WO2007012807A2 - Structures d'adn - Google Patents

Structures d'adn Download PDF

Info

Publication number
WO2007012807A2
WO2007012807A2 PCT/GB2006/002606 GB2006002606W WO2007012807A2 WO 2007012807 A2 WO2007012807 A2 WO 2007012807A2 GB 2006002606 W GB2006002606 W GB 2006002606W WO 2007012807 A2 WO2007012807 A2 WO 2007012807A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
single stranded
template
molecule
substrate
Prior art date
Application number
PCT/GB2006/002606
Other languages
English (en)
Other versions
WO2007012807A3 (fr
Inventor
Giles Davies
Paul Anthony Tosch
Christoph Walti
Original Assignee
University Of Leeds
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0515413A external-priority patent/GB0515413D0/en
Priority claimed from GB0602625A external-priority patent/GB0602625D0/en
Application filed by University Of Leeds filed Critical University Of Leeds
Publication of WO2007012807A2 publication Critical patent/WO2007012807A2/fr
Publication of WO2007012807A3 publication Critical patent/WO2007012807A3/fr

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

  • the invention relates to a process for the production of single stranded nucleic acid and its use in the formation of higher order structures comprising DNA.
  • nucleic acid molecules are well suited for this purpose due to the inherent self recognition of each of the complementary strands of nucleic acid.
  • Linear strands of DNA can self assemble into a range of structures based on the linear sequence information contained in the nucleotide base composition. It is relatively easy to incorporate branching of a double helix by modifying the sequence of a pair of nucleic acid strands to be non-complementary over a specific region of the helix resulting in no annealing in that region of non- complementarity. This can be achieved with anaki level of accuracy to provide nano-scale structures that are stable, predictable and regular and in large quantities. In this way DNA structures can be built of virtually any conformation, for example cages, tubes, nets, scaffolds and other complex three dimensional structures.
  • nano-scale structures may be fabricated on self-assembled DNA scaffolds. It is recognised that traditional “top down” lithographic methods for manufacturing microelectronics will soon reach their fundamental limitations and new “bottom up” techniques will be required to provide circuit layouts on a nano- scale.
  • the self assembly properties of DNA nanostructures as scaffolds may provide the necessary complexity to construct and position nano-molecular scale electronic devices and wiring.
  • DNA sequences can be modified to incorporate specific sequences that bind peptides, proteins and other nucleic acids (e.g. gene therapy vectors) that have therapeutic applications. Further applications in biological systems include the use of DNA nano-structures as binding partners on substrates for the binding of proteins/enzymes involved in macromolecular synthesis.
  • nucleotide composition of DNA can be chemically modified to incorporate modified bases and sugars to alter the properties of the molecule.
  • modified bases and sugars for example, the incorporation of peptidic linkages between bases to form so called peptide nucleic acids.
  • an in vitro method for the production of a single stranded DNA template comprising the steps of: i) forming a preparation comprising: a nucleic acid template molecule; a thermostable DNA polymerase; at least two oligonucleotide primer molecules adapted to anneal to said template; and polymerase chain reaction components including nucleoside triphosphates and an agent to protect the 5' nucleotide of the amplified template molecule; ii) amplifying said nucleic acid template by a polymerase chain reaction to form a double stranded nucleic acid template wherein the template is modified at the 5' nucleotide of the template to protect the digestion of the template by a non-processive 5'-3'exonuclease; iii) contacting the preparation with a non-processive 5 '-3' exonuclease that digests the template to a single stranded DNA molecule; iv)
  • non-processive 5 '-3' exonuclease is T7 gene 6 exonuclease.
  • T7 gene 6 exonuclease hydrolyzes double stranded DNA non-processively in the 5'- 3' direction from either 5' phosphoryl or 5' hydroxyl nucleotides. It also degrades nucleotides at gaps and nicks of double stranded DNA from the 5' termini and RNA from RNA-DNA hybrids in a 5 '-3' direction.
  • the method of the invention allows the production of single stranded template DNA, the sequence of which is modified to allow complementary base pairing between single stranded nucleic acid to form higher order structures that may be linear, branched or circular and of nanometre dimensions.
  • said modification is the incorporation of phosphorothioate to protect the 5 'nucleotide of the template.
  • said agent is chemically or physically cleaved from said single stranded template.
  • said agent is cleaved with restriction enzyme following hybridisation with a complementary oligonucleotide that comprises a restriction enzyme recognition site for said restriction enzyme.
  • said agent is cleaved with a restriction enzyme following generation of a double stranded region by hybridisation with a complementary oligonucleotide, or by any other method, that comprises a restriction enzyme recognition site for said restriction enzyme.
  • nucleoside triphosphates are modified.
  • modified nucleotides encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified nucleotides may also include 2' substituted sugars such as 2'-O- methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; T- fluoro-; 2'-halo or 2;azido- ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
  • 2' substituted sugars such as 2'-O- methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; T- fluoro-; 2'-halo or 2;azido- ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or
  • Modified nucleotides include, by example and not by way of limitation, alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6- methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5 fluorouracil;5- bromouracil;5-carboxymethylaminomethyl-2 thiouracil;5carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine;l-niethyladenine;l- methylpseudouracil;l-methylguanine;2,2 dimethylguanine; 2-methyladenine; 2- methylguanine;
  • Li a preferred method of the invention said template is adapted by the provision of a nucleic acid sequence motif that binds at least one polypeptide molecule.
  • motifs that bind protein or polypeptide molecules include restriction enzymes, DNA binding proteins e.g. histones, transcription factors, DNA polymerases, RNA polymerases, DNA ligases, or an antibody.
  • the motifs may be part of a promoter.
  • Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements.
  • the binding/activity of transcription factors is responsive to a number of physiological/environmental cues which include, by example and not by way of limitation, intermediary metabolites (e.g. glucose, lipids), environmental effectors (e.g. light).
  • Promoter elements also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.
  • a single stranded nucleic acid molecule obtainable or obtained by the method according to the invention.
  • said nucleic acid molecule is at least about 20 nucleotides in length.
  • said nucleic acid molecule is at least about 60 nucleotides in length; at least about 200 nucleotides in length; at least about 400 nucleotides in length; at least about 1000 nucleotides in length; at least about 2000 nucleotides in length; at least about 10000 nucleotides in length; or at least about 50000 nucleotides in length.
  • an in vitro method for the formation of a complex comprising nucleic acid comprising the steps of: i) forming a preparation of at least two single stranded nucleic acid molecules obtained or obtainable by the method according to the invention wherein said molecules are adapted to anneal to one another over at least part of their length; ii) heating said preparation to denature secondary structures in said single stranded nucleic acid; and iii) allowing said single stranded nucleic acid molecules to anneal to one another to form a nucleic acid complex.
  • said preparation comprises at least three single stranded nucleic acid molecules wherein each molecule comprises a region which is able to anneal to a region in at least one other molecule of said three nucleic acid molecules.
  • said nucleic acid molecules include a region complementary td a region found in two of said three nucleic acid molecules.
  • said complex comprises a plurality of single stranded nucleic acid molecules.
  • nucleic acid complex obtained or obtainable by the method according to the invention.
  • a substrate which comprises a surface wherein said surface comprises a nucleic acid complex according to the invention.
  • said substrate is selected from the group consisting of: a metal substrate; a plastics substrate; a glass substrate; a mica substrate; a silicon substrate; a silicon dioxide substrate; a gallium arsenide substrate; a germanium substrate; a diamond substrate; a doped or layered semiconductor substrate or combinations thereof.
  • a device comprising a substrate according to the invention.
  • said device is a product that allows for the directed transport of a molecule or a molecular structure.
  • said molecule or molecular structure is transported by a vehicle that associates with said nucleic acid complex.
  • said vehicle comprises an organic or inorganic molecule or a molecular complex.
  • said vehicle comprises at least one protein.
  • Molecules or molecular structures that are transported by the device of the invention include, but are not limited to, metal-, semiconductor- and insulating nanoparticles; carbon-based materials such as single and multi-walled carbon nanotubes, buckyballs; proteins, nucleic acid complexes and chemical compounds; other inorganic and organic materials; information in various forms.
  • said molecule or molecular structure is integral with said vehicle.
  • said device is adapted for the transport of an electrical charge.
  • An electrical charge includes, but is not limited to, a charge in the form of electrons, holes, electron- or hole-like quasiparticles, protons, and ions, amongst others.
  • said device is adapted to transport data.
  • Data includes, but is not limited to, signalling the occurrence of a specific event, or as means of directing further actions such as triggering catalysis or other chemical reactions at nearby or remote locations.
  • said device is an electronic circuit.
  • said device is a scaffold for the assembly of nanoscale particles and other complexes.
  • the assembly can be, but is not limited to be, 'directed' with spatial and orientational precision at the nanometer scale, i.e. objects are assembled at designed locations on said device.
  • Objects to be assembled include, but are not limited to, metal-, semiconductor- and insulating nanoparticles; carbon-based materials such as single and multi-walled carbon nanotubes, buckyballs; proteins, nucleic acid complexes and chemical compounds; other inorganic and organic materials.
  • said device is a scaffold wherein objects are assembled at random locations along the whole scaffold.
  • said device is a self-assembled, nanoscale object.
  • said device is a scaffold for a subsequent chemical reaction or reactions or catalysis of a chemical reaction or reactions.
  • the reactions or catalysis of reactions occur, but are not limited to occur, with spatial precision at the nanometer scale, i.e. the reaction or catalysis occurs at a designed location or locations on said device.
  • said device is a drug delivery vehicle.
  • composition comprising a drug delivery vehicle according to the invention.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of nucleic acid for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by determining regression of a tumour, decrease of disease symptoms, modulation of apoptosis, etc.
  • the doses of the composition administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • compositions for the administration of compositions
  • administration of compositions to mammals other than humans e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above.
  • a subject as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • compositions may be combined, if desired, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • the pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • the pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of the composition, which is preferably isotonic with the blood of the recipient.
  • This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butane diol.
  • a non-toxic parenterally-acceptable diluent or solvent for example, as a solution in 1, 3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's
  • said device is a diagnostic assay product.
  • Diagnostic assay products typically will include substrates upon which is immobilised an molecule, for example an antigen, antibody or nucleic acid probe, which detects a molecule present in a biological sample the detection of which is desirable because it may be associated with a specific disease or condition.
  • a method to fabricate a surface of a substrate comprising the steps of: i) providing a preparation comprising a nucleic acid complex according to the invention; and ii) bringing into contact the preparation in (i) with a substrate to be fabricated.
  • nucleic acid complex obtainable or obtained by the method according to the invention in the production of electronic circuits.
  • nucleic acid complex obtainable or obtained by the method according to the invention in the delivery of a drug to a subject.
  • kits comprising: a container comprising a single stranded template nucleic acid molecule according to the invention.
  • said template nucleic acid is included in a vector, preferably a plasmid.
  • vectors also include cosmids, mammalian artificial chromosomes, yeast artificial chromosomes and bacteriophage vectors.
  • said kit includes a non processive 5 '-3' exonuclease; preferably said exonuclease is T7 gene 6 exonuclease.
  • said kit further comprises: a thermostable DNA polymerase; at least two oligonucleotide primer molecules adapted to anneal to said template; polymerase chain enzyme reaction components and optionally including nucleoside triphosphates.
  • a method for the isolation of a nucleic acid complex according to the invention comprising providing a nucleic acid complex according to the invention wherein at least one single stranded nucleic acid template comprising said complex is provided with a nucleic acid sequence affinity tag that allows the selection of complexes by affinity purification.
  • Figure 1 Design schematic for 3 way junction a) Schematic of final assembled 3 way junction after annealing, b) Map of Lambda DNA showing regions used as DNA source for the 3-way junction, c) Three single stranded components. Each component contains two regions which are complementary to regions of a neighbouring components (indicated by blue, red, and orange). In addition each component has an upstream 20bp fragment which serves as a unique region leaving a single stranded overhang in the final molecule;
  • FIG. 1 Design methodology for junction fabrication. In the example used in the paper this procedure is followed for all three components given in Figure Ic;
  • FIG. 3 a) T7 Gene 6 exonuclease digested products of A, B, C (ds double stranded undigested controls, ss single stranded T7 gene 6 treated products). Single stranded DNA fragments run slower than double stranded DNA fragments. Double stranded size in base pairs is indicated on the left, b) 2% Agarose Gel shift assay of individual components of the three way junction; [lanes 1-3] individual T7 gene 6 exonuclease digested products; [lanes 4-6] two components of the three way junction annealed; [lane 7] all three components of the three way junction annealed;
  • Figure 4 a) Schematic of method to remove phosphothioate protection groups from T7 gene 6 exonuclease digested components using a complementary oligonucleotide bridge to create an active Nsi l (New England Biolabs) binding site, b) 20% Acrylamide gel of digested products showing removal of phosphorothioate blocking regions: lane 1 ⁇ double stranded (-yds), lane 2 ⁇ single stranded ( ⁇ ss), lane 3 ⁇ single stranded + bridging primer, lane 4 ⁇ single stranded + bridging primer + 5Ou of Nsi I, lane 5 y single stranded;
  • FIG. 5 Atomic force micrograph of three way branched structures on a mica substrate; and Figure 6 A Design schematic for double branched complex after annealing; Figure 6B The temperature decay curve of 1.6 litres of water in a Pyrex beaker O/N after heating to 95 0 C.
  • Figure 6C Atomic force micrograph of a branched DNA structure containing two branch points on a mica substrate.
  • PCR primers were designed for the amplification of each fragment (fragments a & b-RC in Figure 2) such that a linker restriction enzyme (LRE) site is incorporated in the downstream 5' end of the fragment. These restriction sites also later allow for a "checksum" of the assembled construct as individual arms can be removed. In addition, a unique 20 bp sequence was added upstream of all three components to serve as a functional 5' overhang in the assembled complex. After PCR, both products were digested appropriately and purified.
  • LRE linker restriction enzyme
  • FIG. 2 Amplification and protection with phosphorothioate
  • Figure 2 Purified plasmids (Plasmid Mini, Qiagen) were used as templates to amplify the individual components using Pfu Turbo hotstart DNA polymerase (Stratagene).
  • the forward primer of this reaction contains five phosphorothioate modified nucleotides on the 5' end, as well as a bridging restriction enzyme (BRE) site, which can be used to remove the five phosphorothioates if required.
  • BRE bridging restriction enzyme
  • T7 Gene 6 exonuclease digestion and purification (Figure 2; e-g) Double stranded phosphorothioate protected products were digested with 20 units of T7 exonuclease (New England Biolabs M0263) by incubation for 120 minutes at 37 0 C followed by heat inactivation for 10 minutes at 8O 0 C. Products were then purified using Qiaquick columns (Qiagen) with PB binding buffer, and analysed for complete T7 Exonuclease digestion on a polyacrylamide gel. Removal of pliosphorothioate protection groups
  • DNA was deposited onto mica substrates (Aztech Trading) as follows. Freshly cleaved mica was washed with a drop of deionised water (18.2 M ⁇ cm, Millipore) for lOseconds and excess water was removed. The Mica was the covered in 1OmM MgCl 2 for 10 sec, with the excess then removed. Assembled DNA complexes at approximately 5ng/ ⁇ l in 1OmM MgCl 2 were then allowed to adsorb onto the surface for 5 minutes. The mica substrate was washed twice with ddH 2 O for lOsec and blow-dried under Nitrogen.
  • Imaging was performed using tapping mode AFM (DI Multimode AFM, Veeco, USA) under air, using etched silicon tips (OTESPA, Veeco, USA). Length measurements of the imaged structures were performed using the ImageJ image analysis software (Rasband, 1997-2004). Testing of Functionalised Group availability
  • oligonucleotides corresponding to regions of Bacteriophage lambda DNA are shown in bold and are reflected in their name with U or L denoting the upper and lower strand of lambda DNA, respectively, followed by a number indicating the position on lambda DNA of the 5.
  • nucleotide for the upper strand and the 3' nucleotide for the lower strand are complementary sequences from the upper and lower strand of lambda DNA, respectively.
  • the single branch structure was constructed with components a, ⁇ , and 7.
  • Fragment a (Ua denotes the unique 20bp region shown in italics)
  • Fragment b consists of two regions (bl and b2)
  • Fragment bl (Ub denotes the unique 20bp region shown in italics) Ub-U27086 GATAAGTGGATGCCATCAGGTTGAACTTAACGGGGCATCG
  • Fragment b-RC consists of two regions (bl-RC and b2-RC) Fragment c (Uc denotes the unique 20bp region shown in italics)
  • the double branch structure was constructed with components a, % ⁇ , and e which when annealed will form the complex shown in Figure 6A.
  • Fragment d (Ud denotes the unique 20bp region shown in italics)
  • DNA was deposited onto mica substrates (Aztech Trading) as follows. Freshly cleaved mica was washed with a drop of deionised water (18.2 MUcm, Millipore) for 10 sec and excess water was removed. The mica was then covered in 10 mM MgC12 for 10 sec, with the excess removed. Assembled DNA complexes at approximately 1 ng/ ⁇ l in 1OmM MgC12 were then allowed to adsorb onto the surface for 5 minutes. The mica substrate was washed twice with deionised water for 10 sec and blow-dried under nitrogen. Imaging was performed using tapping mode AFM (DI Multimode AFM, Veeco, USA) under air, using etched silicon tips (OTESPA, Veeco, USA). Length measurements of the imaged structures were performed using the Image J image analysis software.21.
  • the schematic design for the three-arm DNA complex is shown in Figure Ia.
  • the junction comprises three arms, A, B, and C, each composed of an upstream fragment (a, b, and c) and a downstream fragment (a-RC, b-RC, and c-RC).
  • the individual arms A, B, and C were taken from suitable regions of lambda- Bacteriophage DNA (Sigma D0144), as indicated in Figure Ib.
  • the only restrictions imposed on the individual regions was the required length of the arm and that they do not contain recognition sites for a certain set of enzymes which will subsequently be used for modifications of the assembled complex and checksum tests.
  • three components are required, designed such that each fragment of a given component will recognise a fragment of another component.
  • each component contains two fragments which will each form one half of an arm of the DNA complex.
  • each component has a downstream region in reverse complement (RC) orientation to the upstream region of another component, such that: a binds ⁇ and ⁇ , ⁇ binds 7 and a; and 7 binds a and ⁇ .
  • RC reverse complement
  • the only possible conformation that can be formed is the one shown in Figure Ia.
  • the individual steps undertaken to generate the component a are shown in Figure 2.
  • the components ⁇ and ⁇ are obtained in a similar manner, however we note that the fragments b and b-RC of the components ⁇ and ⁇ , respectively, consist of two individual fragments as opposed to just one for all other components.
  • the template for component a was obtained by PCR amplification and subsequent ligation of the two fragments a and c-RC.
  • the ligation product was then cloned into a bacterial vector and sequenced to isolate a pure, error-free template.
  • the double stranded component template was amplified by PCR which also incorporated a further restriction site (BRE) and five phosphorothioates on the 5' end of the upstream strand.
  • BRE further restriction site
  • the results of this PCR are shown on a 5% polyacrylamide gel in Figure 3 a (lane 1; lane 3 and 5 shows the results of the equivalent PCR for components ⁇ and ⁇ ).
  • DNA complexes were self-assembled by mixing each component at l ⁇ ng/ ⁇ .1 in IX hybridisation buffer (Mao et. al., 1999). The component mixture was annealed by slowly reducing the temperature. The results of this annealing process was analysed by gel shift assay on a 2% agarose gel (Figure 3b). Lanes 1, 2, and 3 show the individual conponents, lanes 4, 5, and 6 DNA complexes missing one component, and lane 7 shows the complete, assembled three-arm DNA complex. A substantial increase in apparent size of the product was found upon introducing a further component. We note that there are no visible traces of either individual single stranded component, or incompletely assembled complexes when all components were added (lane 7).
  • Figure 5 shows a typical AFM image.
  • the inset shows a high resolution scan of one of the three arm complexes.
  • a typical atomic force micrograph of this complex is given in Figure 6.
  • Nikiforov TT Rendle RB, Kotewicz ML, Rogers YH.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Composite Materials (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Materials Engineering (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention porte sur un procédé de production de molécules d'ADN simple brin et sur leur utilisation dans la production de complexes d'ADN à l'échelle nanométrique.
PCT/GB2006/002606 2005-07-27 2006-07-13 Structures d'adn WO2007012807A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0515413A GB0515413D0 (en) 2005-07-27 2005-07-27 DNA structures
GB0515413.3 2005-07-27
GB0602625A GB0602625D0 (en) 2006-02-09 2006-02-09 DNA structures
GB0602625.6 2006-02-09

Publications (2)

Publication Number Publication Date
WO2007012807A2 true WO2007012807A2 (fr) 2007-02-01
WO2007012807A3 WO2007012807A3 (fr) 2007-04-19

Family

ID=37487688

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2006/002606 WO2007012807A2 (fr) 2005-07-27 2006-07-13 Structures d'adn

Country Status (1)

Country Link
WO (1) WO2007012807A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133695A3 (fr) * 2010-04-20 2012-03-29 Swift Biosciences, Inc. Matériaux et procédés de fractionnement de l'acide nucléique par piégeage en phase solide et détachement enzymatique
WO2015008091A1 (fr) * 2013-07-19 2015-01-22 Atlas Genetics Limited Amorces modifiées pour la détection et l'amplification d'acides nucléiques
WO2016144755A1 (fr) * 2015-03-07 2016-09-15 President And Fellows Of Harvard College Nanostructures d'adn simple brin
US9796749B2 (en) 2011-08-05 2017-10-24 President And Fellows Of Harvard College Compositions and methods relating to nucleic acid nano- and micro-technology
US9975916B2 (en) 2012-11-06 2018-05-22 President And Fellows Of Harvard College Compositions and methods relating to complex nucleic acid nanostructures
US10099920B2 (en) 2014-05-22 2018-10-16 President And Fellows Of Harvard College Scalable nucleic acid-based nanofabrication
US10604543B2 (en) 2012-07-24 2020-03-31 President And Fellows Of Harvard College Self-assembly of nucleic acid nanostructures

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028088A1 (fr) * 1998-11-10 2000-05-18 Biocrystal Limited Nanocristaux comportant des brins de polynucleotides et leur utilisation pour former des dendrimeres dans un systeme d'amplification de signaux
WO2003091406A2 (fr) * 2002-04-25 2003-11-06 Geneohm Sciences Amplification d'adn pour obtention d'un produit a simple brin de sequence et de longueur definies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028088A1 (fr) * 1998-11-10 2000-05-18 Biocrystal Limited Nanocristaux comportant des brins de polynucleotides et leur utilisation pour former des dendrimeres dans un systeme d'amplification de signaux
WO2003091406A2 (fr) * 2002-04-25 2003-11-06 Geneohm Sciences Amplification d'adn pour obtention d'un produit a simple brin de sequence et de longueur definies

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DONG J ET AL: "Genomic organization and chromosome localization of the newly identified human heparanase gene" GENE, ELSEVIER, AMSTERDAM, NL, vol. 253, no. 2, 8 August 2000 (2000-08-08), pages 171-178, XP004215720 ISSN: 0378-1119 *
MIYACHI H ET AL: "Automated specific capture of hepatitis C virus RNA with probes and paramagnetic particle separation." JOURNAL OF CLINICAL MICROBIOLOGY JAN 2000, vol. 38, no. 1, January 2000 (2000-01), pages 18-21, XP002415973 ISSN: 0095-1137 *
MOGHIMI S MOEIN ET AL: "Nanomedicine: current status and future prospects." THE FASEB JOURNAL : OFFICIAL PUBLICATION OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY. MAR 2005, vol. 19, no. 3, March 2005 (2005-03), pages 311-330, XP002411926 ISSN: 1530-6860 *
NIKOFOROV T T ET AL: "THE USE OF PHOSPHOROTHIOATE PRIMERS AND EXONUCLEASE HYDROLYSIS FOR THE PREPARATION OF SINGLE-STRANDED PCR PRODUCTS AND THEIR DETECTIONBY SOLID-PHASE HYBRIDIZATION" 1 April 1994 (1994-04-01), PCR METHODS & APPLICATIONS, COLD SPRING HARBOR LABORATORY PRESS, US, PAGE(S) 285-291 , XP000601523 ISSN: 1054-9803 cited in the application abstract *
SEEMAN NADRIAN C: "At the crossroads of chemistry, biology, and materials: structural DNA nanotechnology." CHEMISTRY & BIOLOGY. DEC 2003, vol. 10, no. 12, December 2003 (2003-12), pages 1151-1159, XP002411933 ISSN: 1074-5521 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133695A3 (fr) * 2010-04-20 2012-03-29 Swift Biosciences, Inc. Matériaux et procédés de fractionnement de l'acide nucléique par piégeage en phase solide et détachement enzymatique
US9796749B2 (en) 2011-08-05 2017-10-24 President And Fellows Of Harvard College Compositions and methods relating to nucleic acid nano- and micro-technology
US10604543B2 (en) 2012-07-24 2020-03-31 President And Fellows Of Harvard College Self-assembly of nucleic acid nanostructures
US9975916B2 (en) 2012-11-06 2018-05-22 President And Fellows Of Harvard College Compositions and methods relating to complex nucleic acid nanostructures
WO2015008091A1 (fr) * 2013-07-19 2015-01-22 Atlas Genetics Limited Amorces modifiées pour la détection et l'amplification d'acides nucléiques
US11180798B2 (en) 2013-07-19 2021-11-23 Binx Health Limited Modified primers for nucleic acid amplification and detection
US10099920B2 (en) 2014-05-22 2018-10-16 President And Fellows Of Harvard College Scalable nucleic acid-based nanofabrication
WO2016144755A1 (fr) * 2015-03-07 2016-09-15 President And Fellows Of Harvard College Nanostructures d'adn simple brin
US10550145B2 (en) 2015-03-07 2020-02-04 President And Fellows Of Harvard College Single-stranded DNA nanostructures

Also Published As

Publication number Publication date
WO2007012807A3 (fr) 2007-04-19

Similar Documents

Publication Publication Date Title
CN110088299B (zh) 通过纳米孔指导的核酸检测方法
Zhao et al. Isothermal amplification of nucleic acids
WO2007012807A2 (fr) Structures d'adn
Bi et al. Target-catalyzed DNA four-way junctions for CRET imaging of microRNA, concatenated logic operations, and self-assembly of DNA nanohydrogels for targeted drug delivery
US9605304B2 (en) Ultra-stable oligonucleotide-gold and-silver nanoparticle conjugates and method of their preparation
Ducani et al. Noncovalent DNA‐Binding Metallo‐Supramolecular Cylinders Prevent DNA Transactions in vitro
WO2012016357A1 (fr) Dosage à base de micropuces comprenant des particules, destiné à analyser des interactions moléculaires
US20080287668A1 (en) Nanostructures and methods of making
JP2002510465A (ja) 修飾核酸プローブおよびその使用
JPH07503370A (ja) 核酸含有溶液の電気化学的処理による変性方法、および、かかる変性方法を利用する増幅方法、複製方法、検出方法およびキット
JP2010521142A (ja) 遺伝子発現アッセイ
Li et al. Graphene nanoprobes for real-time monitoring of isothermal nucleic acid amplification
Zheng et al. Ultrastable bimolecular G-quadruplexes programmed DNA nanoassemblies for reconfigurable biomimetic DNAzymes
JP2004536317A (ja) 合成結合システムを用いる核酸のための選別および固定化システム
Mariconti et al. Enzymatically active DNA-protein nanogels with tunable cross-linking density
JP2023159377A (ja) 標的ポリヌクレオチドを検出するための方法
JP2002534097A5 (fr)
US10738349B2 (en) Polynucleotide based movement, kits and methods related thereto
AU2019239619B2 (en) Methods and compositions for recombinase-mediated selective cleavage of nucleic acids
Xu et al. DNA origami nanostructures with scaffolds obtained from rolling circle amplification
Russell et al. Formation of silver nanostructures by rolling circle amplification using boranephosphonate-modified nucleotides
Moll et al. Grouping of ferritin and gold nanoparticles conjugated to pRNA of the phage phi29 DNA-packaging motor
Gascón et al. Differential functional behavior of viral ϕ29, Nf and GA-1 SSB proteins
CN103014161B (zh) 一种纳米材料表面进行的杂交链式反应技术的制备方法
Niemeyer et al. Supramolecular DNA-streptavidin nanocircles with a covalently attached oligonucleotide moiety

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06764955

Country of ref document: EP

Kind code of ref document: A2