WO2021224427A1 - Procédés de fabrication de nanostructures d'acide nucléique - Google Patents

Procédés de fabrication de nanostructures d'acide nucléique Download PDF

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WO2021224427A1
WO2021224427A1 PCT/EP2021/062073 EP2021062073W WO2021224427A1 WO 2021224427 A1 WO2021224427 A1 WO 2021224427A1 EP 2021062073 W EP2021062073 W EP 2021062073W WO 2021224427 A1 WO2021224427 A1 WO 2021224427A1
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nucleic acid
nanostructure
molecule
population
enzyme
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Ferdinando RANDISI
Jonathan BURNS
Alessandro Ceccarelli
Robert OPPENHEIMER
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FabricNano Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to methods of making nucleic acid nanostructures, in particular DNA nanostructures.
  • the present invention relates to the generation of nucleic acid nanostructures to a predetermined and non-repeating design.
  • These nanostructures can be put to numerous end uses, of particular interest according to the present invention is as a platform for immobilisation of proteins or other molecules, in particular reactive molecules such as enzymes so that the nanostructure with immobilised molecules can act as a bioreactor, e.g. in the biosynthesis of a target molecule which may be of therapeutic or industrial use.
  • DNA molecules possess a distinct set of mechanical, physical and chemical properties. They can be rigid or flexible and the basic building block is very small, about 2x0.34 nm per base pair. DNA is generally stable, non-toxic, water soluble and available at high purity.
  • Watson- Crick rules of base pairing mean that regions of complementary nucleotide sequence (called ‘domains’) will form non-covalent bonds under hybridising conditions and so DNA molecules can self-assemble in a predictable manner.
  • DNA and RNA Self-assembly of nucleic acids
  • Structures have been designed by building sequence complementarity into DNA strands in such a manner that by pairing up complementary domains, the strands self-organize into a prescribed target structure. From this basic principle, researchers have created diverse synthetic nucleic acid structures such as lattices, ribbons, tubes and finite two-dimensional (2D) and 3D objects with defined shapes.
  • nucleic acid structures promise diverse applications; researchers are using nucleic acid structures and devices to direct spatial arrangement of functional molecules, facilitate protein structure determination, develop bioimaging probes, study single-molecule biophysics, and modulate biosynthetic and cell-signalling pathways.
  • An effective method for assembling nanoscale 2D and 3D shapes is DNA origami, in which a long “scaffold” strand (often a viral genomic DNA) is folded in a predesigned shape via interactions with many short “staple” strands, all of which are single-stranded (e.g. US 8,501,923).
  • DNA origami An alternative to DNA origami is the use of a number of single-stranded oligonucleotides of similar size but unique sequence (“bricks”) which each bind to a small number of neighbours to provide modular self-assembly of nucleic acid nanostructures (e.g. Ke et al. in Science (2012), vol 330, p 1177-1183 and WO 2014/018675).
  • Another alternative is the use of a single strand containing self complementary domains so that it folds upon itself into the target nanostructure without requiring additional strands (e.g. Qi et al in Nature Communications, 9, Article number 4579 (2016)).
  • the final structure is predetermined and enabled through the specific and predictable formation of domains (regions) of base-pairing which result in a 2D/3D structure.
  • the nanostructures are not based on a repeating pattern and contain a large number (e.g. 30 or more, typically 50 or more) of unique domains of base-paired nucleic acid. All known methods of this type are performed with nucleic acid at a concentration in the fairly low nM range.
  • the present inventors believe this may be due to the widely-held belief that the formation of DNA nanostructures would be corrupted at higher concentrations, either misfolding due to the increased propensity for intermolecular reactions over intramolecular reactions or becoming difficult to handle by increased viscosity or DNA precipitation.
  • concentrations used in the art are also consistent with the concentration of DNA in vivo which is typically in the nM range.
  • Nucleic acid structures of the hydrogel type where a small number of different oligonucleotide species combine in repeating motifs, have been generated at much higher concentrations. This is because in such systems there are only a small number of unique binding domains. In hydrogel systems, there are typically just 3 or 4 different domains of 20 bp that form the junctions in the structure. Furthermore, hydrogels do not have a defined target conformation. There are a myriad of possible conformations and topologies where all domains of the DNA strands are fully hybridised, and it is not possible to choose one of these conformations at the time of design. In contrast, when a DNA nanostructure is folded, there is typically only a single target structure which self-assembles.
  • WO2012/151537 describes the production of a pre-determined DNA nanostructure, assembled in a step-wise manner from 8 repetitive tile motifs. Unlike the single-step method of the present invention, the nanostructure of WO2012/151537 is constructed using a more cumbersome approach involving multiple separate annealing steps. Each tile is self-assembled separately first, before the larger final structure is made from a mixture of the tiles.
  • RNA nanostructure comprising 3 core RNA strands which each have 2 two binding domains to form an overall repeating lattice structure. This RNA lattice has only 6 unique binding domains.
  • the present inventors have surprisingly shown that complex multi-domain nanostructures can be made using nucleic acid at higher concentrations. This opens up the possibility for more efficient bioreactor type structures and enzyme assays and generally enhances production of nucleic acid based nanomaterials.
  • enzyme activity is improved by being able to immobilise enzymes on DNA nanostructures that have been folded at high concentration.
  • the present invention provides a method of making a predefined nucleic acid nanostructure, which method comprises combining in a reaction mixture a population of (preferably single-stranded) nucleic acid molecules which population, taken together, is capable of forming at least 30 unique domains of base-paired nucleic acid, wherein the concentration of each nucleic acid molecule within said population is at least 500 nM.
  • the present invention can be considered a “single-step” method as it can be performed in a single reaction vessel, with no transfer of intermediate molecules to a second reaction mixture or vessel.
  • all the domains which form the nanostructure are formed in a single process; the starting population of nucleic acid molecules is combined in one reaction mixture and the nanostructure is formed directly from that starting population of nucleic acids in said reaction mixture (in the reaction vessel).
  • the population of nucleic acid molecules is preferably capable of forming at least 40, at least 60, at least 80 or at least 100 unique domains. In the methods of the present invention these unique domains are formed as the nanostructure is made.
  • the starting population of nucleic acid molecules in the methods of the invention is preferably single-stranded but may include or consist of partially or fully double-stranded molecules.
  • the nanostructure is predefined in that the population of nucleic acid molecules has been designed and/or selected to generate a single target 2D or 3D nanostructure when the reaction mixture is exposed to conditions which enable self- assembly (usually hybridisation - the formation of Watson-Crick base pairs through hydrogen bonding) to take place.
  • the target nanostructure is predetermined and rationally designed.
  • the nanostructure is predefined such that the same population of nucleic acid molecules will always generate the same nanostructure (allowing for minor and tolerable levels of misfolding due to off-target hybridisation, e.g. less than 25%, preferably ⁇ 20%, more preferably ⁇ 10% of nanostructures generated are other than the target structure).
  • the target nanostructure is designed before production in its gross, 2D/3D form.
  • caDNAno is a computer-aided design tool which can help visualise and design the crossover junctions between DNA strands.
  • CanDo and oxDNA are further available tools for the design and checking process.
  • the output of this design phase may conveniently be a set of DNA strands.
  • the predefined nanostructure can be of any 2D or 3D shape and may not have any lines of symmetry or involve any repeating pattern. In other embodiments the predefined nanostructure can have one or more lines of symmetry. It is also possible to design and construct DNA nanostructures which are nanomachines and capable of motion (e.g. as described by Marras et al. in PNAS January 20, 2015 112 (3) 713-718).
  • the nanostructure may preferably be in the form of a tile which is typically two dimensional and may conveniently be square or rectangular. Further preferred nanostructures are known in the art as “meshes”, these may be 2D or 3D and designed with voids within the structure which are larger in at least two dimensions than the cross section of a nucleic acid duplex.
  • a mesh may comprise or consist of polyhedral or triangulated regions of nucleic acid structure. Suitable mesh-type structures are disclosed, for example, by Jun et al. Sci. Adv. 2019; 5:eaav0655.
  • nucleic acid synthesis Any methods known in the art for nucleic acid synthesis may be employed, whether in vivo, in vitro replication or solid-phase synthesis etc. Cell culture techniques may be preferred for generation of scaffold molecules (Bellot et al. Nature Protocols (2013) Vol. 8 No. 4 pp 755-770) and sequences of less than 50 nucleotides may preferably be produced using solid-phase synthesis or by in vitro replication with polymerases.
  • the domains (regions) of base paired nucleic acid are generally and preferably intermolecular, but intramolecular domains are also possible. Each individual nucleic acid molecule will typically take part in the formation of at least 2 domains. The domains join the different nucleic acid molecules together and result in the formation of the predefined 2D or 3D nanostructure.
  • Base pairing follows strict rules, e.g. amongst deoxynucleotides, A binds to T and C binds to G (one letter code). As a result, the two sequences which form each domain can be designed/predicted in advance.
  • the certainty of base pairing means the 2D/3D structure of the nanostructure is predictable and reliable and can be modelled in silico before manufacture.
  • Base pairing rules may be more complex in some situations (e.g., in RNA, or involving DNA secondary structures such as G- quadruplexes or i-motifs), yet still enable prediction and control of self-assembly.
  • the domains of base-paired nucleic acid will typically be 10-40 bp in length more usually 15-30 bp in length, e.g. about 20 bp in length.
  • the number of domains will typically be in excess of 50, 75, 100, 200, 300, 400 or 500. It may be several thousand, e.g. in excess of 1,000, 2,000 or 3,000 but possibly in excess of 10,000, 50,000 or 100,000.
  • Each species of nucleic acid molecule within the reaction mixture which participates in formation of the nanostructure is present at a concentration of at least 500 nM, more preferably at least 750 nM, most preferably at least about 1 mM (i.e. in excess of 900 nM) but may be as high as 2, 5, 10, 25 or 50 pM or more. Suitable ranges include 500 nM to 100 pM, e.g. 500 nM to 5 pM, 10 pM or 20 pM.
  • Each species of nucleic acid within the reaction mixture may be present at the same concentration or certain members of the nucleic acid population may be present at higher concentrations, for example the staple molecules may be present in excess of the scaffold molecule.
  • the total concentration of nucleic acids within the reaction mixture is less than 5mM, more preferably less than 3 mM, most preferably less than 1.5 mM or less than 1.2 mM.
  • the total concentration of nucleic acid in the reaction mixture is around 0.1-1.1 or 0.1-1.2mM, more preferably 0.5-1.1 or 0.5-1.2mM.
  • a total nucleic acid concentration of approximately 1.1 mM would correspond to a reaction mixture comprising 1.2pM of M13mp18 scaffold and 6 pM each of 182 'staple' oligonucleotide.
  • the population of (preferably single-stranded) nucleic acid molecules will contain at least 2 individual species of nucleic acid molecule, i.e. at least 2 different nucleic acid molecules in terms of sequence.
  • the number of different species will be greater than 10, greater than 30, 50 or 80 possibly greater than 100 or 200.
  • Some or all of the species will be synthetic, in that they are not naturally occurring.
  • the population of nucleic acid molecules and the resulting nanostructures may be made of natural or synthetic nucleic acids (DNA, RNA, PNA, LNA, HNA, etc.) or a mixture thereof (e.g. DNA plus RNA hybrids, etc.), preferably the nucleic acid molecules are DNA molecules.
  • the reaction mixture will typically comprise a buffer solution and suitable buffer solutions are well known in the art and described, for example, in Bellot et al. supra (which also contains other suitable technical teaching for generation of nanostructures).
  • Buffer components may include EDTA, acetic acid and Tris and the reaction mixture will preferably contain magnesium ions, e.g. at a concentration of 2- 25 mM, preferably 2-20 mM or 10-15 mM, of a suitable salt, e.g. MgCh.
  • Conditions are known in the art and disclosed in the Examples hereto and, for example, in Bellot et al. supra. Conditions will typically include an initial heating step to a temperature in excess of 70°C, preferably 80°C or more, preferably around 95°C, e.g. for 1-10 minutes. The heating step is then followed by cooling, the cooling may take from 1 minute to 1 week but will typically take 1 to 100 hours, e.g. 2 to 12 or 4 to 8 hours.
  • hybridisation could be achieved by removing denaturing agents, e.g. by dialysis of urea or formamide (Jungmann et al. Journal of the American Chemical Society (2008) 130(31), 10062- 10063).
  • an optional purification step to isolate the nanostructure from excess oligonucleotides, for example using size-exclusion chromatography, size-exclusion filtration or PEG purification.
  • Assembly of the nanostructure may be confirmed by any of a number of conventional techniques, e.g. electrophoresis, atomic force microscopy, electrophoresis or electron microscopy.
  • the nanostructure may be of the DNA origami type (Douglas et al. (2007) PNAS, vol. 104, no. 16).
  • the scaffold molecule In such structures there is a single long polynucleotide, often referred to as the scaffold molecule.
  • the remaining members of the population are oligonucleotides, usually referred to as “staples”, which have at least two (typically 2 or 3) regions of complementarity with corresponding regions of the scaffold molecule.
  • the staples effectively pull the scaffold into the predetermined 2D or 3D structure by bringing regions of the scaffold together which are (generally) not neighbouring (adjacent) in terms of the nucleotide sequence of the scaffold .
  • the population of nucleic acid molecules may comprise or consist of:
  • nucleic acid molecule which is single-stranded and has at least 800 nucleotides, preferably 1,000-10,000 nucleotides.
  • This molecule may be a naturally occurring, modified naturally occurring or synthetic sequence.
  • it may be the 7,249 base circular ssDNA from the well-known bacteriophage M13 (known as M13mp18 - Rothermund, Nature [2006], 440, 297-302) or derivatives thereof, e.g. the 59 base extended 7,308 base sequence (Douglas et al. Nature [2009] 459, 414-418); and
  • oligonucleotides of different sequences typically at least 30, 50, 80, 100 or 150 different oligonucleotides, e.g. around 200 (180-220) oligonucleotides.
  • the oligonucleotides are typically 20- 100 nucleotides in length, preferably 30-60 nucleotides in length.
  • the design of the sequences of the staples determines how the scaffold is folded into a 2D/3D nanostructure.
  • the nucleic acid of (i) is known as the scaffold and of (ii) as the staples.
  • the number of unique domains will typically be in excess of 200, preferably in excess of 300, 400 or 500, e.g. 300-1000, preferably 500-800 domains.
  • Each staple will typically take part in the formation of 2 or 3 domains with regions of the scaffold molecule.
  • the scaffold molecule will take part in the formation of many domains along its entire length, typically in excess of 300, 400 or 500, e.g. 300-1000, preferably 500-800 domains.
  • the oligonucleotides (ii) may be present in excess, preferably at 1.5-5 times, e.g. 2-4, 2-3, 2 or 3 times the concentration of the nucleic acid molecule (i).
  • the nanostructure may be of the DNA brick type (Ke et al. supra).
  • the population of nucleic acid molecules may comprise or consist of: a plurality of oligonucleotides of different sequence, typically 50-1000, preferably 100-1000, e.g. 300-600 different oligonucleotides.
  • the oligonucleotides are typically 20-100, preferably 30-60, more preferably 30-40 nucleotides in length, each oligonucleotide participating in the formation of 2 to 4, preferably 4 domains with other oligonucleotides. All of these oligonucleotides (bricks) may be artificial sequences.
  • the entire nanostructure may be folded from a single nucleic acid.
  • the number of domains is determined by the length of the nucleic acid, which is typically between 40-10,000 base pairs, or 2-500 domains.
  • oligonucleotides are fragments of what is referred to herein as a scaffold molecule, e.g. a bacteriophage (or bacteriophage derived) sequence, which has been divided into oligonucleotides referred to herein as the “chopped scaffold”.
  • This set of “chopped scaffold” oligonucleotides may then be combined with a second set of oligonucleotides, e.g. the “staples” referred to herein, in order to form a nanostructure.
  • a scaffold-type sequence may be chopped into sub-sequences of different lengths or of the same or approximately the same length.
  • Nucleic acid nanostructures and preparations containing these nanostructures produced by the methods of the invention are a further aspect of the present invention.
  • Preferred embodiments of features of the nanostructures are discussed herein in the context of the methods of the invention and apply, mutatis mutandis, to this aspect of the invention.
  • the present invention provides a preparation comprising a nucleic acid nanostructure, said nanostructure comprising at least 30 unique domains of base-paired nucleic acid and preferably at least 10 different nucleic acid molecules, wherein the nanostructure is present in said preparation at a concentration of at least 500 nM.
  • the preparation is typically aqueous and may comprise a buffer solution, e.g., as described elsewhere herein.
  • nucleic acid molecule or sequence is defined by the nucleotides covalently bonded (one to another) within it.
  • nucleic acid molecules of use in the methods, structures and preparations of the invention are linear but may be branched or circular.
  • the nanostructures of the invention are not formed through the generation of new covalent bonding but by folding of the starting molecules into predefined 2D or 3D shapes, typically through hydrogen bonding, preferably through hybridisation of single-stranded nucleic acid molecules to form domains of base pairing.
  • the present invention provides a method of making a nucleic acid nanostructure, said nanostructure comprising at least 30 unique domains of base-paired nucleic acid and at least 10 different nucleic acid molecules, which method comprises combining in a reaction mixture a population of nucleic acid molecules, wherein the concentration of each nucleic acid molecule within said population is at least 500 nM.
  • a method of making a nucleic acid nanostructure comprising at least 30 unique domains of base-paired nucleic acid and at least 10 different nucleic acid molecules, which method comprises combining in a reaction mixture a population of nucleic acid molecules, wherein the concentration of each nucleic acid molecule within said population is at least 500 nM.
  • the present invention provides a method of making a nucleic acid nanostructure, which method comprises combining in a reaction mixture a population of nucleic acid molecules which population, taken together, is capable of forming at least 30 unique domains of base-paired nucleic acid, wherein the concentration of each nucleic acid molecule within said population is at least 500 nM.
  • a method of making a nucleic acid nanostructure comprises combining in a reaction mixture a population of nucleic acid molecules which population, taken together, is capable of forming at least 30 unique domains of base-paired nucleic acid, wherein the concentration of each nucleic acid molecule within said population is at least 500 nM.
  • the principle of the present invention namely high concentration folding of nucleic acid to form a predefined nanostructure, may also be employed using a single nucleic acid molecule that folds upon itself. In this scenario all domains formed are intramolecular.
  • Such systems are described, for example, by Han et al. in Science (2017) 358, 1402 and eaao2648.
  • the present invention provides a method of making a predefined nucleic acid nanostructure, said nanostructure comprising at least 30 unique domains of base-paired nucleic acid, which method comprises exposing a nucleic acid molecule, present in a reaction mixture at a concentration of at least 500 nM, to conditions suitable for self-assembly of the nanostructure from said nucleic acid molecule.
  • the entire nanostructure is formed from a single species of nucleic acid.
  • the reaction mixture contains only a single species of nucleic acid molecule.
  • the nanostructures and preparations of the present invention may be used in any scenario where a nucleic acid material or substrate is required, in particular to provide a substrate or platform which can have reactive molecules immobilised thereon.
  • Reactive molecules include an enzyme or a substrate for an enzymatic reaction or a molecule with binding affinity for a target molecule of interest (analyte), e.g. an antibody, antibody fragment or other binding partner; or a mixture of said reactive molecules.
  • the nanostructures may be used to provide a nanoreactor (bioreactor) with one or more enzymes immobilised thereon, e.g. to perform an assay function or for the production of metabolites of interest.
  • a whole enzymatic pathway may be immobilised on the nanostructure leading to an increase in efficiency as compared to multi-step enzymatic reactions occurring in solution.
  • the present invention provides a nanostructure or preparation of the invention wherein one or more reactive molecules as defined herein (e.g. enzymes) have been immobilised on the nanostructure.
  • one or more reactive molecules as defined herein e.g. enzymes
  • the present invention provides a method of making a functionalised nanostructure, said method optionally comprising a method of making a nanostructure, said method as defined herein, and comprising immobilising on a nanostructure produced by the method of the invention a reactive molecule such as an enzyme or a substrate for an enzymatic reaction or a molecule with binding affinity for a target molecule of interest (analyte), e.g. an antibody, antibody fragment or other binding partner; or a mixture of said reactive molecules.
  • a reactive molecule such as an enzyme or a substrate for an enzymatic reaction or a molecule with binding affinity for a target molecule of interest (analyte), e.g. an antibody, antibody fragment or other binding partner; or a mixture of said reactive molecules.
  • the present invention provides a method of generating a metabolite of interest or assaying for a target molecule of interest, said method comprising contacting either i) a sample which may contain said target molecule, or (ii) a precursor of said metabolite, with a nanostructure or preparation of the invention wherein one or more enzymes have been immobilised on the nanostructure.
  • a “precursor” is a substrate for one of the enzymes and is converted, directly or indirectly, into the metabolite of interest.
  • One or more enzymes refers to one or more different enzymes.
  • Figure 1 A) is a sketch of a single layer DNA tile nanostructure; each cylinder represents a dsDNA duplex, a set of duplexes are arranged in parallel to form the tile; B) is a sketch of a DNA mesh nanostructure, again each cylinder represents a dsDNA duplex.
  • Figure 2 shows screenshots from the CAD tool caDNAno for A) a DNA nanostructure composed of a long ssDNA scaffold and many short ssDNA oligonucleotides (DNA origami) and B) a DNA nanostructure composed of many short ssDNA oligonucleotides (i.e. , a mixture of chopped scaffold and staple oligonucleotides).
  • Figure 3 shows images from atomic force microscopy (AFM) of DNA tiles;
  • A) is a DNA origami tile (scaffold of 7249 nt) at a folding DNA concentration of 0.5 mM, the image taken after size-exclusion chromatography;
  • B) is a chopped scaffold DNA tile (in which the scaffold molecule has been broken down into 32 nt oligonucleotides) at a folding DNA concentration of 10 pM and with no purification;
  • C) is the same as B) but at a folding DNA concentration of 1 pM;
  • D) is the same as B) but at a folding DNA concentration of 0.1 pM.
  • Figure 4 shows images from atomic force microscopy (AFM) of DNA mesh;
  • A) is a DNA origami mesh (scaffold of 7249 nt) at a folding DNA concentration of 0.05 pM;
  • B) is a chopped scaffold DNA mesh (in which the scaffold molecule has been broken down into 200 nt oligonucleotides) at a folding DNA concentration of 0.05 pM with the image taken after size-exclusion chromatography.
  • Figure 5 shows images from atomic force microscopy (AFM) of a design D5 DNA origami tile (scaffold of 7249 nt) at a folding DNA concentration of A) 0.1 pM;
  • the scaffold sequence was a circular ssDNA M13mp18 bacteriophage genome (Tilibit Type p7249, 2ml_ 400nM, M1-12).
  • the oligonucleotides were purchased from IDT who generate them by solid phase synthesis.
  • the oligonucleotides were purchased from IDT who generate them by solid phase synthesis. The same scaffold sequences were used but divided into oligonucleotides and then mixed with the same set of staples as for the origami method.
  • Example 1 The sequences as used in Example 1 are recited at the end of this Example.
  • DNA strands were mixed in 40 mM tris, 20 mM acetic acid, 1 mM EDTA, 12.5 mM MgCh, (pH 8.3).
  • the magnesium concentration was optimised to enable self- assembly but prevent aggregation of DNA (2-50mM is typical).
  • the DNA was heated to 95°C to eliminate native secondary structure then cooled slowly to encourage self-assembly of the nanostructure.
  • a typical annealing protocol was followed as shown in Table 1.
  • the rate of cooling can be optimised for each different nanostructure and can take between 1 minute and 1 week.
  • Nanostructures may be assembled isothermally without an annealing step.
  • the assembly of the DNA nanostructures was confirmed via agarose gels and atomic force microscopy (AFM), though other techniques are possible (e.g., automated electrophoresis or electron microscopy).
  • AFM atomic force microscopy
  • Example agarose gel protocol 1 % Agarose (Life Technologies) was dissolved in 1 * of Tris-acetate EDTA (TAE) from ultrapure deionised water and microwaved to dissolve. After agarose was dissolved and cooled, 5 pi SybrSAFE DNA stain per 100 mL was added to the agarose gel cast using the BIORAD agarose gel kit. 2 mI 50 nM DNA samples were added to 6 mI gel loading dye, before loading 5 mI of this solution on the gel. Gel was run in 1 x TAE running buffer, at 120 V, for 40 minutes, at 4 degrees, before imaging with UV light on Azure c150.
  • Example AFM protocol 1 % Agarose (Life Technologies) was dissolved in 1 * of Tris-acetate EDTA (TAE) from ultrapure deionised water and microwaved to dissolve. After agarose was dissolved and cooled, 5 pi SybrSAFE DNA stain per 100 mL was added to the agarose gel cast using the BIORAD
  • Samples were imaged in fluid using a Bruker multimode 8 AFM with tip E of an MSLN cantilever using peak force tapping mode on mica. Sample volume between 1-5 mI was added to the mica surface for sample adhesion, before adding up to 100 mI 1 x TAE, 12.5 mM MgCh. Typically between 0.5 to 3 pm 2 area was imaged with 100-150pN force and 1.5-3 Hertz.
  • the DNA nanostructures were optionally purified from any excess strands used during assembly via size-exclusion chromatography, though other techniques are possible (e.g., size-exclusion filtration, PEG precipitation, etc.).
  • the DNA origami nanostructures were annealed with an excess of oligonucleotides, while the chopped scaffold nanostructures contained an excess of free, unassembled oligonucleotides. This excess can be removed by size-exclusion chromatography with a AKTA pure 25 L.
  • a Superdex 200 Increase 10/300 GL column was used to separate DNA nanostructures from oligonucleotides at 4 °C, using a 500 mI manual injection, and a running buffer 1 x TAE pH 8.3, 12.5 mM MgCh at 0.7 mL/minute. The fractions were collected and the concentration of DNA nanostructure after purification estimated by absorbance at 260nm.
  • Example 2 origami folding at a concentration of up to 1.2uM scaffold
  • the DNA components were concentrated using the Eppendorf Concentrator plus at 30°C, V-AQ program.
  • M13 scaffold was concentrated from 400 nM until dry, and then resuspended in MilliQ water to a concentration of 8 pM.
  • the scaffold formed a gel at room temperature and was melted at 72°C for pipetting using an Eppendorf Thermomixer C.
  • a staple mastermix was prepared mixing 30 pi of each isomolar staple, each of the mixes were evaporated to a final concentration of 6 pM for each staple.
  • Three origami mixes for each design were prepared to a final scaffold concentration of 1200 nM, 300 nM, and 100 nM.
  • the staple concentration was 4.16 times higher than the scaffold (respectively 5000 nM, 1248 nM, and 416 nM).
  • Each sample was prepared to a final volume of 100 pi.
  • Each sample was prepared in a MilliQ water and Magnesium Chloride 12.5 mM buffer.
  • the Eppendorf Mastercycler X50s was used to anneal the origami with a 9 hours- long protocol programmed as following:
  • a sequence list is provided below.
  • Atomic force microscopy was performed on a Bruker Multimode 8 using Bruker MSLN-E probes. 1 pi of water-diluted or undiluted sample was deposited on a freshly cleaved mica followed by 100 mI of MgCI2 12.5 mM.
  • the sample was then analyzed using the ScanAsyst in-liquid protocol.
  • the images obtained were finally elaborated and exported using Nanoscope Analysis 1.7 software.

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Abstract

La présente invention concerne un procédé de fabrication d'une nanostructure d'acide nucléique prédéfinie, le procédé comprenant la combinaison dans un mélange réactionnel d'une population de molécules d'acide nucléique qui, pris ensemble, est capable de former au moins (30) domaines uniques d'acide nucléique appariés aux bases, la concentration de chaque molécule d'acide nucléique dans ladite population étant d'au moins (500) nM et tous les domaines uniques étant formés en une seule étape dans le mélange réactionnel. L'invention concerne en outre des nanostructures d'acide nucléique produites par les procédés de la présente invention et des préparations comprenant lesdites nanostructures. L'invention comprend également un procédé de fonctionnalisation des nanostructures de la présente invention.
PCT/EP2021/062073 2020-05-06 2021-05-06 Procédés de fabrication de nanostructures d'acide nucléique WO2021224427A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202119023D0 (en) 2021-12-24 2022-02-09 Fabricnano Ltd Immobilised enzyme

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