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  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
  • C12N2310/151—Nucleic acids forming more than 2 strands, e.g. TFOs more than 3 strands, e.g. tetrads, H-DNA
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/50—Physical structure
  • C12N2310/52—Physical structure branched

Definitions

• the present invention relates to nucleic acid based molecular structures that bind to other molecular entities, especially entities other than nucleic acids themselves.
• the invention relates to nucleic acid based molecular structures with pharmaceutical activity through binding to specific molecular targets and thereby influencing disease states.
• the invention also relates to nucleic acid based molecular structures with diagnostic utility.
• compositions of matter that are able to specifically alter the activity of particular proteins or modulate the expression of particular gene products.
• molecules able to form specific binding interactions with other molecules and especially for such molecules to exhibit specific binding within the in vivo milieu.
• RNA molecules of defined unit length For libraries of nucleic acids, the technical approach has in general been the use of single-stranded RNA or DNA molecules of defined unit length.
• the basis of binding to a target molecule is not pre-configured and may be dependent on secondary structure formation within the DNA (or RNA) molecule itself facilitating binding to the other molecular entity (Bock L.C. et al 1992 Nature 355: 564-566; Kubrik, M.F. et al 1994 Nucleic Acids Res. 22: 2619-2626).
• the creation of nucleic acid molecules containing pre-configured tracts of secondary (or high order) structure for therapeutic and diagnostic utility has not been previously attempted, and is the object of the present invention.
• the present invention relates to novel high order nucleic acid structures and to novel uses of such structures.
• RNA molecules capable of facilitating enzymatic process such as polynucleotide kinase activity have been evolved by iterative cycles of selection (Lorsch J.R. & Szostak J.W.
• nucleic acid structures include "branched-DNA” (Horn, T. & Urdea, M. S. 1989, Nucleic-Acids-Res. 17: 6959-6967) whereby one or more regions of a DNA molecule ("probe") which hybridizes to a complememtary nucleic acid molecule can themselves be subjected to hybridization to other DNA molecules in order to amplify the amount of DNA associated with the DNA probe.
• probe DNA molecule
• the complexes described by Horn and Urdea (ibid) are linearly extended whereby newly hybridized nucleic acid molecules are not designed to hybridise to molecules previously annealed but to other incoming new molecules to form branched-DNA structures. Indeed, such complexes are simply designed to form as many branches as possible in order to provide more points for annealing of a signaling nucleic acid probe.
• the geometric objects are closed structures fabricated using iterative processes involving restriction enzymes and DNA ligation. Nodal points in the figures may be fixed by cross-over junction between stands to give a rigid form, or more flexible branches achieved by cross-annealing disparate stands.
• the prior art does not include geometric structures which are not closed (i.e. ends ligated).
• the prior art does not include geometric structures which include regions of modified nucleic acids and does not include geometric nucleic acid structures conjugated to other molecular entities.
• the prior art does not include the use of libraries of randomised or semi-randomised nucleic acid structures.
• nucleic acid molecules with potential and or actual therapeutic activity operate either as antisense molecules, triplex reagents or as RNA molecules with endoribonuclease activity ("ribozymes").
• ribozymes RNA molecules with endoribonuclease activity
• the modality of the therapeutic nucleic acid is as a modulator of protein expression by a mechanism of action that reduces or blocks protein translation.
• the specificity of target binding in all of these cases is nucleic acid to nucleic acid.
• a distinctive and inventive feature of the present invention is the use of a high-order nucleic acid structure with binding activity to a target molecule.
• nucleic acid structures especially Aptamers
• ribozymes Certain other nucleic acid molecules, especially ribozymes, have been identified as having enzymatic activities with potential pharmaceutical importance.
• pharmaceutical utility has not been considered although US. Pat No 5,278,051 speculates possible utility as solubilising agents or controlled release vehicles for small molecule therapeutics.
• a first aspect of the present invention relates to novel high order nucleic acid based structures, particularly open geometric structures. Furthermore, the invention also relates to the utility of such structures as pharmaceutical and/or diagnostic agents. The invention also relates to high order nucleic acid based structures including nucleotides with modifications. The invention also relates to high order nucleic acid based structures including regions of randomised or semi- randomised nucleotides. The invention also relates to high order nucleic acid based structures conjugated to other molecular entities such as proteins.
• Structures of the present invention exploit the Watson-Crick base pairing rules in engineering regions of double stranded structure.
• Single-stranded nucleic acid molecules have the ability to anneal (hybridise) to other single-stranded molecules by virtue of complementarity between the bases. Whilst such base annealing of two single-stranded molecules usually leads to a linear double- stranded molecule, other structures can be produced for example hairpin loops where one molecule has internal base-pair complementarity and circles where both ends of each single-stranded molecule have mutual complementarity.
• single-stranded nucleic acid sequences individual molecules can be designed which can simultaneously anneal to two or more other molecules and if, in turn, these other molecules can also anneal to further molecules including molecules already involved in annealing, then complexes of nucleic acids can be formed.
• the overall dimensions and topology of the double stranded DNA molecule are well understood.
• Double stranded DNA is quite flexible and the helix is able to adopt a number of conformations differing in the angle of rotation between adjacent base pairs along the helix.
• Naturally occurring single stranded nucleic acid molecules such as RNA adopt preferred conformations in solution.
• the conformation is dictated by base-pairing interactions within the same molecule leading to the production of a stabilised structure composed of double stranded stems and single stranded loops.
• the molecules will adopt the conformation of lowest energy and this structure for a known sequence of RNA is capable of prediction by computational approaches (Jaeger J.A. et al 1989 Proc.Natl.Acad. Sci USA 86: 7706-7710). Attempts have been made to produce predictive software for DNA folding and have shown some success (Nielsen D.A. et al 1995 Nucleic Acids Res. 23: 2287-2291).
• a typical globular protein such as myoglobin with molecular weight 17kDa has a size in its longest dimension of 3nm.
• a larger globular protein such a bovine serum albumin with molecular weight 68kDa is 5nm in its longest dimension (Cohen C, in Wolstenholme G.E.W. & O'Connor M. (eds), Ciba Foundation Symposium, London, J & A Churchill, 1966).
• the diameter of the double stranded helix is in itself 2nm and DNA strands of a small number of base pairs such as 100, would achieve a contour length approaching 30nm.
• the density of DNA or any other nucleic acid molecule is much less than a typical protein, the topology the DNA molecule, even in its most structured native form as a double helix, could readily cover large parts of the exposed surface of almost any protein molecule.
• the topology of the usually monofilament DNA were so altered, the DNA could occupy a large area of space in a manner more akin to a much higher density protein molecule.
• nucleic acid structures composed of multiple interconnected strands each of only short ( ⁇ 50) nucleotide tracts can readily result in structures with overall dimensions in the range 10-500 nm. It is a particular objective of the present invention to provide for such a formulation of nucleic acid molecule. Structures of the present invention are based on the creation of DNA or RNA molecules with secondary structures formed as a result of the interaction of two or more molecules of nucleic acid or, alternatively, as a result of interaction of different defined segments within individual molecules of nucleic acid.
• the informational content of DNA or RNA is exploited not as a coding entity for expression of a therapeutic protein, nor as a blocking entity for nucleic acid metabolism and gene expression (anti-sense) but to direct assembly of a molecular structure of particular shape in three-dimensions.
• the present invention includes nucleic acid molecules, particularly synthetic DNA molecules, which form three-dimensional (non-planar) molecular structures by specific base pairing within the molecules in the set.
• the DNA molecules are designed to have 1 or more regions of sequence ("domains") that can anneal to other molecules in the set ultimately to form a composite three- dimensional nucleic acid structure.
• domains regions of sequence
• an approximate cuboid structure can be formed by the self-annealing of 6 synthetic DNA molecules each containing 4 domains of complimentarity whereby each molecule interacts with 4 other molecules and whereby each molecule acts effectively like an individual side of a 6-faced cube.
• the structure is open (not covalently closed), flexible and in particular further embodiments amenable to modification by the addition of other functional or structural groups.
• nucleic acid molecules each comprising 2 or more domains of self- complementary sequence enabling the nucleic acid molecule to fold upon itself and to interact with each other to form a particular three-dimensional molecular structure via specific base-pairing events.
• chemical instability of unmodified DNA molecules has been a significant problem for uses such as therapeutic.
• Several approaches are now available for protecting DNA molecules from degradation by enzymatic attack.
• modified phosphodiester backbones methylphosphonate, phosphorothioate, peptide nucleic acids
• capping 5' and or 3' termini using phosphoramedite, phosphorothioate or phosphorodithioate linkages It is a particular objective of the present invention to exploit modified or non-natural nucleic acids in the high order nucleic acid based structures.
• a particularly desired feature is the increased flexibility in binding specificity achieved by use of mixed chemistry and alternative non-natural nucleic acid backbones.
• the nucleic acid sub-units of a high order nucleic acid structure of the present invention may be homotypic or heterologous in nature, for example DNA containing tracts of RNA. It is known that tracts of RNA within a DNA helix alter the coiling in solution (Wang, A. et al, 1982 Nature, 299: 601-04). The ability to offer conformational diversity within a localised tract of nucleic acid may be significant in altering binding specificity to the target protein, and this phenomenon is known in the art where the binding specificity a thrombin aptamer was dependent on a short tract of highly ordered tertiary structure (Griffin, L. et al 1993 Gene 137:25-31).
• non-natural phosphate backbone analogues may be exploited to enhance stability and also alter the binding specificity to the desired target protein.
• Latham et al (Latham, J.A. et al 1994 Nucleic-Acids-Res. 22: 2817-22) provide an example whereby the modified nucleotide, 5-(1- pentynyl)-2'-deoxyuridine was used in place of thymidine in a pool of random oligonucleotides.
• the present invention includes molecules composed of tracts of single stranded nucleic acid, interspersed with tracts of double stranded structure, and other chimeric molecules synthesised to contain different chemical sub-structure but joined exploiting conventional base-pairing rules. Such structures may also combine molecules of DNA and RNA.
• Higher order molecular structures of the present invention are assembled from individual or multiple nucleic acid molecules according to any scheme present in the art and may include synthetic nucleic acid species or fragments from much larger molecules such as recombinant plasmids.
• the structures may be built following self-folding (auto-assembly) or facilitated folding of a single linear molecule of DNA.
• Facilitated folding may be mediated by proteinacious entities (enzymes such as ligase, topoisomerase, endonuclease, polymerase) or via interaction with non-protein physiochemical conditions (pH, temperature, ionic conditions).
• the molecule may be assembled by interaction with molecules bound to a solid matrix, or whist the DNA undergoing folding into a higher order structure is tethered or anchored in space during all or part of the assembly process.
• a second aspect of the present invention is the provision of libraries of nucleic acid molecules formed to contain a range of semi-random molecules some of which may possess a desired topology capable of interacting in a specific manner with a target molecule.
• libraries of nucleic acid molecules featuring a guide framework to facilitate assembly of a common structural sub-unit. Within each sub-unit a randomised tract of sequence is incorporated maximising library diversity and potential functional utility with respect to activity in a selective binding assay.
• an embodiment whereby a library formed from mixtures of n separate populations (sets) of synthetic DNA molecules (sub-units) is exploited.
• the population size of the synthetic nucleic acid sub-units is large and dictated by the degree of randomisation present within a variable segment of the sub-unit.
• Further sub-unit diversity is inbuilt in other embodiments by variation of the positioning of the variable domain, variation in the number of variable domains (by interspersion with tracts of fixed sequence) and variation in the length of any given variable domain. It is preferred that n separate population of sub-unit are mixed in a single cycle of annealing to create a library of multiple nucleic acid structures and individual sequence diversity. It will be obvious that other embodiments may include multiple cycles of annealing and multiple values of the whole integer number n.
• a particular feature of the library under this scheme is the ability to modulate the degree of complexity of inter-subunit interaction by judicious design and placement of the complementary or guide sequence tract.
• a third aspect of the present invention is the novel utility of high order nucleic acid based structures, especially for pharmaceutical and diagnostic use.
• these structures are capable of binding to a specific target molecule, commonly a protein or proteinaceous target molecule.
• a specific target molecule commonly a protein or proteinaceous target molecule.
• the target is a protein complex comprised of multiple protein sub-units such as a cell surface receptor, collectively bound by a molecule of the first aspect of the invention.
• Other embodiments of the third aspect include the binding to a cellular target or cell species identified by an ability to bind a molecule of the first aspect.
• Target or target complex containing non-protein components for example carbohydrate or lipid components of the cell and in particular of the cell surface.
• Protein, carbohydrate and lipid entities and or complexes thereof may be disease specific entities or present as normal components of a tissue or cell.
• the target or target complex would include viral particles or viral derived components such as capsid proteins or host derived components of the viral coat.
• the target or target complexes may include metallic ions or other inorganic chemicals or chemical groups in their composition and may be naturally occurring or introduced by treatments with exogenous agents.
• Target receptors may include those such as the IL-2 receptor or other cytokine receptors such as receptors for IL-3, M-CSF, GM-CSF and numerous others.
• surface molecules such as the IgE receptor whereby blockade of IgE binding together with blockade of a cross- linking activation event at the receptor would be a highly desired outcome.
• Other surface molecules including members of the cluster differentiation (CD antigens) series are desired targets for disease modulation and in particular in respect of diseases of auto-immune component.
• the invention is designed to have particular widespread application in the field of therapeutic molecules.
• Molecular structures of the invention are desired to agonise or antagonise particular receptors or enzymatic processes for therapeutic benefit whilst contributing none of the disadvantages of conventional protein therapeutics such as immunogenicity.
• the invention therefore extends to a method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of the molecular structure.
• the invention also extends to the use of such structures in in vivo and in vitro diagnosis.
• a fourth aspect of the present invention comprises high order nucleic acid based structures with modified nucleotides included in the structures.
• it is particularly desired to impose diversity by the derivitisation of the sub-units of the library and or by the inclusion of modified bases during their synthesis (thiolated bases, biotinylated bases, epsilon-amino derivatised bases etc.).
• modified bases thiolated bases, biotinylated bases, epsilon-amino derivatised bases etc.
• the high order nucleic acid structures will also contain modified nucleotides capable of conferring particular desired properties to the structure additional to features providing stability or binding modulation as above.
• Such additional desired modifications may be embodied under the first or second aspects of the invention and include the use of hydrophobic tracts, the inclusion of psoralen or acridine groups, linking haptenic group such as biotin or linking to different charged side chains such as amino groups or carboxyl groups to provide facilitated binding to a particular target molecule.
• such groups may act as points for attachment of other molecules such as further nucleic acid molecules or proteins such as an antibody or an enzyme.
• a desired feature of molecules of the invention will be high stability in vitro and in vivo.
• the chemical composition of the nucleic acid structures is highly influential but also the physical size of the molecule requires control to minimise shear damage in solution and maximise functional utility in vivo.
• the preference of the invention is for multi-chain nucleic acid structures constructed from generally small ( ⁇ 80mer) sub-units.
• the exploitation of a structure composed of larger sub-units may be desired and equally fall into the scope of the present invention.
• a fifth aspect of the present invention comprises high order nucleic acid based structures attached to other molecular entities.
• this aspect includes nucleic acids attached at one or more specific sites to one or more specific sites on the other molecular entity whereby specific attachment to the nucleic acid is facilitated by modified nucleotides as in the fourth aspect of the invention.
• this aspect comprises high order nucleic acid based structures attached to pharmaceutically or diagnostically relevant molecular entities whereby the nucleic acid binds to specific molecular targets relating to disease and the attached molecular entity is then used to combat or detect the disease.
• Pharmaceutically relevant entities will include cytokines, Fc portions of antibodies, other antibody-related entities, toxins, enzymes, drugs and pro-drugs, receptor agonists or antagonists, receptor molecules themselves (especially ligand binding domains), radioisotopes, pharmaceutically active nucleic acids, drug transport vesicles such as liposomes, live or attenuated microorganisms, light activatable moieties, and other molecular entities which induce a vaccination effect. Diagnostically relevant entities will particularly include radioisotopes, light activatable moieties such as those producing a chemiluminescent signal, fluorochromes, enzymes, and signal transport vesicles such as beads.
• the invention comprises the following objects:
• a three-dimensional poly-nucleic acid structure composed of multiple interconnected strands of nucleic acid molecules or segments thereof by specific base pairing interaction of two or more molecules, characterized in that the structure is not covalently closed.
• a corresponding poly-nucleic acid structure characterized in that the structure is formed by two or more nucleic acid molecule strands.
• a corresponding poly-nucleic acid structure characterized in that the structure is formed by three or more nucleic acid molecule strands.
• a corresponding poly-nucleic acid structure characterized in that said structure is a cube or has essentially the form of a cube.
• a corresponding poly-nucleic acid structure wherein the cuboid structure is formed by six nucleic acid molecule strands, wherein each molecule strand acts like an individual side of the 6-faced cube.
• a corresponding poly-nucleic acid structure wherein said structure includes nucleic acid molecules which are composed of tracts of single stranded nucleic acid, interspersed with tracts of double stranded structure.
• a corresponding poly-nucleic acid structure according to any of the claims 1 - 8, wherein each nucleic acid strand has less than 80, preferably less than 50, nucleotides. • A corresponding poly-nucleic acid structure, wherein said structure has the assembly (A1 +B1+C1)+(A2+B2+C2) as depicted in Figure 1.
• a poly-nucleic acid structure as defined above wherein said structure contains sub-units which are composed of a variable randomized sequence tracts in order to get semi-random molecules or segments thereof capable of interacting with a target molecule.
• a three-dimensional poly-nucleic acid structure containing sub-units which are composed of multiple interconnected strands of nucleic acid molecules or segments thereof by specific base pairing interaction of two or more molecules, wherein said structure is covalently closed and contains sub-units which are composed of a variable randomized sequence tracts in order to get semi-random molecules or segments thereof capable of interacting with a target molecule.
• variable sequence composition is achieved by a one or more modifications of nucleotides within the sequence.
• a corresponding poly-nucleic acid structure wherein said other molecule is a protein, an enzyme, a iipoprotein, a glycosylated protein, an immunglobuline or a fragment thereof. • A corresponding poly-nucleic acid structure, wherein said other molecule is a nucleic acid.
• a pharmaceutical composition comprising a poly-nucleic acid structure as defined above and in the claims optionally together with suitable carriers, excipients and diluents and / or other pharmaceutically effective compounds.
• FIGURE 1 A first figure.
• oligonucleotide sub-units IL2R-1 and IL2R-2 comprising DNA structure with binding activity to IL-2 receptor.
• oligonucleotide sub-units TB-R1 and TB-R2 comprising DNA structure with binding activity to human thrombin.
• Library A comprised molecules of structure:
• Library B comprised molecules of structure:
• sequences were designed to facilitate cross-annealing and represent sub- units of a structure library formed by mixing and cross-annealing of different sub- units according to the scheme of the present invention.
• Oligonucleotide (sub-unit) libraries were synthesised with phosphorothioate linkages to maximise stability in the presence of serum factors and purified by HPLC. Purified oligonucleotides were obtained from GenoSys Biotechnologies (Cambridge, UK). A DNA structure library was assembled using a single cycle of cross-annealing. Sub-unit libraries A and B were denatured, mixed and annealed at a temperature of 37°C in a solution of 50mM Tris pH 7.4, 100mM NaCI, 5mM EDTA. Mixing of sub-unit libraries A and B was conducted at equimolar concentration (1 DM). In other experiments mixing was conducted using different molar ratios. Assembly of the subunits was verified by gel electrophoresis.
• the DNA structure library was screened for structures able to bind the extra cellular domain of the IL-2 receptor (IL2R). This was conducted using soluble recombinant 1L2R prepared according to published methods (Meidel, M.C. et al 1988 Biochem. Biophys. Res. Commun. 154: 372-379; Meidel, M. C. et al 1989, J. Biol. Chem. 264: 21097-21105). Recombinant IL2R was covalently bound to surface activated magnetic beads using protocols recommended by the supplier (Bangs Labs, Fishers, IN, USA). The IL2R-beads were used as an affinity surface to select binding structures from the DNA structure library.
• IL2R IL-2 receptor
• IL2R-beads were reacted with the library under a number of experimental conditions including the presence of chaotrophic salts in control reactions.
• the library (DNA) concentration was approximately l OOnmol in annealing solution as above.
• Binding moiecuies were recovered by polymerase chain reaction (PCR) directly from the beads following extensive washing cycles with a solution of 75mM Tris.HCL, 200mM NaCI, 0.5% N-octylglucoside pH8.0.
• the PCR was conducted using the primer PRA1 (5'-AGTCCCAAGCTGGCT) to recover the library A component using standard reagent systems and conditions.
• primers PRB1 (5'GACTTCACGATGGAG) and PRB2 (5'GTCAGAATGTGAATA) were used to recover the library B component.
• the PCR products were cloned and sequenced using standard reagent systems and procedures.
• sequences provided in Figure 2 were purified and assembled as previously, and used in a cellular assay for IL-2 antagonism.
• TALL-104 (ATCC# CRL-11386) is a human T-cell leukaemia cell line. The cells grow in suspension culture and require IL-2 for optimal growth. The cells may be grown for short period without IL-2 but their growth is significantly reduced. Cells were grown in Iscoves modified Dulbeccos medium (Life Technologies, Paisley, UK) with 50-100u/ml recombinant human IL-2 (Life Technologies, Paisley, UK) and supplemented with 10% (v/v) heat inactivated foetal calf serum. Cells were cultured in an atmosphere of 8-10% C0 2 .
• Dilutions of the annealed IL2-R1/IL2- R2 DNA preparation and a control DNA sample containing random sequence of identical contour length were prepared in culture medium containing IL-2.
• a parallel dilution series was prepared using medium lacking IL-2.
• the dilution series ranged from 50 DM DNA to 390nM DNA.
• Assays were performed using sub-confluent TALL-104 cells plated the preceding day in 96 well micro-titre dishes. Cells were collected by centrifugation, washed with pre-warmed (37°C) phosphate buffered saline and the DNA containing medium added for 48hours. Treatments were carried out in quadruplicate.
• Proliferation was assessed at the end of the 48hour period in a colou metric assay using a commercially available tetrazolium compound and following instructions provided by the supplier (Promega, Southampton, UK). Microtitre plates were read at 540nm.
• the library described in example 1 was used to select for a DNA structure able to bind to human thrombin.
• the library was screened using a human thrombin preparation (Sigma, Poole, UK) linked to surface activated magnetic beads as per example 1.
• Thrombin-beads were reacted with the DNA structure library as per example 1 except the post binding wash was conducted in a solution of 20mM Tris acetate, pH7.4, 140mM NaCI, 5mM KCI, 1mM MgCI 2 .
• Binding molecules were recovered directly from the beads by PCR using reactions and primer sets as for example 1.
• the PCR products were cloned and sequenced using standard reagent systems and procedures.
• a number of sequences were recovered and identified as originating from sub- unit library A and sub-unit library B. Of these, one pair was synthesised using phosphorothioate chemistry as before. Oligonucleotides TB-R1 and TB-R2 (sequences provided in Figure 3) were purified and assembled. The TB-R1/TB- R2 complex was used in a thrombin inhibition assay. Clotting time was measured using a fibrometer at 37°C and adult human plasma freshly prepared from a healthy donor. The extent of thrombin inhibition was determined using a thrombin standard curve plotting clotting time versus thrombin concentration. Clotting time was measured over three logs of DNA structure in the assay.
• the library described in example 1 was used to select for a DNA structure able to bind to a recombinant soluble CD4 (rsCD4) preparation.
• the DNA structure library was screened using a CD4 preparation (BioDesign, Saco, ME, USA) immobilised to activated magnetic beads as previously. Library screening, washing and selection by PCR was as described for example 2.
• a single oligonucleotide pair originating from the A and the B sub-unit libraries was synthesised and assembled.
• the structure was used to inhibit binding of anti- CD4 monoclonal RPAT4 (Serotech, Abingdon, UK) in an enzyme linked immuno absorbant assay (ELISA).
• 96 well ELISA plates were coated overnight with a 0.2mg/ml solution of rsCD4 in coating buffer (0.05M carbonate-bicarbonate buffer pH9.0) at 4°C. Plates were washed extensively using TBS-T (tris-buffered saline pH ⁇ .O .05% (v/v) Tween 20) and test and control DNA structures were diluted (1 :2) across the plate in TBS from a starting concentration of 100DM. The plates were incubated for 40 minutes at 37°C and washed with TBS. A 100ng/mi preparation of antibody RPAT4 in PBS was added to the plate and incubated for 40 minutes at 37°C.
• coating buffer 0.05M carbonate-bicarbonate buffer pH9.0

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• 2000
  • 2000-11-13 CN CN00815433A patent/CN1390253A/zh active Pending
  • 2000-11-13 EP EP00983130A patent/EP1228201A1/en not_active Withdrawn
  • 2000-11-13 KR KR1020027006140A patent/KR20020059727A/ko not_active Application Discontinuation
  • 2000-11-13 CZ CZ20021472A patent/CZ20021472A3/cs unknown
  • 2000-11-13 BR BR0015484-9A patent/BR0015484A/pt not_active IP Right Cessation
  • 2000-11-13 SK SK604-2002A patent/SK6042002A3/sk not_active Application Discontinuation
  • 2000-11-13 AU AU20001/01A patent/AU782880B2/en not_active Ceased
  • 2000-11-13 MX MXPA02004727A patent/MXPA02004727A/es unknown
  • 2000-11-13 RU RU2002113755/04A patent/RU2002113755A/ru unknown
  • 2000-11-13 CA CA002391084A patent/CA2391084A1/en not_active Abandoned
  • 2000-11-13 HU HU0203914A patent/HUP0203914A2/hu unknown
  • 2000-11-13 PL PL00358811A patent/PL358811A1/xx unknown
  • 2000-11-13 JP JP2001538503A patent/JP2003522524A/ja active Pending
  • 2000-11-13 WO PCT/EP2000/011197 patent/WO2001036624A1/en not_active Application Discontinuation
• 2002
  • 2002-05-10 NO NO20022231A patent/NO20022231L/no unknown
• 2003
  • 2003-07-03 HK HK03104743.9A patent/HK1052529A1/zh unknown

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