JP2003522524A - Higher order nucleic acid type structure - Google Patents

Abstract

  (57) [Summary] The present invention relates to a nucleic acid-type molecular structure that binds to another molecular entity (in particular, an entity other than the nucleic acid itself). In particular, the present invention relates to nucleic acid-based molecular structures that have pharmacological activity by binding to specific molecular targets and thereby affecting disease states. The present invention also relates to nucleic acid-type molecular structures having diagnostic utility.

Description

Detailed Description of the Invention

The present invention relates to a nucleic acid-type molecular structure that binds to another molecular entity (in particular, an entity other than the nucleic acid itself). In particular, the present invention relates to nucleic acid-type molecular structures that have pharmaceutically activity by binding to specific molecular targets and thereby affecting disease states. The present invention also relates to nucleic acid-type molecular structures having diagnostic utility.

There is a great need to provide compositions capable of specifically altering the activity of particular proteins or modulating the expression of particular gene products. Specifically, a molecule capable of forming a specific binding interaction with another molecule,
In particular, there is a need for such molecules that exhibit specific binding in the in vivo environment.

In response to such demands, various methodologies have been developed which allow the production of libraries of different types of molecules for which such compositions are selected. A related example includes the exceptional specificity of binding found in antibody molecules. Currently, several important technologies exist for developing monoclonal antibodies and their recombinant derivatives. These techniques provide numerous therapeutic agents as well as many diagnostic and research tools. All such products are protein molecules and, as such, can only be produced using biological systems. Another fully synthetic binding molecule has been produced. These are molecules selected from a diverse library of similar or altered molecules of the same chemical type, generally with screening systems that provide surrogates of the desired therapeutic target or particular aspects of its activity. The molecule selected for use. Screening an enormous chemical library of small molecules has heretofore been the classical route for developing small molecule pharmaceuticals, but now libraries of synthetic peptides and synthetic nucleic acid molecules are also of great potential. Screened for therapeutically useful therapeutic agents.

In the case of nucleic acid libraries, the technical approach is now generally to use single-stranded RNA or DNA molecules of defined unit length. The basis for binding to the target molecule is not predetermined and may depend on the formation of secondary structure within the DNA (or RNA) molecule itself that facilitates binding to other molecular entities (Block). , LC et al., 1992, Nature, 355.
: 564-566; Kubrik, M .; F. 1994, Nucleic A
cids Res. 22: 2619-2626). Making nucleic acid molecules containing pre-configured regions of secondary (or higher order) structure for therapeutic and diagnostic utility has not previously been attempted, and therefore this Is the object of the present invention.

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

Several types of higher order nucleic acid constructs are known in the prior art. One type of such nucleic acid is called an aptamer and differs from the molecule of the present invention in its size, production and topological complexity. Methods involving either in vivo generation or selection from vast random library pools have been applied to the development of both RNA and DNA aptamers. RNA molecules that can facilitate enzymatic processes such as polynucleotide kinase activity have been generated by repetitive cycles of selection (Lorsch, JR & Szostak, J. et al.
W. , 1994, Nature, 371:. 31~36), and single-stranded DNA molecule short to allow for highly specific inhibition of human phospholipase A ₂ is selected (Bennett, C.F et al., 1994, Nucleic Acids
Res. 22: 3202-3209). Apopters based on either RNA or DNA that can bind to human thrombin and inhibit some of its function have been independently developed by other researchers (Bock, LC et al., 1992,
Nature, 355: 564-566; Kubrik, M .; F. Other, 1994
, Nucleic Acids Res. 22: 2619-2626).

Other types of higher order nucleic acid constructs include “branched DNA” (Horn, T. & U.
rdea, M .; S. , 1989, Nucleic-Acids-Res. , 17
: 6959-6967) are included. This allows one or more regions of a DNA molecule (“probe”) to hybridize to a complementary nucleic acid molecule, by itself, to amplify the amount of DNA associated with the DNA probe and thus to other DNA molecules.
It can be subjected to hybridization to the molecule. But Horn and U
The complex described by rdea (ibid.) grows linearly so that the newly hybridized nucleic acid molecule does not become hybridized to the previously annealed molecule, but is added to the others. It hybridizes to new molecules that are present, forming a structure of branched DNA. In fact, such a complex is simply designed to form as many branches as possible in order to provide more than one annealing point for the signal-producing nucleic acid probe.

Structures of other geometries brought about by the base pairing properties of nucleic acids are used in the fields of materials science and nanotechnology (Aliviatos, AP;
1996, Nature, 382: 609-611; Mao, C .; Other, 20
00, Nature, 407: 493-496; Yurke, B .; Other, 2000
, Nature, 406: 605-608). Sophisticated technical methods for the production and analysis of higher order nucleic acid-type constructs containing quadrilateral, cubic, octahedral and triangular motifs intricately linked in a grid have been described (Chen, J. et al., 198
9, J. Am. Chem. Soc. , 111: 6402-6407; Zhang.
1994, J. et al. Am. Chem. Soc. , 116: 1661-1669;
US Pat. No. 5,278,051; US Pat. No. 5,468,851; US Pat. No. 5,386,020 & US Pat. No. 6,072,044). These geometric shapes are closed structures manufactured using an iterative process involving restriction enzymes and DNA ligation. The nodes in these features can be fixed by cross-joints between stands to give a rigid morphology, or can be more flexible branches achieved by dissimilar stands that anneal to each other. Prior art is
It does not contain non-closed geometric structures (ie the ends are connected). The prior art does not include geometric structures containing regions of modified nucleic acid, and does not include geometric nucleic acid structures attached to other molecular entities. Prior art is
It does not involve using a library of randomized or semi-randomized nucleic acid constructs.

It is recognized that for the novel use of higher order nucleic acid constructs, it is known in the art to utilize "lower order" nucleic acids as therapeutic and diagnostic molecules themselves. Specifically, there are numerous examples of nucleic acid molecules that have potential therapeutic activity and / or actual therapeutic activity. These are as antisense molecules, triplex reagents, or as RNA molecules with endoribonuclease activity (“ribozymes”),
Works with either. In all of these methods, the use of therapeutic nucleic acids is as a modulator of protein expression by a mechanism of action that reduces or prevents protein translation. The specificity of target binding in all of these cases varies from nucleic acid to nucleic acid. These features (translational regulation, nucleic acid to nucleic acid binding) are in contrast to the uses of the present invention. A unique and inventive feature of the invention is the use of higher order nucleic acid constructs that have binding activity for a target molecule.

Other “lower” nucleic acid constructs, especially aptamers, have been tested as therapeutic and diagnostic molecules. Certain other nucleic acid molecules, especially ribozymes, have been identified as having enzymatic activity with potential pharmaceutical importance. In the case of closed geometry structures, US Pat. No. 5,278,051 speculates its possible utility as a solubilizer or sustained release vehicle for small molecule therapeutics, but it has no pharmaceutical utility. Is not considered.

The first aspect of the present invention relates to a novel higher order nucleic acid type structure, specifically to an open geometric structure. Furthermore, the invention also relates to the utility of such structures as pharmaceutical and / or diagnostic agents. The present invention also relates to higher order nucleic acid-type structures that include nucleotides with modifications. The invention also relates to higher order nucleic acid based structures comprising regions of randomized nucleotides or semi-randomized (semi-randomized) nucleotides. The invention also relates to higher order nucleic acid type structures attached to other molecular entities such as proteins.

The structure of the present invention utilizes the Watson-Crick base pairing rule in the engineered region of the double-stranded structure. Single-stranded nucleic acid molecules have the ability to anneal (hybridize) to other single-stranded molecules due to the complementarity between the bases. Such base annealing of two single-stranded molecules usually results in a linear double-stranded molecule, while it can result in a variety of other structures. For example, hairpin loops can be created if one molecule has internal base pair complementarity, and a ring can be created if the ends of each single-stranded molecule are complementary to each other. By effectively designing single-stranded nucleic acid sequences, it is possible to design individual molecules that can simultaneously anneal to two or more other molecules, and then these other molecules also in annealing. Various complexes of nucleic acids can be formed if they can anneal to additional molecules, including those already involved.

The overall size and topology of double-stranded DNA molecules is well understood. Double-stranded DNA molecules are extremely flexible and helices can assume a number of conformations that differ in the angle of rotation between adjacent base pairs along the helix. Naturally occurring single-stranded nucleic acid molecules such as RNA adopt various selected conformations in solution. Conformation is determined by base pairing interactions within the same molecule that result in the production of a stabilized structure composed of double-stranded stems and single-stranded loops. The molecule adopts the lowest energy conformation, and this structure for known sequences of RNA can be predicted by computational methods (Jaeger, JA et al., 1989, Proc. Natl. Acad. .Sc
i. USA, 86: 7706-7710). Various attempts have been made to produce predictable software for DNA folding, with some success (Nielsen, DA et al., 1995, Nucleic Ac.
ids Res. 23: 2287-2291). Although size comparisons of nucleic acid and protein molecules explain the striking differences in the structural arrangement between these two types of molecules, the idea underlying the present invention is also supported. A typical globular protein, such as myoglobin with a molecular weight of 17 kDa, has a longest dimension of 3n.
has a size that is m. Larger globular proteins, such as bovine serum albumin with a molecular weight of 68 kDa, have a longest dimension of 5 nm (Cohe
n, C.I. Wolstenholme, G .; E. W. &O'Connor, M
． In (ed.), Ciba Foundation Symposium, L
ondon, J & A Churchill, 1966). The diameter of the double-stranded helix is basically 2 nm, and DNA with a small number of base pairs (100 base pairs, etc.)
The chains reach a feature length close to 30 nm. Thus, although the diversity of DNA or any other nucleic acid molecule is far less than typical proteins, the topology of DNA molecules is almost arbitrary, even in their most structured natural form as a double helix. It can easily cover most of the exposed surface of the protein molecule. In particular, if the topology of normal monofilamentous DNA was so altered, the DNA could occupy most of the space in a manner more similar to much larger density protein molecules. An assembly of nucleic acid constructs composed of multiple interconnected strands each having only a region of short (<50) nucleotides has an overall size of 10 nm to 50 nm.
It can be appreciated that structures in the 0 nm range can easily be produced. Defining such a formulation of nucleic acid molecules is one particular object of the present invention.

The structure of the present invention is a secondary structure formed as a result of the interaction of two or more nucleic acid molecules or the interaction of different defined portions (segments) within each nucleic acid molecule. Based on the production of DNA or RNA molecules having In the present invention, the content of DNA or RNA information is not a coding entity for expressing a therapeutic protein or a blocking entity (antisense) for nucleic acid metabolism and gene expression, but a specific shape in three dimensions. Used to drive the assembly of molecular structures.

The present invention includes nucleic acid molecules (in particular, synthetic DNA molecules) that form a three-dimensional (non-planar) molecular structure by the formation of specific base pair within the molecule in the assembly. In particular, such DNA molecules anneal to other molecules within the assembly to ultimately form a complex three-dimensional nucleic acid structure, which results in one or more sequence regions (" Domain "). Under this scheme, a nearly cubic structure contains four domains, each of which is complementary,
Each molecule interacts with four other molecules, and can be formed by the self-annealing of six synthetic DNA molecules, each acting effectively like the individual edges of a hexahedral cube. The structure is open (not covalently closed), flexible and, in certain further embodiments, easy to modify by the addition of other functional or structural groups.

In one higher order nucleic acid type structure of the present invention, the nucleic acid molecule is allowed to fold itself,
And various nucleic acid molecules are provided, each containing two or more domains of self-complementary sequences capable of interacting with each other to form a particular three-dimensional molecular structure by a particular base pairing event. It It has been recognized that in certain applications, chemical instability of unmodified DNA molecules is a major problem for the use of therapeutic agents and the like. Several methods are currently available to protect DNA molecules from degradation by enzymatic attack. These include the use of modified phosphodiester backbones (methylphosphonates, phosphorothioates, peptide nucleic acids),
Alternatively, it is generally included to use a phosphoramidite bond, a phosphorothioate bond or a phosphorodithioate bond to cap the 5 ′ or 3 ′ end. The use of modified nucleic acids, i.e. non-natural nucleic acids, in higher order nucleic acid type structures is one particular object of the present invention. Moreover, a particularly desirable feature is the increased flexibility in binding specificity achieved by the use of various chemistries and alternative non-natural nucleic acid scaffolds.

The nucleic acid subunits of the higher order nucleic acid constructs of the invention may be homologous or heterologous (eg, the DNA-containing region of RNA) in reality. RNA regions within the DNA helix are known to alter coil formation in solution (Wang, A. et al.,
1982, Nature, 299: 601-04). The ability to bring about conformational diversity within the localized region of nucleic acids can be important in altering the binding specificity for a target protein, and the binding specificity of thrombin aptamers is associated with higher quaternary structure. This phenomenon, which was dependent on the short region of (Gr), is known in the art (Gr
ifin, L .; 1993, Gene, 137: 25-31). In addition, non-naturally occurring phosphate backbone analogs can be utilized to increase stability and also alter binding specificity for the desired target protein. Latham et al. (Latham, JA et al., 1994, Nucleic-A.
cids-Res. , 22: 2817-22) are modified nucleotides 5
-(1-Pentynyl) -2'-deoxyuridine provides an example where thymidine was used in a pool of random oligonucleotides. In the present invention, a molecule composed of regions of single-stranded nucleic acid interspersed with regions of double-stranded structure, and synthesized to contain different chemical sub-structures Other chimeric molecules that are linked using Such a structure is also a DNA
Molecules and RNA molecules can be combined.

The higher order molecular structures of the present invention are assembled from individual nucleic acid molecules or multiple nucleic acid molecules according to any scheme existing in the art, and thus synthetically derived from much larger molecules such as recombinant plasmids. Nucleic acid species or fragments. Such structures can be assembled according to the self-folding (self-assembly) or facilitated folding of one linear molecule of DNA. Facilitated folding is mediated by proteinaceous entities (enzymes such as ligases, topoisomerases, endonucleases, polymerases) or through interactions with non-protein physicochemical conditions (pH, temperature, ionic conditions) Can be done. Alternatively, and / or in combination with the above, such molecules can be assembled by interaction with molecules bound to a solid matrix, or DNA undergoing folding into a conformation is part of the assembly process. Or it can be assembled while being bound or fixed in space at some times.

A second aspect of the invention is a nucleic acid formed to contain a variety of semi-random molecules, some of which may have a desired topology capable of interacting with a target molecule in a specific manner. To provide a library of molecules. This aspect of the invention includes a library of nucleic acid molecules featuring a guiding framework that facilitates assembly of common structural subunits. Within each subunit, a sequence of randomized regions is incorporated, maximizing library diversity and potential functional utility with respect to activity in selective binding assays. In a second aspect of the invention, an embodiment is utilized in which the library is formed from a mixture of n different populations (sets) of synthesized DNA molecules (subunits). In this aspect, the population size of the synthesized nucleic acid subunits is large and is determined by the degree of randomization present in the variable segment of the subunit. Additional subunit diversity is achieved by altering the position of variable domains, by varying the number of variable domains (by interspersing regions of fixed sequence), and by any given It is incorporated into other embodiments by varying the length of the variable domains. To obtain a library of multiple nucleic acid constructs and individual sequence diversity, it is preferred to mix n different populations of subunits in one cycle of annealing. Clearly, other embodiments may include multiple cycles of annealing and multiple values of the total integer n. The specific characteristics of the library under this scheme are:
Careful design and placement of complementary or guide sequence regions can control the degree of complexity of interactions between subunits.

A third aspect of the invention is the novel utility of higher order nucleic acid type constructs, particularly for pharmaceutical and diagnostic uses. In this aspect, these structures are capable of binding to a particular target molecule, generally a protein or proteinaceous target molecule. Where the preferred embodiment comprises a single protein target, the target is a protein complex composed of multiple protein subunits such as cell surface receptors and the molecules of the first aspect of the invention are collectively Further embodiments are contemplated that are bound protein complexes. Other embodiments of the third aspect include binding to a cell target or cell type identified by the ability to bind the molecule of the first aspect. Further embodiments include binding to a target or target complex containing non-protein components, such as cell (particularly cell surface) carbohydrate or lipid components. The protein, carbohydrate and lipid entities and / or their complexes may be disease-specific entities or may be present as normal constituents of tissues or cells. The target or target complex includes a viral particle or virus-derived component such as a capsid protein, or a host-derived component of the viral core. The target or target complex can include metal ions or other inorganic chemicals or groups of chemicals in the composition and can be naturally occurring or introduced by treatment with an exogenous agent. it can. Targeted receptors include IL-2 receptors or other cytokine receptors (IL-3, M-
Receptors such as CSF, GM-CSF, etc.) are included. Similarly,
It is a highly desirable result to block IgE binding as well as block co-ordinated activation events at the receptor by surface molecules such as the IgE receptor. Other cell surface molecules, including members of the cluster differentiation lineage (CD antigens), are desirable targets for disease regulation, and particularly for disorders of the autoimmune component.

The present invention has widespread specific application in the field of therapeutic molecules. It is desirable that the molecular structures of the present invention act as agonists or antagonists for specific receptors or enzymatic processes for therapeutic benefit while at the same time not causing any of the drawbacks of conventional protein therapeutics such as immunogenicity. Be done. Therefore,
The present invention extends to methods of treating or preventing diseases or conditions. This method
Administering to the subject an effective amount of the molecular structure. The invention also extends to the use of such structures in in vivo and in vitro diagnostics.

A fourth aspect of the invention comprises a higher order nucleic acid type structure in which the modified nucleotide is contained within the structure. Apart from, or in addition to, the diversity provided by the second aspect above, modified bases (thiolated bases, by derivatizing subunits of the library and / or during their synthesis). It is particularly desirable to generate diversity by including biotinylated bases, ε-amino derivatized bases, etc.). Thus, a very diverse library with diversity at both the level of sequence composition and length is obtained. Such parameters can be fixed within the range defined for different libraries and different target applications. The higher order nucleic acid structure also contains modified nucleotides that, in addition to the features that provide stability or regulation of binding, as described above, can impart certain desired properties to the structure. Such additional desired modifications, which may be specifically exemplified under the first or second aspects of the invention, include the use of hydrophobic regions, the inclusion of psoralen or acridine groups, biotin and the like. It may include linking hapten groups, or linking to various charged side chains such as amino or carboxyl groups to provide facilitated attachment to a particular target molecule. In a further preferred embodiment, such groups may act as attachment points for other molecules such as additional nucleic acid molecules or proteins (such as antibodies or enzymes).

A desirable feature of the molecules of the invention is their great stability in vitro and in vivo. The chemical composition of nucleic acid constructs has a great influence, but the physical size of the molecule also serves to minimize shear damage in solution and to maximize its functional utility in vivo. Need control. Thus, the advantage of the present invention is generally for multi-stranded nucleic acid constructs constructed from small (<80 mer) subunits. Alternatively, larger subunits (> 80 mer)
The use of a structure consisting of) is also desirable and may be included within the scope of the invention as well.

A fifth aspect of the invention includes higher order nucleic acid type structures that are attached to other molecular entities. Specifically, this aspect includes a nucleic acid bound at one or more specific sites to one or more specific sites existing on the surface of other molecular entities. This facilitates specific binding to nucleic acids with modified nucleotides as in the fourth aspect of the invention. In particular, this embodiment includes higher order nucleic acid type structures attached to a pharmaceutically or diagnostically relevant molecular entity. Thereby, the nucleic acid binds to a particular molecular target associated with the disease, and then the attached molecular entity is used to treat or detect the disease.

Pharmaceutically related entities include cytokines, Fc portions of antibodies, other antibody related entities, toxins, enzymes, drugs and prodrugs, agonists or antagonists of the receptor, the receptor molecule itself (particularly , Ligand binding domains), radioisotopes, pharmaceutically active nucleic acids, drug-transporting vesicles such as liposomes, live or attenuated microbes, components that can be activated by light, and other molecular entities that induce vaccination effects. Is included. Diagnostically relevant entities include radioisotopes, components that can be activated by light, such as components that give rise to chemiluminescent signals, fluorescent chromophores, enzymes, and signal-transporting vesicles, such as beads.

In summary, the present invention comprises: 3 consisting of a plurality of strands in which nucleic acid molecules or parts thereof are interconnected by specific base pairing interactions of two or more molecules. A dimensional polynucleic acid structure, wherein the structure is not covalently 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 the structure is formed by three or more nucleic acid molecule chains. A corresponding polynucleic acid structure, characterized in that the structure is cubic or has essentially a cubic morphology. A corresponding polynucleic acid structure in which a cubic structure is formed by six nucleic acid molecular chains, each of which acts like an individual side of a hexahedral cube. • A corresponding polynucleic acid structure, in which each nucleic acid sequence comprises two or more domains capable of annealing to other molecules in the assembly. A corresponding polynucleic acid construct, in which each nucleic acid sequence comprises four domains. A corresponding polynucleic acid structure, wherein the structure comprises a nucleic acid molecule composed of regions of single-stranded nucleic acid interspersed with regions of double-stranded structure. -The polynucleic acid structure according to any of claims 1 to 8, wherein each nucleic acid strand has less than 80, preferably less than 50 nucleotides. The structure is the assembly (A1 + B1 + C1) + (A2 + B2 + C2) shown in FIG.
) Corresponding polynucleic acid structure. A corresponding polynucleic acid structure, in which the structure contains subunits composed of regions of variable randomized sequence, in order to obtain semi-random molecules or parts thereof capable of interacting with the target molecule body. A three-dimensional polynucleic acid structure composed of a plurality of interconnected strands of nucleic acid molecules or parts thereof by specific base pairing interactions of two or more molecules, said structures being covalently bound A polynucleic acid construct containing subunits composed of variable randomized sequence regions in order to obtain semi-random molecules or parts thereof that are closed in nature and are capable of interacting with a target molecule. -A polynucleic acid construct as defined above, wherein the sequence composition and length are variable. • A corresponding polynucleic acid construct, wherein the composition of the variable sequence is achieved by modification of one or more of the nucleotides within the sequence. • A polynucleic acid structure as defined above, wherein the structure contains nucleic acid sites or groups capable of binding or attaching to another molecule or solid matrix.・ Other molecules are proteins, enzymes, lipoproteins, glycosylated proteins,
A corresponding polynucleic acid construct which is an immunoglobulin or a fragment thereof.
A corresponding polynucleic acid structure, in which the other molecule is a nucleic acid. • A corresponding polynucleic acid structure, wherein said molecule is pharmaceutically effective. A pharmaceutical composition comprising a polynucleic acid construct as defined above and in the claims, optionally together with suitable carriers, excipients and diluents and / or other pharmaceutically active compounds. Use of the corresponding polynucleic acid structure as a diagnostic agent. -Use of a polynucleic acid construct as defined above to provide a library for varying various specifically randomized topologies to obtain various functionalities and / or activities.

EXAMPLES The present invention is illustrated by the following examples, which are not to be considered limiting in scope.

Example 1 A method of inhibiting an IL2-dependent cell line using a DNA construct selected from a DNA construct library.

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

Library A contains a molecule of the following structure: 5′AGTCCCAAGCTGGCT (N) ₁₃ CTCCATCGTGGAAGT.
CAGCCACGCTTTGGACT.

[0031]   Library B contains molecules of the following structure:   5'GACTTCACGATGGAGGTCAGAATGTGAATA (N) _Ten TATTCACATTCTGAC.

These sequences facilitate the mutual annealing of the subunits of the structure library formed by mixing and mutual annealing of the various subunits according to the scheme of the present invention, and the subunits of such structure libraries. Designed to represent a unit.

A library of oligonucleotides (subunits) was synthesized using phosphorothioate linkages to maximize stability in the presence of serum factors and HPLC
Purified by. The purified oligonucleotide was labeled with GenoSys Biot.
Obtained from technologies (Cambridge, UK). The DNA structure library was assembled using one cycle of mutual annealing. Subunit libraries A and B are denatured, mixed, and 50 mM Tris
Annealing was carried out at a temperature of 37 ° C. in a solution consisting of 100 mM NaCl (pH 7.4), 5 mM EDTA. Subunit libraries A and B were mixed at equimolar concentrations (1 DM). In other experiments, mixing was done using different molar ratios. Subunit assembly was confirmed by gel electrophoresis.

A DNA construct library was screened for constructs capable of binding to the extracellular domain of the IL-2 receptor (IL2R). This is the published method (M
eidel, M .; C. 1988, Biochem. Biophys. Res
． Commun. 154: 372-379; Meidel, M .; C. Others, 198
9, J. Biol. Chem. 264: 21097-21105). Recombinant IL2R was covalently bound to surface-activated magnetic beads using the protocol recommended by the supplier (Bangs Labs, Fishers, IN, USA). IL2R beads were used as an affinity surface to select binding structures from DNA structure libraries. IL2R beads were reacted with the library under a number of experimental conditions, including the presence of chaotropic salts in control reactions. The library (DNA) concentration was about 100 nmo in the annealing solution as described above.
It was l. Bound molecules are bound to 75 mM Tris. After a thorough wash cycle with a solution of HCl, 200 mM NaCl, 0.5% N-octylglucoside (pH 8.0), the beads were directly recovered from the beads by polymerase chain reaction (PCR). PCR was performed using the primer PRA1 (5′-AGTCCCA) to recover the library A components using standard reagent systems and conditions.
AGCTGGCT). In another reaction, PRB1 (5'GACT
TCACGATGGGAG) and PRB2 (5'GTCAGAATGTGAAT)
The library B component was recovered using the primers of A). PCR products were cloned and sequenced using standard reagent systems and procedures.

A large number of sequences were recovered and identified as originating from subunit library A and subunit library B. Of these, one pair was synthesized using phosphorothioate chemistry as described above. IL2-R1 and IL2
The -R2 oligonucleotide (sequence is shown in Figure 2) was purified and assembled as described above and used in a cellular assay for IL-2 antagonism.

TALL-104 (ATCC # CRL-11386) is a human T cell leukemia cell line. The cells grow in suspension culture but require IL-2 for optimal growth. The cells can be grown for a short time in the absence of IL-2,
Its growth is significantly reduced. Cells are treated with 50-100 u / ml recombinant human IL-
Including 2 (Life Technologies, Paisley, UK), 1
Grow in Iscobus modified Dulbecco's medium (Life Technologies, Paisley, UK) supplemented with 0% (v / v) heat-inactivated fetal bovine serum. The cells were cultured under an atmosphere of 8-10% CO ₂ . Dilutions of the annealed IL2-R1 / IL2-R2 DNA preparations and control DNA samples containing random sequences of the same outline length were prepared in culture medium containing IL-2. Parallel dilution series were prepared using medium without IL-2. The dilution series ranged from 50 DM DNA to 390 nM DNA. The assay was performed using TALL-104 cells near confluence plated the day before in 96 well microtiter dishes. Cells were harvested by centrifugation, washed with pre-warmed (37 ° C.) phosphate buffered saline,
DNA containing medium was added for 48 hours. Treatments were performed in quadruplicate. Proliferation was assessed in a colorimetric assay at the end of the 48 hour period using commercially available tetrazole compounds and following the instructions provided by the supplier (Promega, Southampton, UK). The microtiter plate was read at 540 nm.

The results showed that the annealed DNA preparations inhibited the growth of the TALL-104 cell line under conditions where the individual synthetic oligonucleotides of IL2-R1 and IL2-R2 were inactive. .

Example 2 Method of Selecting DNA Structures that Bind Human Thrombin The library described in Example 1 was used to select DNA structures that can bind human thrombin. The library was screened using a human thrombin preparation (Sigma, Poole, UK) coupled to surface activated magnetic beads according to Example 1. Thrombin beads were reacted with the DNA construct library according to Example 1, but the post-binding washes were washed with 20 mM Tris acetate (p.
H7.4), was carried out 140mM of NaCl, KCl in 5 mM, a solution consisting of 1mM of MgCl _2. Bound molecules were recovered directly from the beads by PCR using the reaction and primer set as in Example 1. PCR products were cloned and sequenced using standard reagent systems and procedures.

A large number of sequences were recovered and identified as originating from subunit library A and subunit library B. Of these, one pair was synthesized using phosphorothioate chemistry as described above. TB-R1 and TB-R
Two oligonucleotides (sequences shown in Figure 3) were purified and assembled. TB
The -R1 / TB-R2 complex was used in the thrombin inhibition assay. Clotting times were measured using a fibrometer at 37 ° C. and freshly prepared adult human plasma from a healthy donor. The degree of thrombin inhibition was determined using a thrombin standard curve in which the clotting time vs. thrombin concentration is plotted. Clotting time is
Three logarithms of DNA structure were measured in the assay.

The results showed inhibition of coagulation activity in the presence of TB-R1 / TB-R2 DNA complex.

[0041]   Example 3   A method for selecting a DNA construct that binds to recombinant soluble CD4.

Using the library described in Example 1, recombinant soluble CD4 (rsC
D4) A DNA construct was selected that could bind to the preparation. The DNA construct library was prepared by immobilizing CD4 preparations (BioDes) immobilized on activated magnetic beads as described above.
(Ign, Saco, ME, USA). Library screening, washing, and PCR selection were as described for Example 2. One oligonucleotide pair from the A and B subunit libraries was synthesized and assembled. This construct was used as an anti-CD4 monoclonal PRAT4 (Serot) in an enzyme-linked immunosorbent assay (ELISA).
ech, Abingdon, UK).

A 96-well ELISA plate was coated with coating buffer (0.
0.2m of rsCD4 in 05M carbonate-bicarbonate buffer, pH 9.0)
Coated with g / ml solution overnight. Plates were washed extensively with TBS-T (tris buffered saline (pH 8.0), 0.05% (v / v) Tween 20) and test DNA constructs and control DNA. The construct was diluted with TBS in the plate from a starting concentration of 100 DM (1: 2).
. The plates were incubated at 37 ° C for 40 minutes and washed with TBS. P
A 100 ng / ml preparation of antibody RPAT4 in BS was added to the plate and 3
Incubated at 7 ° C for 40 minutes. The plate was washed. The bound RPAT4 was then bound to alkaline phosphatase labeled sheep anti-mouse preparation (Si
gma, Poole, UK) and Sigma Fast O as chromogenic substrate
Detected using PD (Sigma, Poole, UK). In some assays, DNA was incubated with RPAT4 monoclonal. The staining intensity was read using a plate reader and the signals were compared between test and control wells. The results show that in the presence of the DNA construct, rsC
It was shown that the binding of RPAT4 to D4 was significantly inhibited.

[Brief description of drawings]

FIG. 1 is a diagram showing the step-by-step assembly of an open cubic nucleic acid construct from six individual single-stranded molecules designated as A ₁ , A ₂ , B ₁ , B ₂ , C ₁ and C ₂ . Is. Assembly proceeds by conventional antiparallel base pairing between nucleic acid molecules. Shown are the dimeric intermediates formed between the B ₁ and C ₁ molecules as well as the dimeric intermediates formed between the B ₂ and C ₂ molecules. Shown are the trimer intermediates formed between the A ₁ , B ₁ and C ₁ molecules, as well as the trimer intermediates formed between the A ₂ , B ₂ and C ₂ molecules. Assembly of the cubic structure is achieved by the association of the two trimer components and is shown as the molecule (A1 + B1 + C1) + (A2 + B2 + C2).

FIG. 2 is a diagram showing the sequences of IL2R-1 and IL2R-2, which are oligonucleotide subunits containing a DNA structure having binding activity for the IL-2 receptor.

FIG. 3 shows the sequences of TB-R1 and TB-R2 of oligonucleotide subunits containing a DNA structure having a binding activity to human thrombin.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 33/566 G01N 33/566 C12N 15/00 ZNAA (81) Designated country EP (AT, BE) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA ( AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Frankfurter Str. 250, D-64293 Darmstadt, Federal Repeat of Germany (72) Inventor Kerr, Frank, J. UK 23 8 X-X Aberdeen Valmedy The Holding Birchria (No house number) (72) Inventor Carter, Graham UK 21 7 7XB Aberdeenshire By New Macelong Long Hills Cottage (No house number) F term ( Reference) 4B024 AA01 AA11 AA20 CA01 HA11 HA17 4B063 QA19 QR32 QR81 QS34 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA04 NA14 ZA54 ZB01 ZB21

Claims (22)

[Claims] 1. A three-dimensional polynucleic acid structure composed of a plurality of chains in which a nucleic acid molecule or a portion thereof is interconnected by a specific base-pairing interaction of two or more molecules. A polynucleic acid structure characterized in that is not covalently closed. 2. The polynucleic acid structure according to claim 1, wherein the structure is formed by two or more nucleic acid molecule chains. 3. The polynucleic acid structure according to claim 2, wherein the structure is formed by three or more nucleic acid molecule chains. 4. The polynucleic acid structure according to any one of claims 1 to 3, wherein the structure is a cube or has an essentially cubic morphology. 5. The polynucleic acid structure according to claim 3, wherein the cubic structure is formed by six nucleic acid molecule chains, each of which acts like an individual side of a hexahedral cube. 6. The polynucleic acid structure according to claim 1, wherein each nucleic acid sequence comprises two or more domains capable of annealing to other molecules in the assembly. 7. The polynucleic acid structure according to claim 6, wherein each nucleic acid sequence comprises four domains. 8. The polynucleic acid structure according to claim 1, wherein the structure comprises a nucleic acid molecule composed of a region of single-stranded nucleic acid interspersed with regions of double-stranded structure. 9. The polynucleic acid structure according to claim 1, wherein each nucleic acid chain has less than 80 nucleotides. 10. The polynucleic acid structure of claim 9, wherein each nucleic acid strand has less than 50 nucleotides. 11. The structure is the assembly (A1 + B1 + C1) + shown in FIG.
The polynucleic acid structure according to claim 1, which has (A2 + B2 + C2). 12. The structure contains subunits, which are composed of regions of variable randomized sequence, in order to obtain semi-random molecules or parts thereof capable of interacting with a target molecule. The polynucleic acid structure according to any one of 1 to 11. 13. A three-dimensional polynucleic acid structure composed of a plurality of interconnected strands of nucleic acid molecules or portions thereof by specific base-pairing interactions of two or more molecules, said structure comprising: Polynucleic acid structure containing subunits composed of variable randomized sequence regions in order to obtain semi-random molecules or parts thereof whose body is covalently closed and which can interact with target molecules body. 14. The polynucleic acid structure according to claim 1, wherein the composition and length of the sequence are variable. 15. The polynucleic acid structure of claim 14, wherein the composition of the variable sequence is achieved by modification of one or more nucleotides within the sequence. 16. The polynucleic acid structure of any of claims 1-15, wherein the structure contains nucleic acid moieties or groups capable of binding or attaching to another molecule or solid matrix. 17. The other molecule is a protein, enzyme, lipoprotein, glycosylated protein, immunoglobulin or fragment thereof.
6. The polynucleic acid structure according to item 6. 18. The polynucleic acid structure according to claim 16, wherein the other molecule is a nucleic acid. 19. The polynucleic acid structure according to claim 16, wherein the molecule is pharmaceutically effective. 20. A pharmaceutical composition comprising the polynucleic acid construct of claim 19, optionally together with suitable carriers, excipients and diluents and / or other pharmaceutically effective compounds. object. 21. Use of the polynucleic acid structure according to any one of claims 1 to 18 as a diagnostic agent. 22. The poly according to claim 12 or 13 for providing a library for varying various specifically randomized topologies to obtain various functionalities and / or activities. Use of nucleic acid constructs.