CN1390253A - High order nucleic acid based structures - Google Patents

Abstract

The present invention relates to nucleic acid based molecular structures that bind to other molecular entities, especially entities other than nucleic acids themselves. In particular, 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.

Description

Advanced Nucleotype Structure

The present invention relates to nucleic acid-type molecular structures associated with other molecular entities, especially entities other than the nucleic acid itself. In particular, the present invention relates to nucleic acid-based molecular structures that are pharmaceutically active by binding to specific molecular targets and thereby affecting disease states. The invention also relates to nucleic acid-based molecular structures having diagnostic utility.

There is a great need to provide compositions of matter that can specifically alter the activity of a particular protein or modulate the expression of a particular gene product. Molecules capable of forming specific binding interactions with other molecules are particularly desirable, especially such molecules capable of exhibiting specific binding in an in vivo setting.

In response to these needs, methods have been developed enabling the preparation of various molecular libraries from which such compositions of matter are selected. A related example is the very sensitive binding specificity found in antibody molecules, and several efficient techniques are currently available for the development of monoclonal antibodies and their recombinant derivatives. A large number of therapeutic drugs and many diagnostic and research tools are available. All such preparations are protein molecules and thus can only be produced using biological systems. On the other hand, fully synthetic binding molecules have been produced. These binding molecules are molecules selected from diverse libraries of similar or variant molecules of the same chemical class, and are generally selected using a screening system for a surrogate for the desired therapeutic target or for some aspect of its activity. The screening of huge small molecule chemical libraries has always been the classic way to develop conventional small molecule drugs, and currently synthetic peptide libraries and synthetic nucleic acid molecule libraries are also used to screen potentially effective drugs.

For nucleic acid libraries, the general technical approach is to use single-stranded RNA or DNA molecules of known unit length. Binding to target molecules is not pre-configured, but may depend on secondary structure formation within the DNA (or RNA) molecule itself that facilitates binding to other molecular entities (Bock LC et al. 1992 Nature 355 :564-566; Kubrik, MF et al. 1994 Nucleic Acids Res. 22 :2619-2626). No previous attempts have been made to prepare nucleic acid molecules of therapeutic and diagnostic utility that contain prosequences of secondary (or higher) structural configuration, which is the object of the present invention.

The present invention relates to novel higher order nucleic acid structures and novel uses of such structures.

Several types of higher order nucleic acid structures are known in the art. One such nucleic acid is called an aptamer, and differs from the molecules of the invention in terms of size, preparation and topological complexity. Methods involving in vitro evolution or selection from large random libraries are suitable for the development of both RNA and DNA aptamers. RNA molecules capable of promoting enzymatic processes such as polynucleotide kinase activity have evolved through repeated cycles of selection (Lorsch JR and Szostak JW1994 Nature 371 :31-36), and short-term genes that inhibit human phospholipase _A2 with high specificity have been screened. Single-stranded DNA molecules (Bennett CF et al. 1994 Nucleic Acids Res. 22 :3202-3209). Other researchers have independently developed RNA-type aptamers or DNA-type aptamers capable of binding and inhibiting certain functions of human thrombin (Bock LC et al. 1992 Nature 355 : 564-566; Kubrik, MF et al. 1994 Nucleic Acids Res. 22 : 2619-2626).

Other types of higher order nucleic acid structures include "branched DNA" (Horn, T. and Urdea, MS1989, Nucleic-Acids-Res. 17 :6959-6967), whereby one or more regions of a DNA molecule hybridize to complementary nucleic acid molecules ("probes") can themselves hybridize to other DNA molecules so as to amplify the amount of DNA bound to said DNA probes. However, the complexes described by Horn and Urdea (supra) extend linearly, so that new hybrid nucleic acid molecules designed do not hybridize to previously annealed molecules, but instead hybridize to other new molecules imported to form branched DNA structures. In fact, such complexes are designed to form as many branches as possible in order to provide more annealing sites for the signaling nucleic acid probes.

Other geometries provided by the base-pairing properties of nucleic acids have been exploited in the fields of materials science and nanotechnology (Aliviatos, AP et al. 1996 Nature 382:609-611; Mao C. et al. 2000 Nature 407 :493-496; Yurke, B. et al. 2000 Nature 406 :605-608). Elegant technical methods for the production and analysis of higher-order nucleic acid-type structures, including quadrilateral, cuboctahedral, and triangular motifs connected into a grid, have been described (Chen J. et al. 1989, J.Am.Chem.Soc. 111 1994, J.Am.Chem.Soc. 116 :1661-1669; US Patent No. 5,278,051; US Patent No. 5,468,851; US Patent No. 5,386,020 and US Patent No. 6,072,044). The geometries are closed structures assembled using an iterative method involving restriction enzymes and DNA ligation. Nodes in the structure can be anchored by interchain cross-links that create a rigid structure or by cross-annealing distinct branches to obtain more flexible branches. The prior art does not cover unclosed (ie end-connected) geometries. The prior art does not include geometric structures comprising modified nucleic acid regions, and does not include geometric nucleic acid structures in combination with other molecular entities. The prior art does not include the use of random or semi-random libraries of nucleic acid structures.

With regard to novel uses of higher order nucleic acid structures, it is known in the art to employ "lower order" nucleic acids per se as therapeutic and diagnostic molecules. In particular, there are many examples of nucleic acid molecules with potential and or actual therapeutic activity. These molecules function as antisense molecules, triplex agents, or as RNA molecules with endoribonuclease activity ("ribozymes"). In all these molecular appearances, the mode of therapeutic nucleic acid belongs to the modulator of protein expression by the mechanism of action of attenuating or blocking protein translation. In all these cases, the target binding specificity is nucleic acid to nucleic acid binding specificity. These features (translation regulation, nucleic acid to nucleic acid binding) are different from the present model. A particularly inventive feature of the present invention is the use of higher order nucleic acid structures that are active in binding to target molecules.

Other "lower" nucleic acid structures, especially aptamers, have been tested for use as therapeutic and diagnostic molecules. Certain other nucleic acid molecules, especially ribozymes, have been identified as having potential pharmaceutical value because of their enzymatic activity. For closed geometry, while US Patent No. 5,278,051 speculates on its possible utility as a solubilizer or a controlled release vehicle for small molecule therapeutics, it does not consider its pharmaceutical utility.

A first aspect of the invention relates to novel higher order nucleic acid-type structures, in particular open geometry structures. Furthermore, the invention also relates to the use of such structures as pharmaceutical and/or diagnostic agents. The invention also relates to higher order nucleic acid-type structures comprising modified nucleotides. The invention also relates to higher order nucleic acid structures comprising regions of randomized or semi-randomized nucleotides. The invention also relates to higher order nucleic acid-based structures conjugated to other molecular entities such as proteins.

The structures of the present invention utilize the Watson-Crick base pairing rules in engineered regions of the double-stranded structure. Single-stranded nucleic acid molecules can anneal (hybridize) with other single-stranded molecules by virtue of the complementarity between bases. Although such base annealing of two single-stranded molecules typically produces linear double-stranded molecules, other structures can also be produced, such as hairpin loops where one molecule has internal base-pair complementarity; When the two ends of each single-stranded molecule are complementary to each other, a circular structure can be generated. Using single-stranded nucleic acid sequence strategy design, it is possible to design a single molecule that can simultaneously anneal to two or more other molecules. Nucleic acid complex.

The overall spatial structure and topology of double-stranded DNA molecules are well known. Double-stranded DNA molecules are quite flexible, and the double helix of DNA molecules can adopt many conformations with different rotation angles between adjacent base pairs along the helix. Naturally occurring single-stranded nucleic acid molecules such as RNA adopt an optimal conformation in solution. The conformation is dependent on base-pair interactions within the same molecule, resulting in a stable structure consisting of a double-stranded stem and a single-stranded loop. The molecule will adopt the lowest energy conformation, and this lowest energy structure of known RNA sequences can be predicted computationally (Jaeger JA et al 1989 Proc. Natl. Acad. Sci USA 86 :7706-7710). Attempts have been made to produce software that predicts DNA folding, with some success (Nielsen DA et al. 1995 Nucleic Acids Res. 23 :2287-2291). The comparison of the spatial structures between nucleic acid molecules and protein molecules shows that the structural arrangement differences between these two types of molecules are very significant, and support the basic idea of the present invention. A typical globular protein such as myoglobin with a molecular weight of 17 kDa has a longest dimension of 3 nm. Larger globular proteins such as bovine serum albumin with a molecular weight of 68 kDa have a longest dimension of 5 nm (Cohen C. in Wolstenholme GEW and O'Connor M. (eds.), Ciba Foundation Symposium, London, J&A Churchill, 1966) . The diameter of the double-stranded helix itself is 2 nm, and the stretched length of a DNA chain with a small number of base pairs (for example, 100) is about 30 nm. Thus, while the density of DNA or any other nucleic acid molecule is much lower than that of a typical protein, the topology of the DNA molecule, even its most common natural structure, the double helix, allows the DNA molecule to readily cover large areas of the exposed surface of almost any protein molecule. part. Especially if the topology of common monofilament DNA is also changed in this way, said DNA can occupy most of the spatial regions in a way more similar to much denser protein molecules. Known to assemble nucleic acid structures consisting of multiple interconnected strands, each strand being a very short (<50) stretch of nucleotide sequence, structures with an overall dimension of 10-500 nm can be readily obtained. A particular object of the present invention is to provide such nucleic acid molecule preparations.

The structural basis of the present invention is to prepare DNA or RNA secondary structure molecules formed due to the interaction of two or more nucleic acid molecules or due to the interaction of different defined segments in each nucleic acid molecule. In the present invention, the information content of DNA or RNA is not used as a coding entity to express a therapeutic protein, nor as a blocking entity (antisense) for nucleic acid metabolism and gene expression, but to direct the assembly of a specific three-dimensional shape molecular structure .

The present invention includes nucleic acid molecules, especially synthetic DNA molecules, that form three-dimensional (non-planar) molecular structures through specific base pairing in their constituent molecules. Specifically, a DNA molecule is designed to contain one or more sequence regions ("domains") that can anneal to other molecules in its group, ultimately forming a complex three-dimensional nucleic acid structure. In this scheme, an approximate cubic structure can be formed by self-annealing of 6 synthetic DNA molecules, each containing 4 complementarity domains, so that each molecule interacts with 4 other molecules, and each Each molecule functions effectively like the sides of a six-sided cube. The structure is open (not covalently closed), flexible and especially suitable for modification by addition of other functional groups or structural groups.

In an advanced nucleic acid-type structure of the present invention, a plurality of nucleic acid molecules each comprising two or more self-complementary sequence domains are provided, so that the nucleic acid molecules can self-fold and pass specific bases The pairing process interacts to form specific three-dimensional molecular structures. For certain applications, the known chemical instability of unmodified DNA molecules has been a considerable problem for uses such as therapeutic drugs. Several methods are currently used to protect DNA molecules from enzymatic attack and degradation. These methods typically involve the use of modified phosphodiester backbones (methyl phosphate, phosphorothioate, peptide nucleic acids), or the use of phosphoramidite, phosphorothioate, or phosphorodithioate linkages at the 5' or 3' Cap the ends. A particular object of the present invention is the use of modified nucleic acids or non-natural nucleic acids in said higher-order nucleic acid-type structures. Furthermore, a particularly desirable feature is increased flexibility in terms of binding specificity achieved through the application of hybrid chemistry and substitution of non-natural nucleic acid backbones.

The nucleic acid subunits of the higher order nucleic acid structures of the present invention may be natural homotype or heterologous nucleic acid subunits, such as DNA containing RNA sequence segments. Segments of RNA sequences within a DNA helix are known to alter helix formation in solution (Wang, A. et al., 1982 Nature 299 :601-04). The presence of conformational diversity in defined nucleic acid sequence segments may be important in changing the binding specificity to target proteins, a phenomenon known in the art that thrombin aptamer binding specificity depends on short segments of higher tertiary structure ( Griffin, L. et al. 1993 Gene 137 :25-31). In addition, non-natural phosphate backbone analogs can be used to enhance stability and also alter binding specificity to a desired target protein. Latham et al. (Latham, JA et al. 1994 Nucleic-Acids-Res. 22 :2817-22) provide an example whereby the modified nucleotide 5-(1-pentynyl)-2 '-deoxyuridine instead of thymidine. The present invention includes molecules composed of single-stranded nucleic acid sequence segments interspersed with double-stranded structural sequence segments, as well as other chimeric compounds that contain different chemical substructures but are linked by conventional base pairing rules. molecular. Such structures may also contain DNA molecules and RNA molecules in combination.

Higher order molecular structures of the invention may be assembled from single or multiple nucleic acid molecules according to any protocol in the art, and may include various synthetic nucleic acids or fragments from very large molecules such as recombinant plasmids. The structure can be constructed following self-folding (auto-assembly) or facilitated folding of a single linear DNA molecule. Facilitated folding can be mediated by protein entities (enzymes such as ligases, topoisomerases, endonucleases, polymerases) or by interaction with non-protein physicochemical conditions (pH, temperature, ionic conditions). On the other hand, or in combination with the methods described above, molecules can be assembled by interacting with molecules bound to a solid substrate, or DNA that folds into higher order structures during all or part of the assembly is spatially defined or anchored. Certainly.

A second aspect of the present invention is to provide libraries of nucleic acid molecules containing a wide variety of semi-random molecules, some of which may have a desired topology capable of interacting with a target molecule in a specific manner. Included in this aspect of the invention are libraries of nucleic acid molecules characterized by a guiding backbone that facilitates assembly of common structural subunits. The incorporation of random sequence segments within each subunit maximizes library diversity and potential functional utility with respect to activity in selective binding assays. In the second aspect of the present invention, an embodiment of a library formed by mixing n independent populations (sets) of synthetic DNA molecules (subunits) is employed. In this regard, the population of synthetic nucleic acid subunits is large, depending on the degree of randomization present within the variable segments of the subunits. Subunit diversity is further constructed in other embodiments by varying the position of the variable domains, varying the number of variable domains (with intervening fixed sequence segments), and varying the length of any given variable domain. Preferably, n independent subunit populations are mixed in one annealing cycle to generate libraries with multiple nucleic acid structures and individual sequence diversity. Obviously other embodiments may include multiple annealing cycles and multiples of the integer n. In this approach, a particular feature of the library is the ability to modulate the complexity of interactions between subunits through rational design and placement of complementary or leader sequence segments.

A third aspect of the present invention is the novel use of higher-order nucleic acid-type structures, especially pharmaceutical and diagnostic uses. In this regard, these structures are capable of binding to specific target molecules, typically proteins or proteinaceous target molecules. Where preferred embodiments include a single protein target, further embodiments are contemplated wherein the target is a protein complex (e.g., a cell surface receptor) composed of multiple protein subunits, and the protein complex serves as A whole is bound by the molecule of the first aspect of the invention. Other embodiments of the third aspect include binding to cellular targets or various cells identified by the ability to bind to molecules of the first aspect of the invention. Further embodiments include binding to a cell, especially a cell surface target or target complex including non-protein components such as carbohydrate or lipid components. Protein, carbohydrate and lipid entities and or complexes thereof may be disease specific entities or normal constituents of tissues or cells. The target or target complex includes a viral particle or a virus-derived component (eg, capsid protein) or a host component of the capsid. The target or target complex may include in its composition metal ions or other inorganic chemicals or chemical groups, and may be naturally occurring or introduced by treatment with exogenous agents. Target receptors may include, for example, IL-2 receptor or other cytokine receptors such as IL-3 receptor, M-CSF receptor, GM-CSF receptor, and many others. Also, IgE surface molecules that block the activation process of crosslinking, such as by blocking IgE binding to the IgE receptor, would be highly desirable. Other surface molecules, including members of the cluster of differentiation (CD antigens) family, are desirable targets for modulating disease, especially autoimmune component diseases.

The present invention is designed to have particularly wide application in the field of therapeutic molecules. The molecular structures of the present invention are expected to agonize or antagonize specific receptors or enzymatic processes to produce a therapeutic effect without the disadvantages of conventional protein drugs such as immunogenicity. Accordingly, the present invention extends to methods for treating or preventing a disease or condition comprising administering to a subject an effective amount of said molecular structure. The invention also extends to the use of such structures in in vivo and in vitro diagnostics.

A fourth aspect of the present invention comprises higher order nucleic acid-type structures containing modified nucleotides within said structure. It is particularly desirable to confer diversity by derivatization of said library subunits and or by including modified bases (thiolated bases, biotinylated bases, ε-amino derivatized bases, etc.) during their synthesis, This may be independent of or concurrently with the diversity conferred by the second aspect above. Thus, a highly diverse library with diversity both at the level of sequence composition and at the level of sequence length is obtained. Such parameters can be fixed within defined ranges for different libraries and for different targets. Higher order nucleic acid structures may contain, in addition to the stability or binding modulation features described above, modified nucleotides that impart specific desirable properties to the structure. Such additional desired modifications may be implemented in the first or second aspects of the invention, including the use of hydrophobic sequence stretches, inclusion of psoralen or acridine groups, attachment of hapten groups such as biotin or different charged Groups attached to side chains such as amino or carboxyl groups to facilitate binding to specific target molecules. In a further preferred embodiment, such groups can be used as attachment points for other molecules, eg other nucleic acid molecules or proteins such as antibodies or enzymes.

A desirable feature of the molecules of the invention is high stability both in vitro and in vivo. Not only is the chemical composition of the nucleic acid structure highly influential, but the physical size of the molecule needs to be controlled to minimize shear damage in solution and maximize functional utility in vivo. For this reason, the present invention is preferably a multi-stranded nucleic acid structure constructed from ordinary small (<80mer) subunits. On the other hand, it may be desirable to employ structures composed of larger subunits (>80mers), which are also within the scope of the present invention.

A fifth aspect of the invention includes higher order nucleic acid-based structures linked to other molecular entities. Specifically, this aspect includes a nucleic acid linked to one or more specific sites on another molecular entity at one or more specific sites, and the modified nucleotides of the fourth aspect of the present invention facilitate binding to said nucleic acid. specific connection. This aspect specifically includes higher order nucleic acid-based structures linked to pharmaceutically or diagnostically relevant molecular entities such that said nucleic acid binds to specific molecular targets associated with a disease, said linked molecular entities then being used to combat or detect said disease.

Pharmaceutically relevant entities include cytokines, Fc portions of antibodies, other antibody-related entities, toxins, enzymes, drugs and prodrugs, receptor agonists or antagonists, the receptor molecule itself (especially the ligand-binding domain) , radioisotopes, pharmaceutically active nucleic acids, drug delivery vehicles such as liposomes, live or attenuated microorganisms, photoactive moieties, and other molecular entities that induce vaccine effects. Diagnostically relevant entities include, inter alia, radioisotopes, photoactive moieties such as those that generate a chemiluminescent signal, fluorescent dyes, enzymes and signal transporters such as microbeads.

In general, the present invention includes the following objectives:

A three-dimensional polynucleic acid structure consisting of multiple interconnected strands of nucleic acid molecules or segments thereof interacting through specific base pairing interactions of two or more molecules, characterized in that the structure is not covalent closed.

· A corresponding polynucleic acid structure, characterized in that the structure is formed by two or more nucleic acid molecule chains.

· A corresponding polynucleic acid structure, characterized in that said structure is formed by three or more nucleic acid molecular chains.

• A corresponding polynucleic acid structure, characterized in that said structure is cuboidal or substantially cuboidal in form.

• A corresponding polynucleic acid structure, wherein the cuboidal structure is formed by 6 nucleic acid molecular chains, wherein each molecular chain acts as a side of a six-sided cube.

• A corresponding polynucleic acid structure, wherein each nucleic acid sequence comprises two or more domains that can anneal to other molecules within its group.

• A corresponding polynucleic acid structure, wherein each nucleic acid sequence comprises four domains.

• A corresponding polynucleic acid structure, wherein said structure comprises a nucleic acid molecule consisting of a single-stranded nucleic acid sequence segment and a double-stranded structural sequence segment interposed in said single-stranded nucleic acid sequence segment.

- A corresponding polynucleic acid structure according to any one of claims 1-8, wherein each nucleic acid strand is less than 80 nucleotides, preferably less than 50 nucleotides.

• A corresponding polynucleic acid structure, wherein said structure has the assembly (A1+B1+C1)+(A2+B2+C2) shown in Figure 1 .

• A polynucleic acid structure as defined above, wherein said structure contains subunits consisting of variable randomized sequence segments in order to obtain semi-random molecules or segments thereof capable of interacting with target molecules.

A three-dimensional polynucleic acid structure containing subunits consisting of multiple interconnected strands of nucleic acid molecules or segments thereof interacting through specific base pairing interactions of two or more molecules, wherein the structure is co- The valency is closed and contains subunits composed of variable randomized sequence segments in order to obtain semi-random molecules or segments thereof capable of interacting with target molecules.

• A polynucleic acid structure as defined above, wherein said sequence composition and sequence length are variable.

• A corresponding polynucleic acid structure, wherein said variable sequence composition is obtained by one or more modifications of nucleotides within said sequence.

• A polynucleic acid structure as defined above, wherein said structure contains a plurality of nucleic acid sites or groups which can be bound or linked to other molecules or to a solid substrate.

• A corresponding polynucleic acid structure, wherein said other molecule is a protein, an enzyme, a lipoprotein, a glycosylated protein, an immunoglobulin or a fragment thereof.

• A corresponding polynucleic acid structure, wherein said other molecule is a nucleic acid.

• A corresponding polynucleic acid structure, wherein said molecule is a pharmaceutically effective molecule.

A pharmaceutical composition comprising a polynucleic acid structure as defined above, and optionally suitable carriers, excipients and diluents and/or other pharmaceutically effective compounds .

- Use of a corresponding polynucleic acid structure as a diagnostic agent.

• A use of a polynucleic acid structure as defined above to provide a library of topological diversity affecting specific randomization in order to obtain different functions and/or activities.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1

The diagram shows the step-by-step assembly of six single-stranded molecules called A ₁ , A ₂ , B ₁ , B ₂ , C ₁ and C ₂ into an open cubic nucleic acid structure. Assembly occurs by conventional antiparallel base pairing between nucleic acid molecules. The dimerization intermediates formed between molecules B ₁ and C ₁ and B ₂ and C ₂ are shown. Trimeric structures formed between molecules A ₁ , B ₁ and C ₁ and A ₂ , B ₂ and C ₂ are shown. Assembly of the cuboidal structure is accomplished by linking the two trimer parts, denoted as the molecule (A ₁ +B ₁ +C ₁ )+(A ₂ +B ₂ +C ₂ ). figure 2

Sequences of oligonucleotide subunits IL2R-1 and IL2R-2, including DNA structures with binding activity to IL-2 receptors. image 3

Sequences of oligonucleotide subunits TB-R1 and TB-R2, comprising a DNA structure with binding activity to human thrombin.

Example

The invention is illustrated by the following examples, which should not be construed as limiting the invention in any way.

Example 1 A method for inhibiting IL2-dependent cell lines using a DNA construct selected from a DNA construct library.

Two libraries of synthetic DNA molecules are synthesized, each library containing a randomized sequence region.

Library A contains the following structural molecules:

5'AGTCCCAAGCTGGCT(N) ₁₃ CTCCATCGTGAAGTCAGCCAGCTTTGGACT

Library B contains the following structural molecules:

5'GACTTCACGATGGAGGTCAGAATGTGAATA(N) ₁₀ TATTCACATTCTGAC

These sequences are designed to facilitate cross-annealing and represent subunits of a library of structures formed by mixing and cross-annealing different subunits according to the protocols of the present invention.

Libraries of oligonucleotides (subunits) with phosphorothioate linkages were synthesized for maximum stability in the presence of serum factors and purified by HPLC. Purified oligonucleotides were obtained from GenoSys Biotechnologies (Cambridge, UK). A library of DNA constructs is assembled using a single cycle of cross-annealing. Subunit libraries A and B were denatured, pooled and annealed in a solution of 50 mM Tris pH 7.4, 100 mM NaCl, 5 mM EDTA at 37°C. Pooling of subunit libraries A and B was performed at equimolar concentrations (1 D M). In other experiments, different molar ratios were used for mixing. Assembly of the subunits was confirmed by gel electrophoresis.

A library of DNA structures was screened for structures capable of binding to the extracellular domain of the IL-2 receptor (IL2R). This screening adopts according to published method (Meidel, MC etc. 1988 Biochem.Biophys.Res.Commun.. 154 :372-379; Meidel, MC etc. 1989, J.Biol.Chem. 264 :21097-21105) preparation Soluble recombinant IL2R was performed. Recombinant IL2R was covalently bound to surface-activated magnetic beads using the protocol suggested by the supplier (Bangs Labs, Fishers, IN, USA), and IL2R-magnetic beads were used as an affinity surface to select bound structures from the DNA structure library . IL2R-magnetic beads were reacted with the library in controlled reactions under various experimental conditions including the presence of chaotropic salts. The library (DNA) concentration was approximately 100 nmol in the annealing solution as described above. After extensive wash cycles with 75 mM Tris.HCl, 200 mM NaCl, 0.5% n-octylglucoside pH 8.0, bound molecules were recovered directly from the beads by polymerase chain reaction (PCR). The PCR was performed using the primer PRA1 (5'-AGTCCCAAGCTGGCT) using standard reagent systems and conditions to recover the library A fraction. In separate reactions, primers PRB1 (5'GACTTCACGATGGAG) and PRB2 (5'GTCAGAATGTGAATA) were used to recover the library B fraction. The PCR products were cloned and sequenced using standard reagent systems and methods.

A number of sequences were recovered and identified as being derived from Subunit Library A and Subunit Library B. One pair of sequences was synthesized using phosphorothioate chemistry as previously described. Oligonucleotides IL2-R1 and IL2-R2 (sequence shown in Figure 2) were purified and assembled as previously described and then used for cellular assays of IL-2 antagonism.

TALL-104 (ATCC #CRL-11386) is a human T-cell leukemia cell line. The cells are grown in suspension culture and require IL-2 for optimal growth. The cells could grow for some time in the absence of IL-2, but their growth was significantly slower. Let the cells contain 50-100u/ml recombinant human IL-2 (Life Technologies, Paisley, UK) and supplemented with 10% (v/v) heat-inactivated fetal calf serum Iscoves modified Dulbeccos medium (Life Technologies, Paisley, grown in the UK). Cells were cultured in an atmosphere of 8-10% _CO2 . Dilutions of annealed IL2-R1/IL2-R2 DNA preparations and control DNA samples containing random sequences of the same profile length were prepared in IL-2-containing medium. Parallel dilution series were prepared using medium without IL-2. The dilution series ranged from 50 □M DNA to 390nM DNA. Assays were performed using incompletely confluent TALL-104 cells plated the day before in 96-well microtiter plates. Cells were harvested by centrifugation, washed with pre-warmed (37°C) phosphate-buffered saline, and then treated with DNA-containing medium for 48 hours. Processing was performed in quadruplicate. At the end of 48 hours, proliferation was assessed in a colorimetric assay using a commercially available tetrazolium compound and following the instructions provided by the supplier (Promega, Southampton, UK). Microtiter plates were read at 540nm.

The results showed that the annealed DNA preparation inhibited the growth of the TALL-104 cell line under conditions in which the respective synthetic oligonucleotides IL2-R1 and IL2-R2 were inactive.

Example 2 Method for selecting DNA structures that bind to human thrombin.

The library described in Example 1 was used to select DNA constructs capable of binding human thrombin. The library was screened as in Example 1 using a preparation of human thrombin (Sigma, Poole, UK) bound to surface-activated magnetic beads. Thrombin-magnetic beads were reacted with the DNA structure library according to Example 1, except that washing after binding was performed in a solution of 20 mM Tris acetic acid, pH 7.4, 140 mM NaCl, 5 mM KCl, and 1 mM MgCl ₂ . Using the reaction and primer set as used in Example 1, the bound molecules were directly recovered from the magnetic beads by PCR. The PCR products were cloned and sequenced using standard reagent systems and methods.

A number of sequences were recovered and identified as being derived from Subunit Library A and Subunit Library B. One pair of sequences was synthesized using phosphorothioate chemistry as previously described. Oligonucleotides TB-R1 and TB-R2 (sequence shown in Figure 3) were purified and assembled. The TB-R1/TB-R2 complex was used in the thrombin inhibition assay. Clotting times were determined using a fibrometer at 37°C with freshly prepared adult plasma from healthy donors. The thrombin standard curve drawn by plotting the clotting time against the thrombin concentration was used to determine the degree of thrombin inhibition. In the assay, clotting times are determined for three logarithmic DNA structures.

The results show that coagulation activity is inhibited in the presence of the TB-R1/TB-R2 DNA complex.

Example 3 Method for selecting DNA constructs that bind to recombinant soluble CD4.

The library described in Example 1 was used to select DNA constructs capable of binding recombinant soluble CD4 (rsCD4) preparations. The DNA construct library was screened using CD4 preparations immobilized on activated magnetic beads (BioDesign, Saco, ME, USA) as previously described. Library screening, washing and PCR selection were all as described in Example 2. Single oligonucleotide pairs derived from the A and B subunit libraries are synthesized and assembled. The construct was used to inhibit the binding of anti-CD4 monoclonal RPAT4 (Serotech, Abingdon, UK) in an enzyme-linked immunosorbent assay (ELISA).

A 96-well ELISA plate was coated overnight at 4° C. with 0.2 mg/ml rsCD4 solution in coating buffer (0.05 M carbonate-bicarbonate buffer pH 9.0). Wash each plate thoroughly with TBS-T (tris buffered saline pH 8.0.05% (v/v) Tween 20), the DNA structure to be tested and the control DNA structure are in TBS from the initial concentration of 100 M in the plate Dilutions were performed (1:2). After incubation of the plates at 37°C for 40 minutes, the plates were washed with TBS. A 100 ng/ml formulation of antibody RPAT4 in PBS was added to the plate and incubated at 37°C for 40 minutes. After washing the plates, bound RPAT4 was detected using an alkaline phosphatase-labeled sheep anti-mouse preparation (Sigma, Poole, UK) and the chromogenic substrate Sigma Fast OPD (Sigma, Poole, UK). In some assays, the DNA is co-incubated with RPAT4 monoclonal antibody. Read the color intensity using a plate reader and compare the signal from test and control wells. The results showed that the binding of RPAT4 to rsCD4 was significantly inhibited in the presence of the DNA construct.

Claims (22)

1. A three-dimensional multi-nucleic acid structure, the nucleic acid structure is composed of a plurality of nucleic acid molecular chains connected to each other by nucleic acid molecules or segments thereof through specific base pairing interactions of two or more molecules, characterized in that The structure is not covalently closed. 2. The polynucleic acid structure according to claim 1, characterized in that said structure is formed by two or more nucleic acid molecule chains. 3. The polynucleic acid structure according to claim 2, characterized in that said structure is formed by three or more nucleic acid molecule chains. 4. The polynucleic acid structure according to any one of claims 1-3, characterized in that the structure is cubic or substantially cubic in form. 5. The polynucleic acid structure of claim 3, wherein the cuboidal structure is formed by six nucleic acid molecular chains, wherein each molecular chain acts as a side of a six-sided cube. 6. The polynucleic acid structure of any one of claims 1-5, wherein each nucleic acid sequence comprises two or more domains that can anneal to other molecules within its group. 7. The polynucleic acid structure of claim 6, wherein each nucleic acid sequence comprises four domains. 8. The poly-nucleic acid structure according to any one of claims 1-7, wherein said structure comprises a nucleic acid molecule consisting of a single-stranded nucleic acid sequence segment and a double-stranded structural sequence segment interposed in said single-stranded nucleic acid sequence segment. 9. The polynucleic acid structure of any one of claims 1-8, wherein each nucleic acid strand is less than 80 nucleotides. 10. The polynucleic acid structure of claim 9, wherein each nucleic acid strand is less than 50 nucleotides. 11. The poly-nucleic acid structure according to any one of claims 1-10, wherein the assembly of said structure is (A1+B1+C1)+(A2+B2+C2) as shown in Figure 1 . 12. The polynucleic acid structure of any one of claims 1-11, wherein the structure contains subunits composed of variable random sequence segments, so as to obtain semi-random molecules or segments thereof capable of interacting with target molecules. 13. A three-dimensional polynucleic acid structure consisting of a plurality of nucleic acid molecular chains connected to each other by nucleic acid molecules or segments thereof through specific base pairing interactions of two or more molecules, wherein the structure is Covalently closed structures and containing subunits composed of variable random sequence segments in order to obtain semi-random molecules or segments thereof capable of interacting with target molecules. 14. The polynucleic acid construct of any one of claims 1-13, wherein the sequence composition and sequence length are variable. 15. The polynucleic acid structure of claim 14, wherein said variable sequence composition is achieved by one or more modifications to nucleotides within said sequence. 16. A poly-nucleic acid structure according to any one of claims 1-15, wherein said structure contains a plurality of nucleic acid sites or groups which can be bound or linked to other molecules or to a solid substrate. 17. The polynucleic acid structure of claim 16, wherein said other molecule is a protein, an enzyme, a lipoprotein, a glycosylated protein, an immunoglobulin or a fragment thereof. 18. The polynucleic acid structure of claim 16, wherein said other molecule is a nucleic acid. 19. The polynucleic acid structure of any one of claims 16-18, wherein said molecule is a pharmaceutically effective molecule. 20. A pharmaceutical composition comprising the polynucleic acid structure of claim 19 and optionally suitable carriers, excipients, diluents and/or other pharmaceutically effective compounds. 21. Use of the polynucleic acid structure according to any one of claims 1-18 as a diagnostic agent. 22. Use of a polynucleic acid construct according to claim 12 or 13 for providing a library of topological diversity affecting specific randomization in order to obtain different functionalities and/or activities.

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