wherein the component that anchors the DNA-based structure to the surface has a width of about 30 nm to about 33 nm.

12. The DNA-based structure of any one of claims 1-11, wherein the length of the high aspect ratio component is about 70 nm to about 85 nm.

13. The DNA-based structure of any one of claims 1-12, wherein the width of the high aspect ratio component is about 5 nm to about 10 nm.

14. The DNA-based structure of any one of claims 1-13, wherein the double-stranded DNA target region is about 15 bp to about 25 kb in length.

15. The DNA-based structure of any one of claims 1-14, wherein the double-stranded DNA target region comprises a promoter, an enhancer, an intron, an exon, a telomere, a centromere, a silencer, an insulator, a Long noncoding RNA, a tRNA, or a microRNA.

16. The DNA-based structure of any one of claims 1-15, wherein the surface is a slide, a well, a plastic plate, a microtiter plate, a microfluidic array, or a gel.

17. The DNA-based structure of any one of claims 1-16, wherein the first plurality of fluorescent molecules comprise 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino- 2-phenylindole (DAPI); 5', 5" -dibrom opyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144;IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde;Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate,succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); ATTO 488 or VIC®.

18. The DNA-based structure of any one of claims 1-17, wherein the component that anchors the DNA-based structure to the surface is tagged with a second plurality of fluorescent molecules.

19. The DNA-based structure of claim 18, wherein the second plurality of fluorescent molecules comprise 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino- 2-phenylindole (DAPI); 5', 5" -dibrom opyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC); Eclipse™ (EpochBiosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde;Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); ATTO 488 or VIC®.

20. The DNA-based structure of any one of claims 1-19, wherein the DNA-based structure further comprises a barcode.

21. A method for evaluating DNA binding interactions comprising a. contacting the DNA-based structure of any one of claims 18-20 with at least one DNA-binding agent, wherein the DNA-based structure is anchored to a surface and wherein the at least one DNA-binding agent binds to the doublestranded DNA target region of the DNA-based structure, and b. detecting physical interactions between the at least one DNA-binding agent and the double-stranded DNA target region by determining a shift in position of the first plurality of fluorescent molecules relative to the second plurality of fluorescent molecules.

22. The method of claim 21, wherein the surface is a slide, a well, a plastic plate, a microtiter plate, a microfluidic array, or a gel.

23. The method of claim 21 or 22, wherein the shift in position of the first plurality of fluorescent molecules relative to the second plurality of fluorescent molecules is determined via atomic force microscopy (AFM) or single molecule total internal reflection fluorescence (smTIRF) microscopy.

24. The method of any one of claims 21-23, wherein the at least one DNA-binding agent is a DNA-binding protein, a DNA-binding nucleic acid molecule, a DNA intercalator, or a small molecule.

25. The method of claim 24, wherein the DNA-binding protein is selected from the group consisting of transcription factors, polymerases, ligases, nucleases, restriction enzymes, and helicases.

26. The method of claim 25, wherein the small molecule is a groove binder, a DNA intercalator, a single-strand binder, and a multi-mode binder.

27. The method of claim 24 or 26, wherein the small molecule is distamycin-A, methyl Green, Berenil, netropsin, CC-1065, pentamidine, furamidine, duocarmycins, Hoechst 33342, Hoechst 333580, Hoechst 333258, ethidium bromide, doxorubicin, daunorubicin, daunomycin, cisplatin, carboplatin, oxaliplatin, Polynuclear platinum complexes (e.g., BBR 3464), bleomycin, neocarzinostatin, esperamicins, calichemicins, Dynemicin A, actinomycin D, proflavine, berberine, acridine orange, alkylators, lexitropsins, polyamides, ethyleneimines, nitrogen mustards, methane sulfonates, or thalidomide.

28. The method of claim 24, wherein the DNA-binding nucleic acid molecule is an aptamer, an RNA, or a peptide nucleic acid.

29. The method of any one of claims 21-28, wherein the double-stranded DNA target region comprises nicked DNA or non-nicked DNA.

30. The method of any one of claims 21-29, wherein the double-stranded DNA target region comprises an epigentic modification selected from among methylation, acetylation, deacetylation, phosphorylation, ubiquitination, and sumoylation.

31. The method of any one of claims 21-30, wherein the double-stranded DNA target region is about 15 bp to about 25 kb in length.

32. The method of any one of claims 21-31, wherein the double-stranded DNA target region comprises an AT-rich region, an AC-rich region, an AG-rich region, a GC-rich region, a poly-A region, or a GG-rich region.

33. The method of any one of claims 21-32, wherein the double-stranded DNA target region has a persistence length of about 46.5 nm to about 55 nm.

34. The method of any one of claims 21-33, wherein the double-stranded DNA target region has a melting temperature of about 50°C to about 100°C.

35. The method of any one of claims 21-34, wherein the double-stranded DNA target region comprises A-form DNA, B-form DNA, or Z-form DNA.

36. The method of any one of claims 21-35, wherein the double-stranded DNA target region has a diameter of about 18A to 23 A.

37. The method of any one of claims 21-36, wherein the double-stranded DNA target region comprises at least one bend angle that ranges from about 15° to about 180°.

38. The method of any one of claims 21-37, wherein the double-stranded DNA target region comprises simple plectonemes (e.g., two-start right-handed helix with terminal loops), hyperplectonemes, or toroids (e.g., one-start left-handed helix).

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