Novel method for stabilizing nucleic acid nanostructures

Abstract

The present invention relates to a novel method for stabilizing nucleic acid nanostructures by UV curing, in particular by cross-linking pyrimidine nucleotides.

Description

Technical Field

[0001] The present invention relates to a novel method for stabilizing nucleic acid nanostructures by curing with ultraviolet light, particularly by crosslinking pyrimidine nucleotides.

Background Art

[0002] The present invention relates to a novel method for stabilizing nucleic acid nanostructures, which results in nanostructures having advantageous properties, particularly high structural stability.

[0003] DNA nanotechnology 1-4 enables bottom-up self-assembly of individual three-dimensional (3D) objects having precise sub-nanometer shapes and overall dimensions ranging from nanometers to micrometers 5-16 and molecular weights up to the gigadalton scale 17,18 and enables bottom-up self-assembly of individual three-dimensional (3D) objects having precise sub-nanometer shapes and overall dimensions ranging from nanometers to micrometers and molecular weights up to the gigadalton scale. The resulting objects can be site-specifically functionalized and modified with chemical groups and biomolecules 19-21 and can construct objects that may include mechanisms that result in behavior such as that of a machine. 22-24 Since custom DNA objects have been developed and are successfully used in a variety of applications in basic research, new scientific findings have been obtained, and the ability of DNA nanotechnology to provide useful objects has been established. Examples range from structural biology 25-27 to biophysics 28-33 to optical engineering 34-37 to plasmonics 38-42 to molecular electronics 19,43-45 and extends to molecular electronics. The first step has been initiated to explore the use of designed DNA objects as programmable drugs in medicine. 24,46 A scalable biotechnological approach for generating single-stranded DNA helps to open the way for manufacturing the amounts of DNA objects required for materials and medical applications. 47To find applications in various situations, the designed DNA object needs to be stable for a sufficient amount of time under the desired conditions to achieve the desired application effect. Generally, application in low ionic strength solutions in physiological fluids, other solvents, air, or vacuum, and at temperatures above 50°C is impossible. Therefore, researchers have sought ways to extend the range of conditions under which the designed DNA object remains stable. Chemical bonding 48,49 or enzymatic binding 50 Additional covalent bonds are introduced to the correspondingly modified structure between the strand ends via 8-methoxypsoralen. 62 or oligoidine and oligoidine-PEG copolymer 51,52 Further stabilization can be achieved by adding cofactors such as [specific cofactors]. Despite these advances, there is still a desire to establish a complementary and generally applicable approach to covalent stabilization of DNA nanostructures that does not require the addition of expensive chemically modified strands or cofactors. The possibility of creating additional covalent links at user-defined sites on DNA nanostructures would allow for the rational stabilization of the entire structure or parts thereof for use in a wider range of environmental conditions. Furthermore, it may be possible to stably capture conformational states in mechanisms and higher-order aggregates. Here, we present a general and scalable method for site-selectively introducing additional covalent bonds into DNA nanostructures. The target binding site is specified solely by the DNA strand sequence and does not require the introduction of chemical modifications. Our method is generally applicable to a diverse range of DNA nanostructures and works regardless of whether the DNA strand was generated by solid-phase chemical synthesis or using biotechnology processes. 47 .

[0004] To find applications in various situations, the designed DNA object needs to be stable for a sufficient amount of time under the desired conditions to achieve the desired application effect. Generally, application in physiological fluids, other solvents, air, or vacuum, and at temperatures above 50°C, with low ionic strength solutions is impossible. Therefore, researchers have sought ways to extend the range of conditions under which the designed DNA object remains stable. Chemical bonding 48,49 or enzymatic binding 50 Through this, additional covalent bonds are introduced to the correspondingly modified inter-chain structures. DNA nanostructures also include oligolyxins and oligolyxin-PEG copolymers. 51,52 Further stabilization can be achieved by adding cofactors such as [specific cofactors]. Despite these advances, there is still a desire to establish a complementary and generally applicable approach to covalent stabilization of DNA nanostructures that does not require the addition of expensive chemically modified strands or cofactors. The possibility of creating additional covalent links at user-defined sites on DNA nanostructures would allow for the rational stabilization of the entire structure or parts thereof for use in a wider range of environmental conditions. Furthermore, it may be possible to stably capture conformational states in mechanisms and higher-order aggregates. Here, we present a general and scalable method for site-selectively introducing additional covalent bonds into DNA nanostructures. The target binding site is specified solely by the DNA strand sequence and does not require the introduction of chemical modifications. Our method is generally applicable to a diverse range of DNA nanostructures and works regardless of whether the DNA strand was generated by solid-phase chemical synthesis or using biotechnology processes. 47 .

[0005] In other words, despite the fact that many attempts have already been made to address the problem of increasing the stability of nucleic acid nanostructures, there is still a great unmet need to develop novel approaches that lead to the formation of more stable constructs.

[0006] The solution to this problem provided by the present invention, namely the curing of nucleic acid nanostructures by UV-induced crosslinking of pyrimidine nucleotides, has not been achieved or suggested by prior art to date. [Overview of the project]

[0007] The present invention relates to a novel method for stabilizing nucleic acid nanostructures by curing them using ultraviolet light, particularly by crosslinking pyrimidine nucleotides.

[0008] In a first embodiment, the present invention relates to a method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence bound to at least two non-adjacent sequence stretches present on one or more complementary nucleic acid sequences, wherein the method comprises the step of exposing the nucleic acid nanostructure to ultraviolet irradiation, wherein the step of exposing the nucleic acid nanostructure to ultraviolet irradiation results in the formation of at least one chemical bond between two pyrimidine nucleotides, wherein at least one of the two pyrimidine nucleotides is not part of a complementary nucleotide pair contained in a double-helix substructure.

[0009] In a second embodiment, the present invention relates to a method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence bound to at least two non-adjacent sequence stretches present on one or more complementary nucleic acid sequences, wherein the method is characterized by the step of exposing the nucleic acid nanostructure to ultraviolet irradiation.

[0010] In a third embodiment, the present invention relates to a method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least two double-helical substructures, wherein the method is characterized by the step of exposing the nucleic acid nanostructure to ultraviolet irradiation.

[0011] In a fourth embodiment, the present invention relates to a method for enhancing the stability of a non-naturally occurring nucleic acid nanostructure comprising a plurality of double-helical substructures, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence which is part of at least two different double-helical substructures, wherein the method is characterized by the step of exposing the nucleic acid nanostructure to ultraviolet irradiation.

[0012] In a fifth embodiment, the present invention relates to a kit for creating nucleic acid nanostructures comprising at least one copy of a first single-stranded polynucleotide and a set of single-stranded polynucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, where each of the n core sequences is (i) a sequence complementary to a region on the first single-stranded polynucleotide, wherein the region complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0013] In a sixth embodiment, the present invention relates to a kit for creating nucleic acid nanostructures comprising a set of single-stranded oligonucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, where each of the n core sequences is (i) a sequence complementary to the sequence of another member of the set of single-stranded polynucleotides, wherein the region on the other member complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m(iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0014] In a seventh embodiment, the present invention relates to a nucleic acid nanostructure comprising at least one copy of a first single-stranded polynucleotide and a set of single-stranded polynucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer selected from 1 to 40, and each of the n core sequences is (i) a sequence complementary to a region on the first single-stranded polynucleotide, wherein the region complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0015] In an eighth embodiment, the present invention relates to a nucleic acid nanostructure comprising a set of single-stranded oligonucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is independently selected from 1, 2, 3..., 40, and each of the n core sequences is (i) a sequence complementary to the sequence of another member of the set of single-stranded polynucleotides, wherein the region on the other member complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m, and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is independently selected from 1, 2, 3..., 40, and each P is independently selected from thymidine and cytosine residues.

[0016] In a ninth aspect, the present invention relates to a complex nucleic acid nanostructure resulting from the aggregation of two or more nucleic acid nanostructures according to the present invention. [Brief explanation of the drawing]

[0017] [Figure 1] This shows proximal thymidine as a site for crosslinking in DNA nanostructures. (A) The left image shows the chemical structures of two proximal thymidines before UV irradiation. The right image shows a schematic diagram of a six-helical DNA nanostructure characterized by single-stranded thymidines at the strand ends (1), during half-crossover (2), and during full-crossover (3), as well as thymidine loops (4), before UV irradiation. (B) This is the same as (A), but after exposure to light at a wavelength of 310 nm. CPD bonds are shown as ellipsoids.

[0018] [Figure 2]This document presents a conceptual proof of UV crosslinking using multilayer DNA origami. (A) From left to right: Model of brick-like DNA origami objects featuring additional thymidine at all strand ends and all strand crossover locations; laser scan fluorescence image of a 2.0% agarose gel stained with ethidium bromide. Irradiated (310 nm for 135 minutes) and unirradiated samples were incubated for 30 minutes at different temperatures, or incubated for 3 hours at room temperature in double-distilled water containing progressively decreasing concentration of monovalent sodium chloride. p is a pocket; u is unfolded; f is folded; c is a crosslinked stapled strand; s is an uncrosslinked stapled strand; L is a 1 kB ladder; NI and I are unirradiated and irradiated standard samples in folding buffer containing 5 mM MgCl2, respectively. Gel images were auto-leveled, and highlighted areas were auto-leveled twice. Average 2D particle micrograph of irradiated samples in double-distilled water. (B) and (C) are similar to (A), but each has a brick-like DNA origami object characterized by additional thymidine at all strand ends and all strand crossover locations, and 5-T loops, and a pointer object characterized by additional thymidine at all strand ends and all strand crossover locations. See Figure 29 for an auto-leveled gel image of the whole.

[0019] [Figure 3]Assays demonstrating stability under physiological conditions are shown. Laser scan fluorescence images of 2.0% agarose gels stained with ethidium bromide. Crosslinked samples were irradiated at 310 nm for 135 minutes. (A) Brick-like DNA origami objects featuring additional thymidine at all strand ends and all strand crossover sites were incubated in phosphate-buffered saline (PBS) at 37°C for various times. (B) Brick-like DNA origami objects featuring additional thymidine at all strand ends, all strand crossover sites, and 5T loops were incubated in 10% fetal bovine serum (FBS) at 37°C for various times. (C) The brick-like DNA origami objects from (A) were exposed to a set of different nucleases (100 U / ml) at 37°C for 24 hours. Lanes marked "c" show controls in which the samples were dissolved in the corresponding buffer in the absence of nucleases. The brick-like DNA origami samples (D) and (B) were exposed to DNase I (0.4 U / ml) for varying durations at 37°C. Unirradiated and irradiated samples (B) to (D) were alternately placed on a gel. All gel images were automatically leveled.

[0020] [Figure 4] The following shows the cryo-EM structural analysis before and after UV irradiation. (A) Cryo-EM density map of an unirradiated brick-like object with TT motifs 1-3 (electron microscopy database identifier EMD-4354). (B) and (C) Cryo-EM density maps of an irradiated (135 min at 310 nm) brick-like object with TT motifs 1-3 in a buffer containing 5 mM MgCl2 or 150 mM NaCl in phosphate-buffered saline (PBS) buffer, respectively. The electron density threshold was selected so that all crossovers in the uppermost layer were visible, as seen in the side view (electron microscopy database identifiers EMD-0027 and EMD-0028, respectively). (D) Sections along the z-direction obtained from the three density maps shown in (A)-(C) from top to bottom. The first and last sections were selected to determine the torsion angle delta theta. (E) Sections showing the three crossover layers in the reconstructions shown in (A)-(C). (F) Comparison of the overall dimensions of the non-crosslinked variant in 5 mM MgCl2 buffer and the crosslinked variant in PBS buffer.

[0021] [Figure 5] The conformational states and covalent bonds of higher-order aggregates are shown. (A) Schematic diagram of a two-state switch consisting of two rigid rods flexibly connected in the center by a fixed Holliday junction. The cylinders in the model represent double-helix DNA domains, and surface shapes complementary to the morphology are highlighted in light gray and dark gray. The inset shows a magnified view of the blunt-end interface of protruding surface shapes (light gray) and recessed surface shapes (dark gray). Thymidine directly located at the blunt-end region can be crosslinked by UV irradiation. The resulting CPD bond is shown as a light gray ellipsoid. (B) Laser scan fluorescence image of a 2.0% agarose gel stained with ethidium bromide. Switch samples were irradiated at 310 nm for various times and placed on the gel; o and c are particles present in the open and closed states, respectively. (C) Plot of the proportion of crosslinked switch particles as a function of time obtained from the gel of (B). The experiment was performed using triple measurements. The data points represent the mean, and the error bars represent the standard deviation. (D) Example of TEM micrographs; top is an unirradiated sample with particles in an open state, and bottom is an irradiated sample (20 minutes at 310 nm) with particles fixed in a closed conformation state. Scale bar is 100 nm. Inset is an average 2D particle micrograph of cross-linked particles. Scale bar is 20 nm. (E) Top left is a model of a multilayer DNA origami brick that polymerizes to form linear filaments. Field of view of a TEM micrograph recorded under the indicated conditions. Scale bar is 100 nm.

[0022] [Figure 6] The diagram shows the design of brick-like objects (TT motifs 1-3) prepared using caDNAno66. These objects feature 1-thymine-length overhangs at all staple ends. Furthermore, TT motifs were inserted at all crossover sites on each strand. The interface was passivated with polythymine overhangs. The upper right of the inset shows a cross-section of the object designed with a honeycomb lattice.

[0023] [Figure 7] Examples of negatively stained TEM micrographs of brick-like objects (TT motifs 1-4) in different buffers / solvents are shown. Images were high-pass filtered to reduce the staining gradient. Insets show corresponding mean 2D particle micrographs. Scale bar is 20 nm.

[0024] [Figure 8] Fluorescence images of laser scans of 2.0% agarose gels performed in an ice-cold water bath are shown. Brick-like objects with TT motifs 1-3, dissolved in different solvents, were placed on the gel. p is a pocket; u is an unfolded object; f is a folded object; L is a 1kB ladder; R1 is an unirradiated standard in folding buffer containing 30mM MgCl2; R2 is a standard irradiated at 310nm for 135 minutes in folding buffer containing 30mM MgCl2. The gel images were auto-leveled, and the highlighted areas were auto-leveled again.

[0025] [Figure 9] Fluorescence images of laser scans of 2.0% agarose gels performed in a water bath at ambient temperature are shown. The gel and running buffer contained 0.5×TBE without MgCl2. Samples of brick-like objects with TT motifs 1-3 were placed on the gel in the presence of different percentages of DMSO. p is a pocket; u is an unfolded object; f is a folded object; R1 is a non-irradiated standard in a folded buffer containing 30 mM MgCl2. The gel images were automatically leveled.

[0026] [Figure 10] The diagram shows a design for brick-like objects (TT motifs 1-4) prepared using caDNAno66. These objects feature 1-thymine overhangs at all staple ends. Furthermore, TT motifs were inserted at all crossover sites on each strand. Additionally, the helix features 5-thymine loops for interhelical bridging. The interface was passivated with polythymine overhangs. The upper right of the inset shows a cross-section of the object designed with a honeycomb lattice.

[0027] [Figure 11] Fluorescence images of laser scans of 2.0% agarose gels placed in an ice-cold water bath are shown. Various irradiated (135 min at 310 nm) and unirradiated samples of brick-like objects having TT motifs 1-4, characterized by 1-T, 3-T, and 5-T loops, were incubated at various temperatures and placed on the gel. p is a pocket; u is unfolded; f is folded; c is a cross-linked staple; s is an uncross-linked staple; L is a 1 kB ladder; R1 is an unirradiated brick-like object with TT motifs 1-3 in a folding buffer containing 30 mM MgCl2; R2 is an irradiated (135 min at 310 nm) brick-like object with TT motifs 1-3 in a folding buffer containing 30 mM MgCl2. The gel images were auto-leveled, and highlighted areas were auto-leveled again. Arrows indicate bands whose intensity increases with increasing loop length.

[0028] [Figure 12] A blueprint of a pointer object prepared using caDNAno66 is shown. This object features a 1-thymine overhang at every staple end. TT motifs were inserted at all crossover sites on each strand. The interface was passivated with polythymine overhangs. The upper right of the inset shows a cross-section of the object designed with a square grid.

[0029] [Figure 13] Examples of negatively stained TEM micrographs of a pointer object in different buffers / solvents are shown. The images were high-pass filtered to reduce the staining gradient. The inset shows the corresponding mean 2D particle micrograph. The scale bar is 20 nm.

[0030] [Figure 14]Laser-scanned fluorescence images of 2.0% agarose gels placed in a water bath are shown. Brick-like objects with TT motifs 1-3, irradiated for different times and incubated at different temperatures, were placed on the gel. p is a pocket; u is an unfolded object; f is a folded object; c is a cross-linked staple; s is an uncross-linked staple; L is a 1kB ladder; R1 is an unirradiated standard in folding buffer containing 30mM MgCl2; R2 is a standard irradiated at 310nm for 135 minutes in folding buffer containing 30mM MgCl2. The gel images were auto-leveled, and highlighted areas were auto-leveled again. (A) Samples were irradiated in the presence of 5mM MgCl2. (B) and (C) Samples were irradiated in the presence of 30mM MgCl2.

[0031] [Figure 15] Laser-scanned fluorescence images of a 2.0% agarose gel placed in a water bath are shown. Brick-like objects with TT motifs 1-4, irradiated for different times and incubated at different temperatures, were placed on the gel. p is a pocket; u is an unfolded object; f is a folded object; c is a cross-linked staple; s is an uncross-linked staple; L is a 1kB ladder; R1 is an unirradiated standard in a folding buffer containing 30mM MgCl2. The gel images were auto-leveled, and highlighted areas were auto-leveled again.

[0032] [Figure 16]Laser-scanned fluorescence images of 2.0% agarose gels placed in an ice-cold water bath are shown. The gel and running buffer contained 10 mM MgCl2. Cyanine 5-labeled brick-like objects having TT motifs 1-3 and TT motifs 1-4 were subjected to defect analysis using the de-Bruijn assay.58 Unirradiated and irradiated samples were mixed with two cyanine 3-modified oligonucleotides (de-Bruijn probes; final concentration 16 μM) and then placed on the gel. (A) The gel was laser-scanned through two channels. Upper gel: defect channel; cyanine 3 fluorescent agent was excited at 532 nm, and emission was captured at 560-580 nm. Lower gel: structure channel; cyanine 5 fluorescent agent was excited at 635 nm, and emission was captured above 665 nm. p is a pocket and f is a folded object. The gel images were automatically leveled across. (B) A plot of relative defect strength (i.e., the ratio of band strength between defects and structural channels) calculated from the gel of (A).

[0033] [Figure 17] Laser-scanned fluorescence images of 2.0% agarose gel placed in a water bath are shown. Brick-like objects with TT or TC motifs 1-3 were placed on the gel, irradiated for different times and incubated at different temperatures. p is a pocket; u is an unfolded object; f is a folded object; c is a cross-linked staple; s is a non-cross-linked staple; L is a 1kB ladder. The gel images were auto-leveled.

[0034] [Figure 18]Laser-scanned fluorescence images of a 2.0% agarose gel placed in a water bath are shown. Brick-like objects with TT motifs 1-3, irradiated at 365 nm for 135 minutes (A) and 365 nm for 20 hours (B), and incubated at various temperatures, were placed on the gel. p is a pocket; u is an unfolded object; f is a folded object; s is a non-crosslinked staple; L is a 1 kB ladder; R1 is an unirradiated standard in folding buffer containing 30 mM MgCl2; R2 is a standard irradiated at 365 nm for 135 minutes (A) and 20 hours (B) in folding buffer containing 30 mM MgCl2. The gel images were auto-leveled, and the highlighted areas were auto-leveled separately.

[0035] [Figure 19] Laser-scanned fluorescence images of 2.0% agarose gels placed in a water bath are shown. Various variations of brick-like objects with TT motifs 1, 1-2, and 1-3 were placed on the gel. p is a pocket; u is an extended object; f is a folded object; s is a non-crosslinked staple; L is a 1kB ladder. Arrows indicate bands where electrophoretic mobility increases when TT motifs are added to the design. The gel images were auto-leveled.

[0036] [Figure 20] Laser-scanned fluorescence images of a 2.0% agarose gel placed in a water bath are shown. Brick-like objects with TT motifs 1-3 (left) and TT motifs 1-4 (right), incubated for different times with 0.4 U / ml DNase I diluted in DNase I reaction buffer, were placed on the gel. p is a pocket; u is unfolded; f is folded; c is a cross-linked staple; s is a non-cross-linked staple; R1: unirradiated standard; R2: standard irradiated at 310 nm for 135 minutes. Standard samples R1 and R2 were dissolved in DNase I buffer in the absence of DNase I. The gel images were auto-leveled, and the highlighted areas were auto-leveled again.

[0037] [Figure 21]The following shows cryoEM data of brick-like objects with TT motifs 1-3 before crosslinking in folding buffer: (A) Motion-corrected and dose-weighted cryoEM micrographs of a dataset of brick-like objects with TT motifs 1-3 before crosslinking in folding buffer containing 5 mM MgCl2. Scale bar represents 100 nm. A 15-frame dose-resolved movie was acquired with a 2.3 Å magnified pixel size and a total dose of 60 e- / Å2 using an FEI Titan KriosG2 operating at 300 kV. (B) Representative 2D class average showing different directions. Scale bar represents 40 nm. (C) Graphs of various FSC curves showing resolution after sharpening. (D) 3D histogram showing the distribution of particle orientation. (E) Six different figures of the final sharpened map reconstructed from 165,000 individual particles. A B coefficient of -1,000 was used for sharpening.

[0038] [Figure 22] The following shows cryoEM data of brick-like objects with TT motifs 1-3 after crosslinking in a folding buffer: (A) Motion-corrected and dose-weighted cryoEM micrographs of a dataset of brick-like objects with TT motifs 1-3 after crosslinking in a folding buffer containing 5 mM MgCl2. Scale bar represents 100 nm. A 15-frame dose-resolved movie was acquired with a 2.3 Å magnified pixel size and a total dose of 60 e- / Å2 using an FEI Titan KriosG2 operating at 300 kV. (B) Representative 2D class average showing different directions. Scale bar represents 40 nm. (C) Graphs of various FSC curves showing resolution after sharpening. (D) 3D histogram showing the distribution of particle directions. (E) Six different figures of the final sharpened map reconstructed from 95,000 individual particles. A B coefficient of -1,000 was used for sharpening.

[0039] [Figure 23]This shows cryoEM data of brick-like objects with TT motifs 1-3 after crosslinking in phosphate-buffered saline. (A) Motion-corrected and dose-weighted cryoEM micrographs of the dataset of brick-like objects with TT motifs 1-3 after crosslinking in PBS buffer. Scale bar represents 100 nm. A 15-frame dose-resolved movie was acquired with a magnified pixel size of 2.3 Å and a total dose of 60 e- / Å2 using an FEI Titan KriosG2 operating at 300 kV. (B) Representative 2D class average showing different directions. Scale bar represents 40 nm. (C) Graphs of various FSC curves showing resolution after sharpening. (D) 3D histogram showing the distribution of particle orientation. (E) Six different figures of the final sharpened map reconstructed from 57,000 individual particles. A B coefficient of -1,000 was used for sharpening.

[0040] [Figure 24] This shows section-by-section visualizations of the cryoEM map determined from the brick sample. The 3D volume was rotated so that the helical axis is perpendicular to the plane of the figure (denoted as the z direction). The original volume had 400x400x400 pixels with a size of 2.3 Å per pixel. For section analysis, the volume was trimmed to 200x200 in the xy plane and binned in the z direction so that each section had a thickness of 3.35 Å. This corresponds to the contribution of one base pair along the helical direction. Image J was used to create a montage of the 3D volume. (A) Brick variant with thymine at all staple ends and TT motifs at all crossover sites per strand. (B) Brick variant with additional 5-T loops to create interhelical bonds.

[0041] [Figure 25]The following shows cryoEM data of brick-like objects with TT motifs 1-4 before crosslinking in folding buffer: (A) Motion-corrected and dose-weighted cryoEM micrographs of a dataset of brick-like objects with TT motifs 1-4 before crosslinking in folding buffer containing 5 mM MgCl2. Scale bar represents 100 nm. A 15-frame dose-resolved movie was acquired with a magnified pixel size of 2.3 Å and a total dose of 60 e- / Å2 using an FEI Titan KriosG2 operating at 300 kV. (B) Representative 2D class average showing different directions. Scale bar represents 40 nm. (C) Graphs of various FSC curves showing resolution after sharpening. (D) 3D histogram showing the distribution of particle orientation. (E) Six different figures of the final sharpened map reconstructed from 33,000 individual particles. A B coefficient of -1,000 was used for sharpening.

[0042] [Figure 26] The following shows cryoEM data of brick-like objects with TT motifs 1-4 after crosslinking in a folding buffer: (A) Motion-corrected and dose-weighted cryoEM micrographs of a dataset of brick-like objects with TT motifs 1-4 after crosslinking in a folding buffer containing 5 mM MgCl2. Scale bar represents 100 nm. A 15-frame dose-resolved movie was acquired with a 2.3 Å magnified pixel size and a total dose of 60 e- / Å2 using an FEI Titan KriosG2 operating at 300 kV. (B) Representative 2D class average showing different directions. Scale bar represents 40 nm. (C) Graphs of various FSC curves showing resolution after sharpening. (D) 3D histogram showing the distribution of particle directions. (E) Six different figures of the final sharpened map reconstructed from 75,000 individual particles. A B coefficient of -1,000 was used for sharpening.

[0043] [Figure 27] The diagram shows the design of a switch object prepared using caDNAno66. The interface was passivated with a polythymine overhang. The lower left of the inset shows a cross-section of the object designed with a honeycomb grid.

[0044] [Figure 28] The diagram shows a design for a polymerized brick object prepared using caDNAno66. The interface was passivated with polythymine overhangs. The upper right of the inset shows a cross-section of the object designed with a honeycomb lattice.

[0045] [Figure 29] Fluorescence images of laser scans of 2.0% agarose gels placed in a water bath are shown. (A) From left to right: Model of brick-like DNA origami objects featuring additional thymidine at all strand ends and all strand crossover locations; laser scan fluorescence image of 2.0% agarose gel stained with ethidium bromide. Irradiated (310 nm for 135 minutes) and unirradiated samples were incubated for 30 minutes at different temperatures, or incubated for 3 hours at room temperature in double-distilled water containing progressively decreasing concentrations of monovalent sodium chloride. p: pocket; u: spread out; f: folded; c: crosslinked stapled strand; s: uncrosslinked stapled strand; L: 1 kB ladder; NI and I: unirradiated and irradiated standard samples in folding buffer containing 5 mM MgCl2, respectively. Gel images were automatically leveled overall. (B) and (C) are similar to (A), but have brick-like DNA origami objects characterized by additional thymidine at all strand ends and at all strand crossover locations, and 5-T loops, and pointer objects characterized by additional thymidine at all strand ends and at all strand crossover locations. [Modes for carrying out the invention]

[0046] The present invention relates to a novel method for stabilizing nucleic acid nanostructures by curing with ultraviolet light, particularly by crosslinking pyrimidine nucleotides.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention relates.

[0048] In a first embodiment, the present invention relates to a method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence bound to at least two non-adjacent sequence stretches present on one or more complementary nucleic acid sequences, wherein the method comprises the step of exposing the nucleic acid nanostructure to ultraviolet irradiation, wherein the step of exposing the nucleic acid nanostructure to ultraviolet irradiation results in the formation of at least one chemical bond between two pyrimidine nucleotides, wherein at least one of the two pyrimidine nucleotides is not part of a complementary nucleotide pair contained in a double-helix substructure.

[0049] In a second embodiment, the present invention relates to a method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence bound to at least two non-adjacent sequence stretches present on one or more complementary nucleic acid sequences, wherein the method is characterized by the step of exposing the nucleic acid nanostructure to ultraviolet irradiation.

[0050] In this specification, the terms “comprising” and “including” are used in their unrestricted, non-limiting sense unless otherwise specified. Therefore, with respect to such latter embodiments, the term “comprising” includes the narrower term “consisting of.”

[0051] In the context describing the present invention (particularly in the context of the following claims), the terms "a," "an," and "the," and similar references, should be interpreted as encompassing both singular and plural forms unless otherwise specified or unless the context clearly contradicts this interpretation. For example, the term "cell" includes multiple cells, including mixtures thereof. Where the plural form is used for compounds, salts, etc., this should be interpreted as also referring to a single compound, salt, etc.

[0052] In the context of the present invention, the term “nanostructure” refers to a three-dimensional structure formed from a complex of smaller substructures and at least partially regularly arranged structures. In the context of the present invention, smaller substructures include double helix substructures. In certain embodiments, double helix substructures are arranged in a more complex nanostructure by regular connections between different double helix substructures, where the connections are formed by single-stranded nucleic acid sequences and form crossovers between different double helix substructures by being complementary to at least two different sequence stretches on different nucleic acid sequence comparators.

[0053] In the context of the present invention, the term "single-stranded nucleic acid sequence" refers to a single strand of nucleic acid monomer units (nucleosides) linked by a phosphate group, a modified phosphate group, or a phosphate analog (the nucleoside and the phosphate-based linking group together are called a nucleotide). In the case of a deoxyribonucleic acid-based nucleic acid sequence, the nucleic acid monomer is formed from (i) a nucleoside containing a nucleic acid base selected from four nitrogen-containing nucleic acid bases: cytosine [C], guanine [G], adenine [A], or thymine [T], and a sugar called deoxyribose, and (ii) a phosphate group. In the case of a ribonucleic acid-based nucleic acid sequence, the nucleic acid monomer is formed from (i) a nucleoside containing a nucleic acid base selected from four nitrogen-containing nucleic acid bases: cytosine [C], guanine [G], adenine [A], or thymine [T], and a sugar called ribose, and (ii) a phosphate group.

[0054] In this particular embodiment, the single-stranded nucleic acid sequence is based on a deoxyribonucleic acid-based nucleic acid sequence.

[0055] In the context of the present invention, the term "non-adjacent sequence stretch" refers to a stretch of nucleic acid sequences that are not directly linked. A non-adjacent sequence stretch may be located on different nucleic acid sequences or on a single nucleic acid sequence, provided that at least one nucleotide is present between any of the non-adjacent sequence stretches that are not part of any of the other non-adjacent sequence stretches.

[0056] In the context of the present invention, the term "at least one single-stranded nucleic acid sequence bound to at least two non-adjacent sequence stretches" means that the binding occurs via the formation of hydrogen bonds between complementary bases contained in the at least one single-stranded nucleic acid sequence and in the at least two non-adjacent sequence stretches. The binding may be further enhanced by covalent bonds formed between the at least one single-stranded nucleic acid sequence and one, both, or all of the at least two non-adjacent sequence stretches after ultraviolet irradiation according to the present invention.

[0057] In a third embodiment, the present invention relates to a method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least two double-helical substructures, wherein the method is characterized by the step of exposing the nucleic acid nanostructure to ultraviolet irradiation.

[0058] In the context of the present invention, the term “double helix substructure” refers to a subpart of the nanostructure according to the present invention that is in a double helix configuration formed by two complementary nucleic acid sequence stretches. The ends of the double helix substructure may be due to the end of a complementary region, by the fact that (i) one of the two complementary nucleic acid sequence stretches reaches its 3' or 5' end, or (ii) one of the two complementary nucleic acid sequence stretches continues to form another double helix substructure by complementarity to a second non-adjacent sequence stretch on another or different nucleic acid sequence.

[0059] In a fourth embodiment, the present invention relates to a method for enhancing the stability of a non-naturally occurring nucleic acid nanostructure comprising a plurality of double-helical substructures, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence which is part of at least two different double-helical substructures, and the method is characterized by the step of exposing the nucleic acid nanostructure to ultraviolet irradiation.

[0060] In certain embodiments, the non-naturally occurring nucleic acid nanostructures include either a two-dimensional or three-dimensional arrangement of double-helix substructures.

[0061] In a particular embodiment, the non-naturally occurring nucleic acid nanostructure is such that each of the double-helical substructures consists of 10 to 5000 complementary nucleotide pairs, the double-helical substructures can be connected to adjacent double-helical substructures every 7, 8, or 9 bases, and one or more single-stranded oligonucleotides forming the double-helical substructures are part of the same or at least two different double-helical substructures.

[0062] In certain embodiments, the connections between the double helix substructures result in honeycomb, square, or hexagonal packing shapes, or combinations thereof.

[0063] In certain embodiments, at least 85% of the single-stranded oligonucleotides forming the double-helical substructure are part of at least two different double-helical substructures.

[0064] In a particular embodiment, the step of exposing the nucleic acid nanostructure to ultraviolet irradiation results in the formation of at least one chemical bond between two pyrimidine nucleotides.

[0065] In certain embodiments, at least one of the two pyrimidine nucleotides is not part of a complementary nucleotide pair included in the double helix substructure.

[0066] In a particular embodiment, the nucleic acid nanostructure comprises at least one copy of a first single-stranded polynucleotide and a set of second single-stranded polynucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, and each of the n core sequences comprises (i) a sequence complementary to a region on the first single-stranded polynucleotide, wherein the region on the first single-stranded polynucleotide complementary to the nth core sequence is a sequence not adjacent to the regions on the first single-stranded polynucleotide complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0067] In the context of the present invention, the first single-stranded polynucleotide can be considered as a scaffold polynucleotide or a main-chain polynucleotide.

[0068] In the context of the present invention, the value of m=0 is that the core sequence is a pyrimidine nucleotide stretch P at the corresponding end of the nucleic acid sequence. mThis indicates that they are not adjacent to each other. In certain embodiments, at least 50% of the second single-stranded polynucleotides in the set have at least one pyrimidine nucleotide stretch at either the 3' or 5' end. In certain embodiments, at least 75%, particularly at least 80%, at least 85%, or at least 90%, have at least one pyrimidine nucleotide stretch at either the 3' or 5' end. In certain embodiments, at least 35% of the second single-stranded polynucleotides in the set have two pyrimidine nucleotide stretches at both the 3' and 5' ends. In certain embodiments, at least 50%, particularly at least 75%, at least 80%, or at least 85%, have two pyrimidine nucleotide stretches at both the 3' and 5' ends.

[0069] In a particular embodiment, the first single-stranded polynucleotide contains at least 100 nucleotides.

[0070] In a particular embodiment, the first single-stranded polynucleotide has at least 70% sequence identity with respect to the DNA of the filamentous bacteriophage. In a particular embodiment, the single-stranded polynucleotide has at least 80% sequence identity, more specifically at least 85%, more specifically at least 90%, and most specifically at least 95%.

[0071] In a particular embodiment, the fibrous bacteriophage is M13, and more specifically M13mp18.

[0072] In a particular embodiment, the nucleic acid nanostructure comprises a set of single-stranded oligonucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, and each of the n core sequences comprises (i) a sequence complementary to the sequence of another member of the set of single-stranded polynucleotides, wherein the region on the other member complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0073] In a particular embodiment, each m is an integer independently selected from the range of 0 to 5.

[0074] In a particular embodiment, for each of the pyrimidine nucleotide stretches at the 3' and 5' ends, m is either 0 or 1, and P is a thymidine residue.

[0075] In a particular embodiment, the nucleic acid nanostructure is a pyrimidine nucleotide stretch P m This includes one or more insertions, where m is independently selected from the range 1 to 5, and in particular m is independently selected from 1, 3, and 5.

[0076] In a particular embodiment, each of the core sequences consists of x nucleotides, where x is independently selected from integers that are multiples of 7, 8, or 16.

[0077] In the context of the present invention, the value of x is determined by the shape of the DNA double helix. Those skilled in the art can determine the size and number of nucleotides of the double helix that must be present in order to enable the regular arrangement of the double helix substructures within the nanostructure stabilized according to the present invention.

[0078] In a particular embodiment, the ultraviolet irradiation is performed using ultraviolet light with a wavelength in the range of 250 nm to 350 nm.

[0079] In a particular embodiment, the ultraviolet irradiation is performed using a xenon light source (Asahi Spectra MAX303) in Tris buffer at a temperature range of approximately 0 to approximately 25 degrees Celsius, with a liquid guide to connect the light beam to the sample (the distance between the solution surface of the sample and the end of the light guide is less than approximately 5 cm), using the following parameters (sample volume of approximately 5 to 2,000 μl, concentration of nucleic acid nanostructures in the sample of approximately 1 to 500 nM). The sample is exposed to approximately 1 to 10 mW / cm². 2 The subject is exposed to ultraviolet radiation at this intensity for approximately 1 to 6 hours.

[0080] In the context of this invention, the term "about" in combination with a value or range of values ​​indicates that a given value or range of values ​​does not exclude values ​​close to the specifically enumerated value or range of values. In particular, depending on the context, the term "about" includes values ​​within plus or minus 10% of the specified value. In specific embodiments, the term "about" is ignored, and the value or range of values ​​is used as described.

[0081] In a specific embodiment, the step of exposing the nucleic acid nanostructure to ultraviolet irradiation is performed for a period of time required for the reference DNA nanostructure of Example 2, which has been treated under the same conditions to reach a stable state, where the stability is determined by a gel electrophoresis assay as described in Example 2, where the reference DNA nanostructure is (i) untreated under reference conditions of 5 mM Tris, 5 mM NaCl, 1 mM EDTA, and 5 mM MgCl2 at 25°C, and (ii) after ultraviolet treatment, incubated under the required target conditions, particularly at high temperatures, particularly at 90°C, in pure water, under physiological conditions, or in a vacuum, where a stable state is reached as soon as the band of the reference DNA nanostructure exhibits the same electrophoretic mobility as under reference conditions.

[0082] In a fifth embodiment, the present invention relates to a kit for creating nucleic acid nanostructures comprising at least one copy of a first single-stranded polynucleotide and a set of single-stranded polynucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, where each of the n core sequences is (i) a sequence complementary to a region on the first single-stranded polynucleotide, wherein the region complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0083] In a specific embodiment, each m is an integer independently selected from the range of 0 to 5.

[0084] In a specific embodiment, the pyrimidine nucleotide stretch P at the 3' and 5' ends mFor each of these, m is either 0 or 1, and P is a thymidine residue.

[0085] In a specific embodiment, the nucleic acid nanostructure is a pyrimidine nucleotide stretch P m This includes one or more insertions, where m is independently selected from the range 1 to 5, and in particular m is independently selected from 1, 3, and 5.

[0086] In a sixth embodiment, the present invention relates to a kit for creating nucleic acid nanostructures comprising a set of single-stranded oligonucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, where each of the n core sequences is (i) a sequence complementary to the sequence of another member of the set of single-stranded polynucleotides, wherein the region on the other member complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0087] In a specific embodiment, each m is an integer independently selected from the range of 0 to 5.

[0088] In a specific embodiment, the pyrimidine nucleotide stretch P at the 3' and 5' ends m For each of these, m is either 0 or 1, and P is a thymidine residue.

[0089] In a specific embodiment, the nucleic acid nanostructure is a pyrimidine nucleotide stretch P mThis includes one or more insertions, where m is independently selected from the range 1 to 5, and in particular m is independently selected from 1, 3, and 5.

[0090] In a seventh embodiment, the present invention relates to a nucleic acid nanostructure comprising at least one copy of a first single-stranded polynucleotide and a set of single-stranded polynucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer selected from 1 to 40, and each of the n core sequences is (i) a sequence complementary to a region on the first single-stranded polynucleotide, wherein the region complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0091] In a specific embodiment, each m is an integer independently selected from the range of 0 to 5.

[0092] In a specific embodiment, the pyrimidine nucleotide stretch P at the 3' and 5' ends m For each of these, m is either 0 or 1, and P is a thymidine residue.

[0093] In a specific embodiment, the nucleic acid nanostructure is a pyrimidine nucleotide stretch P m This includes one or more insertions, where m is independently selected from the range 1 to 5, and in particular m is independently selected from 1, 3, and 5.

[0094] In an eighth embodiment, the present invention relates to a nucleic acid nanostructure comprising a set of single-stranded oligonucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is independently selected from 1 to 40, and each of the n core sequences is (i) a sequence complementary to the sequence of another member of the set of single-stranded polynucleotides, wherein the region on the other member complementary to the nth core sequence is not adjacent to the regions complementary to the (n-1)th and (n+1)th core sequences, and (ii) a 3'-terminal pyrimidine nucleotide stretch P m (iii) Pyrimidine nucleotide stretch P at the 5' end m , and (iv) optionally, pyrimidine nucleotide stretch P m It consists of one or more insertions, where each m is an integer independently selected from the range of 0 to 40, and each P is independently selected from thymidine and cytosine residues.

[0095] In a specific embodiment, each m is an integer independently selected from the range of 0 to 5.

[0096] In a specific embodiment, the pyrimidine nucleotide stretch P at the 3' and 5' ends m For each of these, m is either 0 or 1, and P is a thymidine residue.

[0097] In a specific embodiment, the nucleic acid nanostructure is a pyrimidine nucleotide stretch P m This includes one or more insertions, where m is independently selected from the range 1 to 5, and in particular m is independently selected from 1, 3, and 5.

[0098] In a specific embodiment, the nucleic acid nanostructure includes one or more ultraviolet-induced bridges between spatially adjacent thymidine and / or cytosine residues.

[0099] In a specific embodiment, the one or more bridges include a cyclobutanepyrimidine dimer and a pyrimidine dimer selected from (6,4)pyrimidine-pyrimidone.

[0100] In a specific embodiment, the one or more bridges are located at the 3' end of a single-stranded oligonucleotide or core sequence, and at the 5' end of a P m Thymidine and / or cytosine residues contained in the stretch, and / or any of the above P m It is located between one of the thymidine molecules in the insert.

[0101] In a specific embodiment, one or more of the bridges are intrahelical bridges between two adjacent single-stranded oligonucleotides or thymidine or cytosine residues at the 3' and 5' ends of a core sequence, which are part of the same or different double-helix substructure of the nucleic acid nanostructure.

[0102] In a specific embodiment, one or more of the bridges are interhelical bridges between thymidine or cytosine residues contained in a single-stranded oligonucleotide or a portion of such a single-stranded oligonucleotide, which are part of two different double-helix substructures of the nucleic acid nanostructure, and particularly between two thymidine residues contained in two of the arbitrary inserts.

[0103] In a specific embodiment, the crosslinks are as follows: from the 3' end of polynucleotide A to the 5' end of polynucleotide B, from the 3' end of polynucleotide A to the 3' end of polynucleotide B, from the 5' end of polynucleotide A to the 5' end of polynucleotide B, from the 3' end of polynucleotide A to the insertion in the core sequence of polynucleotide B, from the 5' end of polynucleotide A to the insertion in the core sequence of polynucleotide B, and from the insertion in the core sequence of polynucleotide A to the insertion in the core sequence of polynucleotide B.

[0104] In a ninth aspect, the present invention relates to a complex nucleic acid nanostructure resulting from the aggregation of two or more nucleic acid nanostructures according to the present invention.

[0105] In a specific embodiment, the aggregate includes one or more ultraviolet-inductive bridges between two or more nucleic acid nanostructures according to the present invention. [Examples]

[0106] The following examples illustrate the present invention without limiting its scope.

[0107] The bottom-up fabrication of custom nanostructures using DNA nanotechnology methods holds great potential for applications in many fields of science and technology. One significant obstacle to these applications is related to the constrained environmental conditions under which DNA objects maintain their structure. Here, we present a general, site-selective, and scalable method for introducing additional covalent bonds to enhance the structural stability of DNA nanostructures. The key concept is the close-proximity arrangement of thymidines within a user-defined DNA nanostructure to rationally create sites for introducing cyclobutanepyrimidine dimer (CPD) covalent bonds via UV irradiation. These additional bonds can be used in a sequence-programmable manner to ligate free strand ends, eliminate strand breaks occurring at crossover sites (i.e., ligate strand breaks at crossover sites), and create additional interhelical connections. As a result, objects covalently crosslinked at user-programmable sites are obtained without the need for chemical modification. Thus, the designed multilayer DNA origami objects can maintain their overall shape and remain stable at temperatures up to 90°C and in pure double-distilled water without the presence of additional cations. Furthermore, these objects exhibit a significantly extended lifetime, i.e., enhanced resistance to nuclease activity. Cryo-electron microscopy (cryoEM) structural analysis of non-crosslinked and crosslinked objects showed that the overall shape and internal network of the crossover were maintained even after irradiation. CryoEM maps of CPD-stabilized multilayer DNA origami objects, determined at physiological ionic intensities, revealed substantial swelling behavior, likely caused by repulsive electrostatic forces, which would lead to decomposition at low ionic intensities without covalent stabilization. Our method opens new avenues for the application of DNA nanostructures under a wider range of conditions and therefore in various fields.

[0108] Example 1 General description of the approach and achieved results Pyrimidine dimers are molecular defects produced by photochemical reactions of DNA. 53Ultraviolet light induces covalent bond formation through reactions at C=C double bonds in thymine (T) or cytosine (C) bases (Figure 1, left). Common products include cyclobutane-pyrimidine dimers (CPDs) containing thymine dimers. (6-4) Minor byproducts such as pyrimidine-pyrimidone and Dewar isomers may also be formed by UV irradiation. These damages are carcinogenic and target the cellular DNA repair mechanisms because they can halt DNA replication and transcription. 54 In 1982, Lewis and Hanawalt reported that CPDs could also be formed from adjacent terminal thymines in separate DNA strands that were combined by templated complementary DNA strands. 55 However, the potential of this finding to solve stability problems in DNA nanotechnology has not been recognized until now. A key concept in our research is the close arrangement of thymidine within user-defined DNA nanostructures to rationally create sites for introducing covalent CPD bonds via UV irradiation. These additional bonds can be used to ligate free strand ends, eliminating strand breaks that occur at crossover sites and creating additional interhelical connections (Figure 1, right).

[0109] The fundamental building blocks of DNA nanotechnology are the double-helix DNA domains. 3,4,56 These domains are formed by hybridizing a set of short, single-stranded staple oligonucleotides into a long, single-stranded scaffold molecule. DNA tile-brick object 16In other types of DNA nanostructures, such as DNA origami and tile-brick objects, double helix domains are formed only between single-strand oligonucleotides. DNA origami and tile-brick objects contain hundreds of single-strand breaks, which is a weakness. This is because the free ends allow for dissolution as well as the formation of entangled double helix domains. To create an option for removing single-strand breaks after the self-assembly of the target object, we prepare DNA strands containing additional thymidines at both ends (Figure 1A, motif 1). The added bases do not participate in Watson-Crick base pairing, but the thymidines are close to the single-strand break sites of the folded object, which allows for the formation of a CPD bond between the two thymidines by irradiation with light at a wavelength of 310 nm.

[0110] In DNA objects, double helix domains are connected to adjacent double helix domains by interhelical connections formed by antiparallel single-strand crossovers, which typically include both half-crossovers and double-crossovers (Figure 1A, motif 2 vs. motif 3). For example, DNA tile-brick objects are connected almost exclusively via half-crossovers, while both types of interhelical connections can occur in DNA origami objects. Crossover sites also represent weak points in DNA nanostructures because the main-strand linkage in the helical direction is interrupted. To create an option for closing weak links after self-assembly, we can add additional unpaired thymidine bases to the stapled strands at crossover sites, as shown in Figure 1A (motifs 2 and 3). By utilizing proximity and re-irradiating with 310 nm light, the formation of CPD bonds covalently linking the strands along the helical direction is induced (Figure 1B, motifs 2 and 3), thus creating another topological obstacle to helix unwinding. When designing DNA objects, strand crossovers are typically located between adjacent double-helical domains where the helical main strands are close to each other. To complement strand crossovers, additional interhelical bonds can be created after the object's self-assembly using light-induced CPD dimer bonding. For this purpose, single-stranded thymidine loops (T-loops) are positioned where the main strands of adjacent DNA helices are roughly aligned (Figure 1A, motif 4). Irradiation at 310 nm can then induce the creation of interhelical covalent bonds (Figure 1B, motif 4).

[0111] Proof of Concept: High Temperature and Low Salt Stability To test our method, we made the design modifications shown in Figure 1 to several variants of the multilayer DNA origami object. We tested the stability of the objects obtained after irradiation with 310 nm light in melting experiments (Figure 2, left) and experiments after removing cations from the solution (Figure 2, right). By inserting additional thymidine at both the ends of all strands and at all strand crossover positions, we created a brick-like multilayer DNA origami with a honeycomb packing shape (Figure 6). 57The object was modified. As observed in gel electrophoresis, the non-irradiated control sample decomposed ("spread out") at approximately 50°C, due to the disappearance of the band indicating the folded object and the appearance of free staple chains (Figure 2A, left gel). In contrast, the irradiated sample maintained its overall shape up to 90°C, judging from the fact that the electrophoretic mobility of the folded object remained almost unchanged. Slight blemishes at high electrophoretic mobility indicate that some chains still separated from the folded object at high temperatures. However, the separated chains had much lower electrophoretic mobility and were therefore larger in mass than the staple chains that emerged from the molten non-irradiated control sample. The high-temperature resistance of the irradiated object and the appearance of higher-mass chains provide evidence that UV irradiation successfully introduced covalent crosslinks to the designed thymidine moieties.

[0112] We also tested the stability of irradiated and unirradiated samples after removing cations from the solution. The buffer was replaced using filtration, and the samples were dissolved in double-distilled water containing progressively decreasing concentrations of monovalent sodium chloride (Figure 2A, right). The irradiated samples remained folded even in double-distilled water without added cations, while the unirradiated control decomposed between 300 and 150 mM sodium chloride (NaCl), as evidenced by a strong mobility shift and the appearance of single-chain particles with high mobility. Transmission electron microscopy (TEM) imaging of the irradiated samples dissolved in pure water revealed particles with the predicted shape (Figure 7). The degree of heterogeneity of the samples in pure water was slightly higher than under high-salt conditions. Electrophoretic analysis after storage in pure water for up to one day showed no change in electrophoretic mobility, and no staple chains separated from the folded material were detected (Figure 8). Therefore, simple design modifications and UV irradiation allow for the stabilization of multilayer DNA origami, which is normally highly cation-sensitive, for use under physiological conditions (approximately 150 mM NaCl) and even lower ionic strength conditions. After UV stabilization, many other harsh environments may also be usable. As a simple demonstration, we dissolved the crosslinking material in an aqueous mixture of dimethyl sulfoxide (DMSO; organic solvent) without added cations (Figure 9).

[0113] As a second example, variants of the brick-like object were prepared and tested, which included not only extra thymidines at all chain ends and crossover positions, but also single-stranded T-loops (motif 4 in Figure 1) (Figure 10). The degree of thermal and cationic stabilization after irradiation (Figure 2B) was similar to that of the design variant lacking single-stranded T-loops. Loops containing 1, 3, and 5 thymidines were tested. The variant with 5-T loops showed a gradually increasing degree of stabilization at higher temperatures (80°C), judging from the band intensity of gel electrophoresis (Figure 11). When the brick variant with the additional 5-T loops for helical bonding was irradiated, a slight increase in electrophoretic mobility was observed compared to the unirradiated control (Figure 2B, lane R2 vs. lane R1), suggesting that the additional helical bonding may result in some degree of compression or mechanical stabilization.

[0114] As a third example, we consider the aforementioned "pointer" object, which is a multilayer DNA origami in the shape of a square lattice packing. 13 We selected and added additional thymidine to all chain ends and all crossover positions (Figure 12). For the brick variant, UV irradiation stabilized this object against exposure to temperatures up to 90°C, and the pointer object could dissolve in pure water without cations (Figure 2C). As is evident from electrophoretic mobility analysis (Figure 2C) and TEM imaging (Figure 13), the unirradiated control pointer sample had already decomposed between 45°C and 50°C, and more than 300 mM NaCl in solution was required for it to remain folded. In the process of establishing our method, we tested several parameters, including exposure time to UV irradiation. Furthermore, we performed the de-Bruijn assay. 58Defect analysis was performed using [a specific method] to evaluate the structural integrity of double helix domains in DNA samples upon UV irradiation. Exposure to our UV configuration for approximately 2 hours resulted in the most efficient stabilization of all tested structures (Figures 14 and 15), with no signs of structural degradation (Figure 16). Shorter irradiation times resulted in incomplete crosslinking, meaning the structures would not survive exposure to temperatures significantly exceeding the pre-UV melting temperature. For irradiation times longer than the optimal duration, structural radiation damage accumulated, reflected in a continuous decrease in the electrophoretic mobility of the samples. Therefore, exposure to UV irradiation follows the Goldilocks principle. Our optimal irradiation time may not necessarily be maintained under other circumstances, as it depends on the details of the UV source and other parameters. However, users can identify the optimal irradiation time using a screening method similar to the one we performed.

[0115] As outlined at the beginning, our method relies on cyclobutane-pyrimidine dimers, which can form not only between TT but also, for example, between TC junctions. As an example, we compared the crosslinking efficiency of brick variants prepared using TT and TC at all chain ends and all chain crossover locations (Figure 17). Based on the amount of structure that withstands exposure to high temperatures after UV exposure, TT bonds form much more efficiently than TC junctions, leading to complete stabilization. Crosslinking worked well with exposure to 310 nm light. It has also been reported that CPD bonds may form upon exposure to 365 nm UVA light (Figure 18), but stabilization was not achieved at longer wavelengths such as 365 nm. 59 .

[0116] Stability under physiological conditions Our UV crosslinking method can be used to substantially enhance the stability of DNA nanostructures, particularly multilayer DNA origami objects, for applications under physiological conditions. As proof, we dissolved brick-like multilayer DNA origami objects containing additional T bases at all staple ends and all crossover positions in phosphate-buffered saline (PBS) and incubated the objects at physiological temperature of 37°C. No degradation was detected in the irradiated and covalently crosslinked samples even after storage in PBS at 37°C for two days (Figure 3A). In contrast, the unirradiated control degraded within minutes after exposure to these conditions. Judging from the degree to which the design variants of the irradiated brick samples remained stable at physiological temperature and ionic intensity, stabilization appeared complete for design variants with additional T bases at all strand ends and all half and full crossover positions. Linking only free strand ends and a subset of crossovers was not sufficiently effective in maintaining the complete structure (Figure 19). In 10% fetal bovine serum (FBS) at 37°C, irradiated brick samples survived for several hours, significantly longer than the unirradiated control (Figure 3B). In serum, the disappearance of the folded structures was likely caused by enzymatic activity rather than the low ionic strength of the solution. Physiological fluids such as serum contain various exonucleases and endonucleases for digesting DNA molecules. To elucidate the activity of various nucleases, we exposed brick-like multilayer DNA origami objects characterized by T at the strand ends, all crossover sites, and T-loops to a panel of such enzymes (Figure 3C). Some enzymes, such as ExoVIII and T7Exo, appeared to be inactive in brick samples by default, regardless of whether they were irradiated or not. However, in the case of others (ExoIe, ExoT, T7Endo, ExoIII, etc.), the introduction of additional covalent bonds by irradiation significantly extended the lifetime of the crosslinked objects compared to the unirradiated control samples. The most active DNA-degrading enzyme was deoxyribonuclease I (DNase I). Kinetic analysis of the digestion of brick-like objects using DNase I at plasma activity level 60 revealed that irradiated and stabilized brick samples were digested much more slowly than unirradiated controls.Band strength analysis revealed an approximately 5-6 times extension of lifespan, from 10 minutes to 60 minutes, under the conditions tested by crosslinking (Figure 3D). Brick variants characterized only by extra T at the chain ends and all crossover locations (lacking interhelical T loops) showed slightly weaker resilience to DNaseI digestion (Figure 20).

[0117] CryoEM structural analysis of UV-crosslinked multilayer DNA origami objects To elucidate the effects of UV irradiation and CPD binding formation on the structure of DNA objects, five electron density maps were exemplary determined using single-particle cryo-EM (Figure 4, A-C). First, single-particle cryo-EM data were collected from a non-irradiated control multilayer brick sample containing additional T at all strand ends and all strand crossover locations (Figure 21). The reconstructed 3D-EM density map revealed the predicted overall rectangular brick-like shape (Figure 4A). However, the object also showed overall torsional deformation (Figure 4D), and the extent of this deformation was due to additional T. 17 This was more pronounced than predicted based on previous analyses of similar brick-like objects lacking this characteristic. Perhaps the added T increases the flexibility of the crossover region, which may influence the helical packing shape. We determined the chirality of the torsional deformation to be right-handed using tilted series of tomography.

[0118] Secondly, we collected single-particle cryoEM data of the brick-like object after UV irradiation (Figure 22). The reconstructed 3D-EM density map again revealed the overall rectangular brick-like shape (Figure 4B). After irradiation, the overall right-handed twist was significantly reduced (Figure 4D). This reduction in twist is thought to be due to the generation of additional covalent bonds at the crossover sites, which reduces the flexibility of the junctions and realigns the helix into a shape closer to the default honeycomb packing design. Previously, Chen and collaborators irradiated a rectangle of monolayer DNA origami with UV to investigate radiation damage and observed a flattening effect that reduced twist. 61However, since Chen and his collaborators' samples were not specifically designed to contain thymidine-thymidine crosslinking sites, the mechanism leading to twist removal may differ from that in our samples.

[0119] Thirdly, we obtained single-particle cryoEM data from irradiated brick-like objects after they were dissolved in physiological ionic strength PBS buffer (Figure 23). The resulting 3D-EM density map again showed an overall rectangular brick-like shape (Figure 4C). A section-by-section comparison of these three cryoEM maps shows that the crossover internal network is preserved after irradiation and exposure to low ionic strength conditions (Figures 4E, 24). The overall aspect ratio of the cryoEM density map determined for crosslinked samples at low (physiological) ionic strength differed from that of the cryoEM density map determined at higher ionic strength in the presence of magnesium (Figures 4A-C and F). The cross-section of the object expanded by approximately 15% under physiological conditions and contracted by approximately 8% in the helical direction. This deformation is likely a result of strong electrostatic repulsion in the PBS buffer, which pushes the helices apart. Without UV irradiation, these forces would normally lead to the decomposition of the object. However, additional covalent CPD binding after UV exposure prevents unwinding and dissociation of the double helix DNA domain.

[0120] Finally, we also collected single-particle cryoEM data of brick-like variants designed with additional T-loops for interhelical bonding, before and after UV irradiation. The resulting 3D-EM density maps again revealed the predicted overall rectangular brick-like shape (Figures 25 and 26, respectively). However, the internal crossover lattice was not as well resolved as in the design variant without the additional T-loops, which we believe is because the molecular heterogeneity in these samples, caused by the presence of the additional flexible T-loops, is more pronounced.

[0121] Covalent conformational states and higher-order aggregates passing through interfaces Targeted introduction of base-paired thymidine makes it possible to covalently crosslink DNA-based mechanisms passing through the binding interface with higher-order aggregates. Here, the already described two-state switch is used. 11 The possibility of locking the conformational state using this method is demonstrated (Figure 27). In the closed state of the switch, the shape-complementary surfaces of the two horizontal bars are in direct contact, and the system is stabilized by base-pair stacking junctions (Figure 5A). The object can be switched between the two states by raising or lowering the temperature or by adding cations such as magnesium chloride.

[0122] We hypothesized that, in the closed state, terminal thymidines directly positioned at the blunt-end base-pair stacking junctions are close enough to allow CPD dimer binding upon UV irradiation. Several such TT stacking junctions were already included in the switch design. Time-resolved analysis of the effect of UV irradiation on the switch in the presence of 30 mM MgCl2 (stabilizing the closed state) shows that after 30 minutes of exposure, approximately 80% of the particles were irreversibly trapped in the closed state. This can be concluded from the band patterns of gel electrophoresis under low ionic strength conditions, where the switch is typically opened with 5 mM MgCl2 (Figure 5, B and C). Thus, CPD binding can also be formed between completely separated double-helix DNA domains that are held in close proximity.

[0123] Furthermore, we demonstrate the potential to stabilize higher-order aggregates using the aforementioned multilayer DNA origami bricks (Figure 28), which oligomerize into linear filaments with high ionic strength via shape-complementary base-pair stacking junctions. 11By default, the filaments dissolve when the ionic strength of the solution decreases again (Figure 5E). By placing TT motifs at base-pair stacking junctions, higher-order filaments can also be covalently stabilized by simple UV irradiation. As a result, the filaments do not dissociate even when exposed again to low ionic strength conditions, as is evident from TEM imaging (Figure 5E). The ability to stabilize specific conformational states or higher-order assemblies can be particularly useful for preparing vessels or mechanisms constructed from many subunits for physiological or low-ionic strength applications. Combining an internal design as shown in Figure 1 with an interfacial bonding scheme as shown in Figure 5 would allow for the generation of subunits and higher-order assemblies that can withstand a wide range of conditions.

[0124] Consideration Users of our method can easily define covalent bonding sites within DNA aggregates by creating TT sequence motifs, where the two Ts do not need to be placed within a double helix domain. Since the bacteriophage-derived scaffold strands themselves already contained multiple TT and AA motifs, the objects studied herein by default featured several sites for CPD binding formation. Novel custom scaffold sequences can be developed in the future to suppress the formation of undesirable CPD dimers upon irradiation and, if desired, to avoid the insertion of extra Ts. By design, these sequences may lack TT motifs and may feature AA motifs at regular intervals corresponding to internal bonding spacing rules for honeycomb or square lattice packing shapes. Tile brick structure 15 DNA objects without a scaffold, such as those mentioned above, can also be specially designed using sequences that selectively position TT motifs at crossovers and strand ends to enable covalent bonding by UV irradiation. Our results show that mere proximity of thymidines is sufficient to template covalent bond formation by UV irradiation. Furthermore, the thymidines do not necessarily need to be located within the double helix to form these bonds.

[0125] The cryoEM maps presented here demonstrate that DNA objects maintain their overall shape even after UV treatment. Our maps also complement the body of structural data for DNA nanotechnology, helping to understand the relationship between design details and the resulting shape. For example, we present multilayer DNA origami cryoEM maps at physiological ionic intensities. Previously, it was not possible to analyze these structures because the objects would "explode" under these conditions. Our cryoEM maps under physiological conditions reveal substantial swelling behavior, which helps to understand the electrostatic contribution to the overall shape. Future designs for physiological conditions will need to consider swelling behavior to generate the shape according to specifications. Our method supports the broad applicability of DNA-based nanotechnology, particularly to structurally complex multilayer 3D DNA objects, which may offer designers an attractive degree of freedom, although they tend to be more sensitive to environmental conditions. Due to their simplicity, programmability of sequences, and scalability, covalent bonding by UV irradiation will help pave the way for applications of DNA nanostructures under a wide range of conditions in various fields.

[0126] Example 2 Materials and methods 2.1. Folding of DNA origami objects The reaction mixtures contained scaffold DNA at a concentration of 20 nM and oligonucleotide chains at a concentration of 200 nM, respectively. The folding buffer contained 5 mM Tris, 1 mM EDTA, 5 mM NaCl (pH 8), and 20 mM MgCl2. The reaction mixtures were subjected to a thermal annealing gradient using a TETRAD (MJ Research, now Bio-Rad) thermal cycling apparatus. Oligonucleotides were purchased from Eurofins MWG (Ebersberg, Germany). The 213 oligos used to create the "brick-like object having TT motifs 1-3" are shown in the array list as sequence numbers 1-213. The 176 oligos used to create the "brick-like object having TT motifs 1-4" are shown in the sequence list as sequence numbers 214-389. The 159 oligos used to create the "pointer" objects are shown in the array list as array keys 390-548. The 206 oligos used to create the "switch" targets are shown in the array list as sequence numbers 549-754. The 211 oligos used to create the "polymerization brick" object are shown in the sequence list as sequence numbers 755-965.

[0127] The table below shows the folding incline used to assemble the object described in this test. [Table 1]

[0128] 2.2 Purification and Concentration of DNA Origami Objects After the folding reaction, all reaction products were purified using a single PEG precipitation. 63 The obtained pellet was dissolved in a folding buffer containing 5 mM MgCl2 (5 mM Tris, 1 mM EDTA, 5 mM NaCl). The final volume was selected to obtain a monomer concentration of 100 nM. The sample was equilibrated overnight in a shaking incubator (Eppendorf Thermomix comfort) at 30°C and 450 rpm. All procedures were performed as described above. 64 .

[0129] 2.3. Ultraviolet irradiation For UV irradiation, a 300W xenon light source (Asahi Spectra MAX-303) equipped with a high transmittance band filter (Asahi Spectra XAQA310) centered at 310nm was used. The light source was connected to the sample by placing it directly on top of the 0.65 ml reaction tube using a light guide (Asahi Spectra). Unless otherwise specified, brick-like samples were irradiated for 135 minutes, pointer samples for 120 minutes, and polymerizing brick samples for 30 minutes. Unless otherwise specified, samples were irradiated in a folded buffer containing 30 mM MgCl2 (5 mM Tris, 1 mM EDTA, 5 mM NaCl).

[0130] 2.4. Ultrafiltration and buffer exchange for concentration All samples (crosslinked and uncrosslinked) were subjected to three ultrafiltrations (500 μl of Amicon Ultra with a 100 kJ cutoff). Ultrafiltration was performed at 20°C and 7 kJ using relative centrifugal force (Eppendorf 5424R). The buffer was replaced with folded buffer (containing 5 mM Tris, 1 mM EDTA, 5 mM NaCl; 5 mM MgCl2), PBS, or twice-distilled water with 300, 150, 100, 50, 25, or 0 mM NaCl added. Samples used for cryo-electron microscopy were concentrated to 1,000 nM.

[0131] 2.5. Gel electrophoresis of DNA origami specimens Unless otherwise specified, samples were electrophoresed on a 2.0% agarose gel containing 0.5x trisborate-EDTA and 5 mM MgCl2 in a gel box immersed in a water or ice bath at a 90V bias voltage for approximately 2 hours. Samples were loaded onto the gel at a monomer concentration of approximately 5 nM. The electrophoresed agarose gels were scanned at a resolution of 25 μm / pixel using a Typhoon FLA 9500 laser scanner (GE Healthcare). The resulting 16-bit TIFF images were analyzed using ImageJ 1.440.

[0132] 2.6. Negative-stained transmission electron microscopy (TEM): Data preparation, acquisition, and processing Samples were adsorbed onto glow discharge, collodion-supported, carbon-coated (10 nm) Cu400 TEM grids (in-house manufactured) and stained with a 2% uranyl formate aqueous solution containing 25 mM sodium hydroxide. Samples were incubated for 15–300 seconds depending on the buffer / solvent used. Samples dissolved in solvents containing low concentrations of positively charged ions were given higher monomer concentrations (50 nM) and longer incubation times. Data were acquired using magnifications from 10,000x to 30,000x. Imaging was performed using various microscopes. See the table below. [Table 2]

[0133] The TEM micrographs used in the figures were filtered to remove long-range staining gradients, and the contrast was automatically leveled (Adobe Photoshop CS6). For 2D image processing, a library of individual particle micrographs was created by particle picking using the RELION-2 picking routine. 65 The average 2D particle microscope images were created using RELION-2. 65 Typically, approximately 2,000 individual particles were averaged out.

[0134] 2.7. Cryo-electron microscopy: Data preparation, acquisition, and processing For brick-like objects containing TT motifs 1-3, concentrations of 700 nM to 850 nM were used. Samples were applied to C-Flat 1.2 / 1.3, 1.2 / 1.3, 2 / 1, or 2 / 2 thickness grids (Protochips). Plunge freezing was performed using an FEI Vitrobot Mark V instrument with a blotting time of 3 seconds, blotting force of -1, and drain time of 0 seconds at 95% humidity and 22°C. For brick-like objects containing TT motifs 1-4, concentrations of 560 nM to 800 nM were used. Samples were applied to C-Flat 1.2 / 1.3, 2 / 1, or 2 / 2 thickness grids. Plunge freezing was performed using an FEI Vitrobot Mark V instrument with a blotting time of 3 seconds, blotting force of -1, and drain time of 0 seconds at 95% humidity and 22°C. Automated data acquisition was performed using a Titan Krios G2 electron microscope (FEI) operating at 300kV, first with a Falcon III direct detector (FEI). EPU was used for single particles, and FEI tomography was used for tilt series acquisition. For all brick-like objects under various conditions, 15 frames, exposure times of 1.5-2 seconds, and 60 e were used. - / Å 2 A movie consisting of the total dose was recorded using a Falcon III (FEI) direct electron detection camera in split mode with a 2.3 Å enlarged pixel size and a calibrated magnification of 29,000x. A defocus value in the range of -1 to -3 μm was used. The recorded video was processed using MotionCor2. 67 Motion correction is performed, followed by setting the contrast transfer function parameters to CTFFIND4.1 68 It is estimated that all subsequent processing steps are performed using RELION-2.1 65,69This was carried out. For each dataset, the automatic picking criterion was calculated from approximately 5,000 manually selected particles. Using the selected particles, multiple rounds of 2D classification without a criterion were performed. The best 2D class average judged by visual inspection was selected. The initial model was created from the bild file created by CanDo. After performing 3D classification multiple times, the class showing the most features was selected for 3D automatic refinement, and subsequently, post-processing was performed to sharpen the refined map using various manually selected B factors. The cryotomograms for verification of the twist direction were obtained using a defocus value of -3 μm at a calibrated magnification of 29,000× corresponding to an enlarged pixel size of 2.3 Å in FEI tomography. The session was set as a bidirectional tilt in 2° increments up to 50°, and the dose per image was about 2e - / Å 2 was set. The obtained tilt series was processed with the IMOD4.9 routine 70 .

[0135] 2.8. Additional Experiments 2.8.1 Experiments Leading to the Results Shown in Figure 2 The sample was folded and PEG-purified, and the MgCl2 concentration was adjusted to 30 mM. After ultraviolet irradiation, ultracentrifugation was used to exchange the buffer with the target buffer / solvent. Before gel electrophoresis, the sample was incubated at room temperature for about 2 - 3 hours. Samples for temperature screening were incubated at the indicated temperatures for 30 minutes. Samples for negative staining TEM were prepared at a monomer concentration of 50 nM and incubated on the grid for 3 - 5 minutes.

[0136] 2.8.2 Experiments Leading to the Results Shown in Figure 3. In Figure 3B, stability screening in folded buffer (5 mM MgCl2) supplemented with 10% fetal bovine serum (not heat-inactivated, Gibco®, A3160801, Thermo Fisher Scientific) was performed at 37 °C for the indicated times at a monomer concentration of 20 nM. The samples were frozen in liquid nitrogen and analyzed using agarose gel electrophoresis. In Figure 3C, all nucleases were purchased from NewEngland Biolabs and used at a concentration of 100 U / mL in the manufacturer's buffer solution. The sample (10 nM) was incubated at 37°C for 24 hours. In Figure 3D, the time course of stability against DNaseI nuclease digestion was examined at 37°C with a monomer concentration of 10 nM in the supplied DNaseI buffer.

[0137] 2.8.3 Experiments leading to the results shown in Figure 5: In Figure 5, the irradiation time screening of the switch was performed using a triple measurement method. The irradiation dose was 25 μl at a monomer concentration of 5 nM. In the analysis of the gel shown in Figure 5B, we calculated the ratio of bands containing closed particles to bands containing both open and closed particles. The grayscale values ​​for each band were obtained by integration. In Figure 5C, the data points represent the mean, and the error bars represent the standard deviation of the three independent experiments. For filament assembly, the monomer was folded and purified using PEG. The pellet was dissolved in folding buffer (5 mM MgCl2) to obtain a monomer concentration of 100 nM. After equilibration, the MgCl2 concentration was adjusted to 20 mM, and the sample was incubated in TETRAD at 40°C for 3 days to obtain filaments. A portion of the sample was irradiated at 310 nm for 30 minutes. The MgCl2 concentration was reduced to 5 mM by adding EDTA.

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[0139] The present invention is not limited in scope by the specific embodiments described herein. In fact, in addition to what is described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0140] All patents, applications, publications, test methods, documents, and other cited materials referenced herein are incorporated herein by reference to the extent permitted under the respective patent laws.

Claims

1. A method for enhancing the stability of a nucleic acid nanostructure that does not exist in nature, wherein the nanostructure comprises at least one single-stranded nucleic acid sequence bound to at least two non-adjacent sequence stretches present on one or more complementary nucleic acid sequences, the method comprising the step of exposing the nucleic acid nanostructure to ultraviolet irradiation, wherein the ultraviolet irradiation is performed using ultraviolet light with a wavelength in the range of 250 nm to 350 nm, the step of exposing the nucleic acid nanostructure to ultraviolet irradiation results in the formation of at least one chemical bond between two pyrimidine nucleotides, at least one of the two pyrimidine nucleotides is not part of a complementary nucleotide pair contained in a double-helix substructure, and the at least one chemical bond is a bridge between the single-stranded nucleic acid sequence and one 3' and / or 5' pyrimidine residue of the complementary nucleic acid sequence, the resulting structure being a cyclobutanepyrimidine dimer.

2. The method according to claim 1, wherein the nucleic acid nanostructure that does not exist in nature comprises a double helix substructure consisting of 10 to 5000 complementary nucleotide pairs, the double helix substructure can be connected to an adjacent double helix substructure every 7, 8, or 9 bases, and one or more single-stranded oligonucleotides forming the double helix substructure are part of the same or at least two different double helix substructures.

3. The method according to claim 2, wherein the connection between the double helix substructures results in a honeycomb, square, or hexagonal packing shape, or a combination thereof.

4. The method according to claim 2 or 3, wherein at least 85% of the single-stranded oligonucleotides forming the double-helical substructure are part of at least two different double-helical substructures.

5. A method according to any one of claims 1 to 4, wherein the nucleic acid nanostructure comprises at least one copy of a first single-stranded polynucleotide and a set of second single-stranded polynucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, and each of the n core sequences comprises (i), (ii), and (iii): (i) A sequence complementary to the region on the first single-stranded polynucleotide, wherein the region on the first single-stranded polynucleotide complementary to the p-th core sequence is a sequence that is not adjacent to the regions on the first single-stranded polynucleotide complementary to the (p-1)-th and (p+1)-th core sequences, where p is an integer selected from 2 to (n-1). (ii) Pyrimidine nucleotide stretch P at the 3' end m , and (iii) 5'-terminated pyrimidine nucleotide stretch P m The above method, wherein P represents a pyrimidine, each m is an integer independently selected from the range of 0 to 40, the total number of m is at least 2, and each P is independently selected from thymidine and cytosine residues.

6. Each of the n core arrays is (iv) Pyrimidine nucleotide stretch P m The method according to claim 5, further comprising the insertion of one or more of the following.

7. The method according to claim 5 or 6, wherein the first single-stranded polynucleotide comprises at least 100 nucleotides.

8. The method according to claim 7, wherein the first single-stranded polynucleotide has at least 70% sequence identity with respect to the DNA of the filamentous bacteriophage.

9. The method according to claim 8, wherein the fibrous bacteriophage is M13.

10. The method according to claim 9, wherein the fibrous bacteriophage is M13mp18.

11. A method according to any one of claims 1 to 4, wherein the nucleic acid nanostructure comprises a set of single-stranded oligonucleotides, each of which consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, and each of the n core sequences comprises (i), (ii), and (iii): (i) A sequence complementary to the sequence of another member of the single-stranded polynucleotide of the set, wherein the region on the other member complementary to the p-th core sequence is not adjacent to the regions complementary to the (p-1)-th and (p+1)-th core sequences, where p is an integer selected from 2 to (n-1). (ii) Pyrimidine nucleotide stretch P at the 3' end m , and (iii) 5'-terminated pyrimidine nucleotide stretch P m The above method, wherein P represents a pyrimidine, each m is an integer independently selected from the range of 0 to 40, the total number of m is at least 2, and each P is independently selected from thymidine and cytosine residues.

12. Each of the n core arrays is (iv) Pyrimidine nucleotide stretch P m The method according to claim 11, further comprising the insertion of one or more of the following.

13. The method according to any one of claims 5 to 10, wherein for each of the pyrimidine nucleotide stretches at the 3' and 5' ends, m is either 0 or 1 and P is a thymidine residue.

14. The method according to any one of claims 5 to 13, wherein each of the core sequences consists of x nucleotides, where x is independently selected from integers that are multiples of 7, 8, or 16.

15. The method according to any one of claims 1 to 14, wherein the ultraviolet irradiation is performed in Tris buffer at a temperature range of about 0 to about 25, using the following parameters (a sample volume of about 5 to 2,000 μl, a nucleic acid nanostructure concentration of about 1 to 500 nM in the sample), using a liquid guide to connect the light beam to the sample (the distance between the solution surface of the sample and the end of the light guide is less than about 5 cm), using a xenon light source (the distance between the solution surface of the sample and the end of the light guide is less than about 5 cm), and irradiating the sample at about 1 to 10 mW / cm². 2 The above method is performed by exposing the subject to ultraviolet irradiation at a certain ultraviolet intensity for approximately 1 to 6 hours.

16. A kit for creating nucleic acid nanostructures comprising at least one copy of a first single-stranded polynucleotide and a set of single-stranded polynucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, where each of the n core sequences comprises (i), (ii), and (iii): (i) A sequence complementary to the region on the first single-stranded polynucleotide, wherein the region complementary to the p-th core sequence is not adjacent to the regions complementary to the (p-1)-th and (p+1)-th core sequences, where p is an integer selected from 2 to (n-1). (ii) Pyrimidine nucleotide stretch P at the 3' end m , and (iii) 5'-terminal pyrimidine nucleotide stretch P m , where each m is an integer independently selected from the range of 0 to 40, each P is independently selected from thymidine and cytosine residues, the total number of m is at least 2, and the 3'-terminal pyrimidine nucleotide stretch P m and the 5'-terminal pyrimidine nucleotide stretch P m one or more pyrimidines of which are used for the formation of cyclobutane pyrimidine dimers, the above kit.

17. Each of the n core arrays is (iv) Pyrimidine nucleotide stretch P m The kit according to claim 16, further comprising the insertion of one or more of the following.

18. A kit for creating nucleic acid nanostructures comprising a set of single-stranded oligonucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer independently selected from 1 to 40, and each of the n core sequences comprises (i), (ii), and (iii): (i) A sequence complementary to the sequence of another member of the set of single-stranded polynucleotides, wherein the region on the other member complementary to the p-th core sequence is not adjacent to the regions complementary to the (p-1)-th and (p+1)-th core sequences, where p is an integer selected from 2 to (n-1). (ii) Pyrimidine nucleotide stretch P at the 3' end m , and (iii) 5'-terminated pyrimidine nucleotide stretch P m Here, each m is an integer independently selected from the range of 0 to 40, each P is independently selected from thymidine and cytosine residues, the total number of m is at least 2, and the pyrimidine nucleotide stretch P at the 3' end m and the 5'-terminus pyrimidine nucleotide stretch P m The above kit, wherein one or more pyrimidines are used to form a cyclobutanepyrimidine dimer.

19. Each of the n core arrays is (iv) Pyrimidine nucleotide stretch P m The kit according to claim 18, further comprising the insertion of one or more of the following.

20. Stretch P of the pyrimidine nucleotide at the 3' and 5' ends m The kit according to any one of claims 16 to 19, wherein for each of the following, m is 0 or 1 and P is a thymidine residue.

21. A nucleic acid nanostructure comprising at least one copy of a first single-stranded polynucleotide and a set of single-stranded polynucleotides, wherein each single-stranded polynucleotide consists of an n-specific sequence comprising n core sequences, where n is an integer selected from 1 to 40, and each of the n core sequences comprises (i), (ii), and (iii): (i) A sequence complementary to the region on the first single-stranded polynucleotide, wherein the region complementary to the p-th core sequence is not adjacent to the regions complementary to the (p-1)-th and (p+1)-th core sequences, where p is an integer selected from 2 to (n-1). (ii) Pyrimidine nucleotide stretch P at the 3' end m , and (iii) 5'-terminated pyrimidine nucleotide stretch P m Here, each m is an integer independently selected from the range of 0 to 40, the total number of m is at least 2, and each P is independently selected from thymidine and cytosine residues, and the 3' terminal pyrimidine nucleotide stretch P m and the 5'-terminus pyrimidine nucleotide stretch P m The nucleic acid nanostructure wherein one or more pyrimidines are used to form a cyclobutanepyrimidine dimer.

22. Each of the n core arrays is (iv) Pyrimidine nucleotide stretch P m The nucleic acid nanostructure according to claim 21, further comprising one or more insertions.

23. The pyrimidine nucleotide P at the 3' and 5' ends m The nucleic acid nanostructure according to claim 21 or 22, wherein for each of the above, m is either 0 or 1, and P is a thymidine residue.

24. A nucleic acid nanostructure according to any one of claims 21 to 23, comprising one or more ultraviolet-induced bridges between spatially adjacent thymidine and / or cytosine residues, wherein the resulting structure is a cyclobutanepyrimidine dimer.

25. The one or more bridges are located at the 3' end of a single-stranded oligonucleotide or core sequence, or at the 5' end of a single-stranded oligonucleotide or core sequence. m Between thymidine and / or cytosine residues contained in the stretch, and / or any of the aforementioned P m A nucleic acid nanostructure according to claim 24, wherein one of the inserts is a thymidine.

26. A nucleic acid nanostructure according to any one of claims 24 or 25, wherein one or more of the bridges are intrahelical bridges between 3' and 5' terminal thymidine or cytosine residues of a core sequence which is part of two adjacent single-stranded oligonucleotides or the same or different double-helix substructures of the nucleic acid nanostructure.

27. A nucleic acid nanostructure according to any one of claims 24 to 26, wherein one or more of the bridges are interhelical bridges between thymidine or cytosine residues contained in a single-stranded oligonucleotide or a portion of the same or different double-helix substructure of the nucleic acid nanostructure, particularly between two thymidine residues contained in two of the optional inserts.

28. A complex nucleic acid nanostructure resulting from the assembly of two or more nucleic acid nanostructures according to any one of claims 24 to 27.

29. The complex nucleic acid nanostructure according to claim 28, wherein the aggregate comprises one or more ultraviolet-inducible bridges between two or more nucleic acid nanostructures according to any one of claims 24 to 27, and the ultraviolet-inducible bridges form a cyclobutanepyrimidine dimer.

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